ruoounu. nnSlunCIS&UINI-rOSIlnS - the Society for Reproductive ...

7JC~fsn/n noruoounu. nnSlunCIS &UINI-rOSIlnSincorporating the Proceedings of theThirty-First Annual Conference of theAustralian Society for ReproducliveBiology

·.....................•...••.........................The organising committee is delighted to welcomeyou to what we hope will be a beautiful Canberraautumn and an interesting, but, we hope, not tooexhausting program designed to clarify in our mindswhere we are heading in the next few years.We are meeting with the Australian Society forReproductive Biology for the first time in twelveyears and Friday will be given over to freecommunications of a largely scientific nature inconjunction with the ASRB, the organisation of whichhas been in the hands of Lyn Hinds. At I 1.00 amthere will be two plenary presentations and at 4.00pm the presentations from our ESHRE and theBritish Fertility Society exchange lecturers.Saturday morning is given over to symposia and theCounselling and Psycho-social free communicationsession with clinical and scientific free communicationsessions in the afternoon.The following morning willbe occupied with three symposia, We hope that theSaturday and Sunday symposia will provide a cleareridea of where we are heading and will be thoughtprovoking.The President's Debate That the Units with theloudest trumpets are the best' will be on Sundayafternoon and prizes will be presented at thecocktail party which will bring the proceedings toan end.I would like to take this opportunity to thank oursponsors without whose generous support it wouldbe impossible to stage these meetings. We thank alsoOUt' overseas speakers who have been selected withthe expectation that they will make a significantaddition to the knowledge that we possess in thiscountry and should complement what has beendesigned as very much a display of our own talent.My thanks to everyone who has assisted in theproduction ofthis meeting and to all speakers who Iam sure will make the meeting a success.Martyn Stafford-BellCONFERENCE CHAIRMAN....................................... '.............•.FSAASRBMartyn Stafford-Bell (choir) Lyn Hinds (choir)Jenny McGregorChris HardyKim RidingMichael HollandChris CopelandBob SeamarkRobert ArmellinFSAlASRB 2000 Conferencec/- Australian Convention and Travel Services (ACTS)GPO Box 2200Canberra ACT 260 I AUSTRALIATelephone 02.6257.3299Facsimile 02.6257.3256Dr R.A.Anderson (UK) British Fertility SocietyExchange lecturer will present on 'Decline of inhibinB in seminal plasma parallels suppression ofspermatogenesis in a male contraceptive study'.Dr Susan Cooper (USA) speaking on 'Avoidingpsychological and ethical pitfalls in gstationalsurrogacy' in the FSA Symposium on Surrogacy.Dr Penelope Kane (Melbourne) ASRB/FSAlecturer on 'Reproductive health needs worldwide;constraints to fertility control'Dr Rick legro (USA) speaking on 'Geneticcauses-new genes' in the FSA Symposium onPolycystic Ovary.Dr Nick Macklon (The Netherlands) speaking on'Stimulation protocols and results-effects on eggand embryo quality, implantation rates and futureimplications' in the FSA Symposium on Embryology.Dr Marilyn Monk (UK) ASRB lecturer on'Regulation of gene expression in early mammaliandevelopment'.Professor Bob Moor (Canberra) ASRB lectureron 'Why chromosomes separate at meioticanaphase'.Dr Helen Picton (UK) ESHRE Exchange lectureron The initiation of human primordial follicle growthin vitro in ultra thin slices of ovarian cortex.•

Professor Roger Short (Melbourne), ASRBFounders Lecturer~ speaking on The future fertility ofmankind'.Arnaud Wattiez (France) speaking on 'Endoscopicsurgery vs larapotomy for endometriosis andftbroids' FSA Symposium on Reproductive Surgery.ASRBWednesday 26 April 20005.00 pm-6.30 pmBal/room I, Hyatt HotelFriday 28 April 2000Saturday 29 April 2000Sunday 30 April 20007.00 am-S.OO pm7.30 am-S.OO pm7.30 am-S.OO pmcommencement of the session in which they arespeaking.They should take the slides with them totheir session. Slides can be collected from thespeaker-s' room.Dr Ryuzo Yanagamachi (USA) ASRB/FSAlecturer on llCSI and ooycte activation'.•..••••..•..••..•.....•..•.•..•.•...•...•••.......•••26 APRil 200010.30 am-5.00 pmScientists in Reproductive Technology (SIRT)WorkshopBal/room 2, Hyatt Hotel4.00 pm-7.00 pmFSA Council MeetingGriffins Lounge, Hyatt HotelTHURSDAY 27 APRIL9.00 am-5.1 0 pmSerono Symposia Australasia InternationalSymposium on "Embr-yos, Embryonic Stem Cellsand Transplantation"Federation Ballroom, Hyatt Hotel8.00 pmFSAlASRB Executives Meeting,Griffins Lounge, Hyatt Hotel28 APRIL 20007.30 amIVF Directors' Meeting and BreakfastGriffins Lounge, Hyatt Hotel8.15 am-5.00 pmANZICA Counsellors & Fertility Nurses ofAustralasia WorkshopRydges Capitol Hill, Cnr Canberra Avenue andNotional Grefe9.00 am-5.00 pmIVF Administr'ation Managers' WorkshopOak Room 2, Hyatt Hotel299.00 am-ILOO amFertility Nurses Executive Committee MeetingOak Room I, Hyatt Hotel30 APRIL 20001.00 pmFSAlASRB Executives Meeting,Griffins Lounge, Hyatt HotelFERTILITY NURSES OF AUSTRALASIAFriday 28 April 20003.30 pm-5.00 pmRydges Capitol Hill HotelANZICA COUNSELLORSSaturday 29 April 20001.00 pm-2.30 pmGriffins Lounge, Hyatt HotelfERTILITY SOCIETY OF AUSTRALIAGENERAL MEETINGSunday 30 April 20002.00 pm-2AS pmFederation Ballroom, Hyatt Hotel...•.•...•.•..•..•.........•••.•....•...•......••....A major industry exhibition is being held inconjunction with the conference. Participants areencouraged to visit the exhibition as the support ofthe exhibitors has greatly assisted in the organisationof the conference. Companies taking part in theexhibition are:IIIIIIIIIIIIIIIIIIIIIIIIIIIIAccess Australia Infertility NetworkAustralian Medical Couches/ApscanBoots HealthcareChemtec AustraliaCook IVFCryologic Pty LtdExcelrayGytech Pty LtdHD Scientific SuppliesOrganon (Aust) Pty LtdSearle HealthcareSerono Australia Pty Ltd.•••.•..•••.•....•......••..•...•...•....•..•••..•..•The registration desk will be staffed in the Atrium ofthe Hyatt Hotel Canberra during the following times:Wednesday 26 April 2000Thursday 27 April 20007.30 am-5.00 pm8.00 am-7.00 pm••••••••••••••••••••••••• ft •••••••••••••••••••••••••••Please check the message board adjacent to theregistration desk daily for messages or for changes oradditions to the program.Conference telephone and fax numbers:Telephone: 02.6270 1234and ask for the FSAIASRB Conference deskFax: 02.6281.5998(efearly mark fax-'please deliver to FSAIASRBconference registation desk)Name badges must be worn for admittance tosessions and social events.Tickets will be issued forthe ASRB Student Barbeque and the ConferenceDinner and will be collected at the door.Lunches and morning and afternoon teas areincluded in the registration fee and will be served inthe Canberra Room, Atrium and Gallery.Mobile telephones must be switched OFF duringscientific sessions and meetings.The speakers' room (Assembly Room) will be openat the following times:Wednesday 26 AprilThursday 27 AprilFriday 28 AprilSaturday 29 AprilSunday 30 April9.00 am-5.00 pm3.00 pm-5.00 pm7.30 am-5.00 pm7.30 am-5.00 pm7.30 am-4.00 pmSpeakers are asked to load their slides into carouselsand check the order and orientation oftheir slidesAT LEAST 30 MINUTES prior to theAPRIL 20007.00 pm-I 1.00 pmASRB Student BarbequeKarmel Room, ANU University Union-cost$40.00-tickets will be coilected-tronsport is notbeing provided.THURSDAY 275.30 pm-7.30 pmASRB and FSA Welcome Reception and FSAConference OpeningHyatt Hotel Canberra-included in registration feefor registrants-cost for guests $35.00.7.30 pmThe 10th Anniversary of Fertility Nurses ofAustralasia dinnerBlock Mountain Tower Restaurant-eost $25.00.Transport is not being provided.8.00 pmThe 10th Anniversary of ANZICACounsellors dinnerMosaics Brasserie, James Court Apartments,Northboure Avenue. Transport is not being provided.FRIDAY 28 APRIL 20007.00 pm-IL30 pmASRB and FSA Conference DinnerGreat Hall, Parliament House. This promises to be amemorable evening. Cost per ticket $60.00, includesfood, drink, entertainment and transport. Note-thisis a subsidised cost for FSA or ASRB member or nonmemberregistrants only. Ail others cost $100.00.Tickets will be collected.Coach transport will depart from conferencehotels from 6.45 pm and return at 10.30 pm and11.30 pm.•

..•.•••....•.•..•..•.....•.•..•.....•..•..•..•...•......••.•..•.•.•.....•.•....•.•••...•......••....•....•.......The following bus transfers will be provided to and from the conference venue. Please note that buses will leavefrom the rear entrance of the Hyatt Hotel. Please allow Ia minutes for buses to arrive from another hotel.DAYWednesday 26 AprilThursday 27 AprilFriday 28 AprilSaturday 29 AprilSunday 30 AprilCONFERENCE HOTELSIII Hyatt Hotel CanberraIII Rydges Canberra III Ursula College.DEPART CONFERENCE HOTELS DEPART HYATT HOTEL5.30 pm7.30 pm8.00 amB.OOam5.45 pm6.15 pm8.00 amBrassey Hotel III Rydges Capital Hill..•........••........•.........•....•.... ,..•......................................•..•.......•...................2000 OFfiCE BEARERS 1988 John Yovich ryvA)President Robert McLachlan (Vic) 1989 Gordon Baker (Vic)Vice President Christine Kirby (SA)1990 Gordon Baker (Vic)1991 Gabor Kovacs (Vic)SecretaryKarin Hammarberg (Vic)1992 John McBain (Vic)TreasurerDonna Howlett (Vic)1993 Leon Clark (NSW)Assistant Secretary Kim Matthews (NSW) 1994 Colin Matthews (SA)...........................•.......................•...........................•...........•.....•.............•.Year1982198319841985198619871988198919901991. 19921993199419951996199719981999Best Clinical Paperby a Recent GraduateD. HurleyD.KeepingB. DowningD. MolloyR. McLachlanH.R. DraperS.ThorntonA. LowerI. CalderonN. McClureU. SunqurtekinJ. EdenB.WatkinsK. MatthewsC. SternS. RobsonN. HusseyBest Scientific Paperby a Young ScientistM. SinosichJ,Cl.lmrninsC. O'NeillP. NayuduJ. CarrollC. HoldenD. GookM.JasperG. FleishmanH. KarusoD. PayneS. MortimerD. PayneM.O'BryanBest Mini-PosterPresentationP. LutjenWGillettc.Y--T SunS. OsbornK~HCuiC. GarrettS. CookeK~HCuiA.VennG. PhillipsonI. van WezelS. FlahertyCouncil Members Peter Benny (NZ) 1995 David Healy (Vic)Joi Ellis (NZ) 1996 David Kay (NSW)Keith Harrison (Qld) 1997 Roger Cook (Vic)Anne Jequier (WA)1998 Richard Fisher (NZ)Gayle Jones (Vic)1999 Ric Porter (NSW)Frank Quinn (NSW)MEMBERSHONORARY MEMBERSJames BrownImmediate Past PresidentHenry BurgerRic Porter (NSW)Lloyd CoxPresident, IVF Director's GroupRobert EdwardsDavid Molloy (Qld)Tim GloverACCESS RepresentativeLachlan Hardy-WilsonSandra Dill (NSW)Bryan HudsonChairman, RTAC Ian Johnston (Vic)Ian JohnstonGeorgeanna JonesYear19891990199119921993199419951996199719981999Best Paper by aNurseS. RandallF. HutchingsE.DurnaR. GilderS. ChamberlainP. HillR.WithersD. SmithS. Brown/G. HolmanC. OwenH. PlummerBest Paper by aCounsellorF. GarnerJ.AndersonK.OkeH. WellsmoreK.OkeBest Paper onPsychosocial IssuesH. PollockS.GriersonA. Rl.lmballS. de LaceyK. Hammarberg•1983 Ian Johnston (Vic) Howard Jones Jr.1984 Warren Jones (SA) Warren Jones1985 Doug Saunders (NSW) John Leeton1986 Tim Glover (Qld) Colin Matthews1987 Stephen Steigrad (NSW) Carl Wood

IIIIIIIIIIIIBest mini-poster presentationBest scientific paper by a young scientistSponsored by Serono - restricted to scientists,35 years or younger or who have graduated inthe last 15 yearsBest clinical paper by a recent graduateSponsored by Organon - restricted to clinicians,35 years or younger" or who have graduated inthe last 5 yearsBest paper by a nurse Sponsored by SeronoBest paper by a counsellor Sponsored bySearle HealthcareBest paper on a psychosocial issueSponsored by Searle HealthcareThe above prizes will be awarded at the CocktailPat"ty to be held on Sunday afternoon, 30 April2000.Both qualitative and quantitative studies will beconsidered for the counselling and psychosocialprizes.To ensure that there is uniform judging for thesix prize categories, a limited number of judgingpanels will be used and eligible presentations will beadjudicated in selected sessions.SELECTION ANDABSTRACTSOfAbstracts were judged anonymously by reviewers onthe clarity and appropriateness of the originality,aims, materials and methods, results, and conclusions.A value was awarded for each of these criteria andthe overall scores were used to select abstracts forpresentation.PRIZESANDTo ensure that there is uniform judging, eligiblefORpresentations will be pre-selected and adjudicated inselected sessions. Prize winners will be announced atthe final cocktail party.The decisions of the judgingpanels will be final and no correspondence will beentered into....................................................•..The FSA conference has been approved for thefollowing number of Category A points in theContinuing Education program ofThe RoyalAustralian College of Obstetricians andGynaecologists. To obtain these points please sign theform available at the registration desk:full attendanceattendance 28 Aprilattendance 29 Aprilattendance 30 AprilChairmanMarilyn RenfreeHonorary SecretaryTina LavranosHonoraryTreasurerMark HarveyCommittee MembersJon CurlewisJohn HearnGraham JenkinJill ShawAnne TurnerClinical RepresentativeRob NormanPostgraduate RepresentativeNatasha RanceCommunications OfficerJim CumminsChair, JSA CommitteeGraeme Martin21 points8 points8 points6 points.........................................'....•..•...•.....................................................•......Year Chairman Treasurer Secretary1969-701970-71 T.J. Robinson R.G.Wales j.R. Goding1971-72 \)1972-73 ft,--1973-74 I.GWhite .].t1;,Btnd~1974-75 Cw. Emmens1975-76 ( ~ W. Hansel1976-77 t 0.1 Baird1977-78 G.M. Stone I.A. Cumming T.O. Glover1978-79 D.M. de Kretser C Tyndale-Biscoe1979-80 £ Ntl~ GThwaites1980-81 tIf GH. McDowell., R.J.•SS.at::am1:1zzi K.P. McNatty1981-82 N.W. Moore B.K. Follett1982-83 B.G Miller 1K. Roberts J.Wilson R.J. Rodgert1,.& C.B.Gow1983-84 J.K. Findlay I~ 7 B.M. Bindon S.P. Flaherty1984-85 i Co. Nancarrow j.M. Cummins A. Bellve C O'Neill1985-86 B.M. Bindon~B.P. Setchell B.j.Waddell1986-87 Nf.I \'l.. G Evans W. Hansel L.j. Wilton,1987-8'8 B.P. Setchell LA Hinds A H,j. Burger A. Stojanoff1988-89 ,i\ ~ J.M. Shelton F.w. Bazer M.B. Harvey1989-90 j.C Rodger GB. Martin GO. Thorburn A.H,Torney1990-91 !(i1991-92 R.J. Scaramuzzi CGTsonisI' P.L. Kaye R.M. Moor H. MassaIi CR. Austin DX Gardner1992-93 L.A. Salamonsen J.K. Findlay S.W.Walkden-NIl N;\ Brown1993-94 A.0.Trounson L.J.Wilton If) Ge. Liggins CM. Markey1994-95 M.P. Hedger I. Huhtaniemi M.J.Hotzel &~l't,\S. McDougall'..:1995-96 R.F. Seamark J.D. Curlewis R.F. Seamark I.vanWezel1996-97 j) ~ J.G Thompson No lecture S. Robinson1997-98 F. Bronson M.jasper1998-99 M.B. Renfree M.B: Harvey1999-2000()..D.M. De KretserM. Panteleoni> ~ 1 Lavranos lanWilmut E. Whiteside2000-2001 ~ Roger Short

NSnot due no potyeystle mtarianQr~elttdnoma.::.,....'I'ulE()UriI·.l:t··~ breastll, uterus" testis, ~us,iIPd pltuit!U't ~.- Prior bypersensitti'ity td Puregon!'_ Any condition in which a pregaaney (in~ ~ulliplepregnan'cy) would bepartlcu1arly haza11d()llJ(e.g, e:memesofwelght disorders and uterine abno~).- Unexplained vaginal b1 7 ed!ng.prepncy.PUREGON® - PRODUCT INFORMATION_Puregonoiscontraindicatedwhen an effective ~nse can-,not be obtained, suchas;- PrimarYovarian failure such as Indicated byhighlevels ofFSH.Organic disorders of the reproductive organs incompatible~ ma\forinationsof theut_ Puregon° should not be used in the elderly or in children,(vi) ~CAUTI()NS_k;I pregnancies occurring after induction of oVulation withgonadotropic preparations there is an increased risk ofmuItiplets.AnliIySls of pooled data does not indicate thllt the use ofgona4l:>trop/nlf lB ovulation induction and medically assisted.reproduction progrll1II8 cani.es an inereased risk of con,genjtallllll1tbrmations.- Priorthe foilL QtrefuI clinic:algeri.if:al. Pllthology.lI11 unacteptable increase in the amount of third SPllCe fluidacMa'pagenaentinclud~ac1n:liQlstration of limitedand serum albumin.t .ofetl1la, is recomq;for inadequate gonadal fuIlction,assessed;tion to deth't>'",!"",,,>,;w,,,,,,,Puregono100IU/o.5DlL Clear glass vials cont:airiib:g,.t6llitropinbeta 100 IU per 0.5 mL ofsolution for injection.Puregono150 lU/0.5DlL Clearglass vials cont3iDlgg.rfollitrQpmbeta 150 IU per 0.5 mL ofsolution for injectioni:Puregono 200 J:U/0.5mL Clearglass vials con~g(g1.\ittOPlnbeta 200 IU per 0.5 mL ofsolution for injPuregono 225 IU/0.5mL Clear glass vialsbeta 225 IU per 0.5 mL of solutiOl)fo~.~jPuregonO 250 IU/0.5mL Clearglassbeta 250 IU per 0.5 mL of solutionforlfi,jt;ctio~\i7f'Instructions for use/handlingThe freeze-dried substance is reconstirutevent.Puregon" solution for injectioJ;lis used lIS }.s.StoragePowderfor injection:Stored in the dark below 30·C (do not ;"",,,,,"'\ ,., ." ".Solution for injection:Stored at 2 to S·C (refrigerate, do not freeze).Shelf·life: All forms - two years whenstoredPuregono may be used until the expirationpackage.Powder for injection: Since an opened ampoule'01';ylal cannotbe resealed in such a way to further guarati~;ttie;jsterilltyof the contents, the solution should be used immediately afterreconstitution.Solutionfor injection: The entire contents ofthe vial shouldbe used in one injection.Vials are single-use only.The Information supplied relatesoruy to I'tiii:gdh-ahd lil:lQl!J

.....•.................................................•......................................................•...........TIME BALLROOM 1 OAK ROOM NTH10.00am-11.45am ASRB Seasonality ASRB ART Male& Neuroendocrinology12 noon-1.00pm Founders Lecture2.00pm-3.30pm4.00pm-4.30pm4.30pm-5.00pm5.00pm-6.30pm2000EVENT BALLROOM 1 BALLROOM 2 OAK ROOM NTH8.30am-10.30am FSNASRB Male FSNASRBReprod. Tract SpermOoctye &Emb. Dev.11.00am-12.30pmSATURDAY 29SUNDAY 3020002000EVENT fED NTH (1) fED STH (2) ALBERT HALL8.30am-1.00pm FSA Symposium on FSA Symposium on FSA Symposium onthe Male Surrogacy Polycystic Ovary Syndrome2.00pm-2.45pm2.45pm-3.45pm4.00pm-5.00pmFSNASRB Plenary LecturesDr R. YanagamachiDr Penny Kane1.30pm-3.30pm FSNASRB FSNASRBOvarian Dev. Ass.UterusReprod.-Female4.00pm-4.40pmJSA PresentationsProf Bob MoorDr Marilyn MonkASRB AGMFSNASRB Plenary LectureDr Helen Picton4.40pm-5.20pm FSNASRB Plenary Lecture ASRB OvarianDr R.A. Andersonfunction &foil. dev.(till 5.45pm)7.00 for 7.30pm fSA/ASRB COMBINED CONfERENCE DINNERGREAT HALL, PARLIAMENT HOUSEEVENT BALLROOM 1 BALLROOM 2 ALBERT HALL8.30am-1.00pm FSA Symposium on FSA Symposium on FSA Free Comm.Embryology Reprod. Surgery Counselling & Psychosocial2.00pm-3.45pm FSA Free Comm. FSA Free Comm. FSA MiniposterSperm & Embryos IVF Admin & Outcomes session(tilI3.15pm)(tilI4.15pm)4.15pm-6.00pm FSA Free Comm. FSA Free CommIVF ClinicalInfertility (Clin.)FSA General MeetingPresident's DebatefiNAL COCKTAIL PARTY·..................................•.......•...................•.......................•.•.•..............•...•..........•BALLROOMTIME1000-1015 Oral1015-1022 Miniposter1022-1030 Miniposter1030-1037 Miniposter1037-1052 Oral1052-1107 Oral1107-1122 Oral1122-1130 Miniposter1130-1137 Miniposter1200-13001300-1400OralAUTHORM. Guerin, J. Deed and C. MatthewsM. Guerin, J. Deed and C. MatthewsM. Guerin, J. Deed and C. MatthewsM. Guerin, J. Deed, R. Woodward,D. Washington and C. MatthewsD. B1ache and G. MartinP. Celi, G.B. Martin, D. Blache,P.E. Vercoe and R.L. TellamM.W. Leihy, G. Shaw, J.D. Wilsonand M.B. RenfreeD.A. Coveney, G. Shaw, M.B. Renfree,P.J. Farmer and J.M. Hutsonl Cheung and J.P. HearnBALLROOM 1-ASRB-=", ~' ~~- t~lV.FOUNDER'S LECTURE (chair-Marilyn Renfree)Professor Roger ShortLUNCH-ATRIUM, CANBERRA ROOM, GALLERYNORTH-ASRBSEASONALITY AND NEUROENDOCRINOLOGY(chairs-Graeme Martin and Geoff Shaw)Afternoon, not morning, exogenous melatonin provides alongnight signal to initiate ovarian activity in the ewe.The circadian pacemaker responds differently to the naturalphotoperiod in a highly and aless seasonal breed of ewe.Advancing a short night signal does not necessarily stop ovarianactivity earlier in the ewe.Oral melatonin administered to cycling mares in autumndoes not advance anoestrus.Asingle intracerebroventricular injection of orexin-A stimulatespUlsatile LH secretion in mature male sheep.Effects of central administration of recombinant bovine leptin onpulsatile LH secretion in male sheep under different feedingregimes.5 d -Androstan-3d, 17B-Diol virilises the developing femalemarsupial genital tract.Calcitonin-like immunoreactivity in the genitofemoral dorsalroot ganglia of the tam mar wallaby, Macropus eugeniiMolecular cloning of wallaby GnRH receptor geneThe future fertility of mankindTIME AUTHOR ASSISTED REPRODUCTIVE TECHNOLOGY-MALE(chairs-Russell Jones and Michael Holland)1000-1015 Oral J.E Smith, RM. McDonald, Motility parameters of fresh and frozen abaloneRD. Roberts, S. Adams and P.A. Pugh (Haliotis iris) sperm.1015-1030 Oral R.K. Browne, J. Clulow and M. Mahony Short-term storage of cane toad (Bufo marin us) sperm withsubsequent cryopreservation.1030-1037 Miniposter J.E Smith, R.M. McDonald, D.K. Berg, Effect of diluent and oviduct conditioned media on the motilityK.S. Sidhu and EC. Moliniaand viability of brushtail possum (Trichosurus vulpecula)epididymal sperm.1037-1045 Miniposter M. Porn mering, R.K. Browne, Non-invasive collection and cryopreservation ofJ. Clulow and M. Mahony amphibian sperm.1045-1052 Miniposter S. Watson, S. M. Junk, S. Huntress, Successful cryopreservation of non-human primatelP. Fletcher and J.L. Yovichspermatozoa.1052-1049 Miniposter A.R Jones and E Piccolo Glycolytic enzyme activity in hypotonically-treated boar •spermatozoa.1100-1115 Oral W. Gu, P.J. Kerr and M.K. Holland, Immune responses in the female rabbit reproductive tract toR Jones, P.A. Janssens andinfluenza HA or rabbit ZPB delivered by recombinantRE Seamarkmyxoma viruses1115-1130 Oral R. Knee, K.W. Beagley, J. Clulow Role of the male reproductive ducts in concentrating spermand R.C. Jonesand IgG antibodies: significant for immunocontraception.1130-1137 Miniposter H. Ecroyd, K.W. Beagley, The entry of immunoglobulin into the male reproductive tractN. Newcombe and R.C. Jones

(Conlinuod).•.•.•.......•.•....•..•.••••..•..•...•.•...•.....•••..•....•...••..•.•.....•••.••....•.•.•.•....••.•..•...•..••...•....•.BALLROOM 1 (ASRB)1400-14151415-14301430-14451445-15001500-15151515-15301530-1600BALLROOM 1 (ASRB)1600-16301630-17001700-18301900-IateJSA PRESENTATIONS (chair-Marilyn Renfree)OralOralOralOralOralOralSPECIAL INVITED LECTURES (chairs-Lyn Hinds and Bob Seamark)Oral *Prof Bob Moor Why chromosomes separate at meiotic anaphase*SPONSORED BY PEST ANIMAL CONTROL CRGA.J. Pask, M. B. Renfree,R.J. Waugh O'Neill, S.L. Layfield,J.A. Marshall Graves and J. L. HarryC.E. Gargett, EL. Ledermanand PAW. RogersL.M. Kilpatrick, D. Xu, I. Kolaand L.A. SalamonsenI.K. Greaves, M. Svartman,M.J. Wakefield, D. Taggartand JAM. GravesL.T. Sebastian, G. Shaw, G.E. Riceand L.J. ParryS.D. Roman, C. Wallaceand K.A. SuttonAFTERNOON TEA-ATRIUM, CANBERRA ROOM, GALLERYAnnual General Meeting of ASRBSTUDENT EVENING-ANU UNION, INCLUDES BUSH BANDThe roles of SOX genes in marsupial sex determination.VEGF, intravascular neutrophils and human endometrialangiogenesis.Progesterone downregulation of matrix metalloproteinase-1(MMP-1) in the human endometrium: evidence for indirecttranscriptional regulation.Non-random arrangement of chromosomes in Sminthopsiscrassicaudata and Macropus eugenii spermProstaglandin Hsynthase-2 gene expression in the endometriumand placenta of the Tammar wallaby.Identifying markers of the mammalian male germ line usingsubtractive hybridisation.Oral Dr Marilyn Monk Regulation of gene expression in early mammalian development......•....•.....................................•........................................................................TIME AUTHOR TITLE: MALE REPRODUCTIVE TRACT/SPERM FUNCTION(chairs-Gordon Baker and John Aitken)0830-0845 Oral B. Lewis and J. Aitken Impact of epididymal maturation on the redox regulatedpathways controlling sperm capacitation.0845-0852 Miniposter M.A. Wade,R.J. Aitken, R.C. Jones Mechanisms of activation of mammalian sperm.and R.N. Murdoch0852-0890 Miniposter D.Y. Liu, M. Martic, G.N. Clarke, Anti-Actin monoclonal antibody inhibits hyperactivation and theI. Grkovic, C. Garrett, M.E. Dunlop zone pellucida induced acrosome reaction of human sperm.and H.W.G. Baker0900-0907 Miniposter M. Lin, C. Scarlett and R.J. Aitken Final construction of sperm acrosome may be anactin-mediated process.0907-0915 Miniposter M.S. Harris and J.C. Rodger Development of PSA-10 monoclonal antibody reactivity withBrushtail possum (Trichosurus vulpecula) spermatozoa duringepididymal maturation.0915-0930 Oral O. Gerdprasert, M.K. O'Bryan, Monocyte chemoattractant protein -1 (MCP-1) is up-regulated inD.P. Nikolic-Paterson, K.L. Sebire, the testes of lipopolysaccharide-treated adult rats: apossibleD.M. de Krester and M.P. Hedger role in monocyte recruitment.0930-0937 Miniposter M. Martic, D.Y. Liu, M.E. Dunlop SNARE proteins, Syntaxin and Munc-18 are present inand H.W.G. Bakerhuman sperm0937-0945 Miniposter H.W.G. Baker, C.J. Stern Azoospermia, Hypospermatogenesis, low FSH, high inhibin Bandand D.M. Robertsonpossible intratesticular genital tract obstruction.0945-0952 Miniposter D. Wright, K. De Boer, J. Persson Analysis of sperm using fish to determine the appropriatenessand C. Robertsof IVF with preimplantation genetic diagnosis in cases of malechromosomal anomalies.0952-1000 Miniposter S. Maddocks, B.P. Setchell Whole body heat-stress of male mice impairs sperm-zonaand J. Yaerampenetration.1000-1007 Miniposter M.G. Katz, D.S. Cram and A.O. Trouson Development of DNA fingerprinting for application in assistedreproductive technology.1007-1015 Miniposter Y. Sistina, R. Trijatmoko, I Setiadi Induced triploid by heat shock in NILEM (Osteochilus hasseltiand R. WidiastutiC.V.) fish.1015-1022 Miniposter R.V. McClean and WG. Breed Glycoprotein maturation of the Brushtail possum sperm plasmamembrane during epididymal transit.1022-1030 Miniposter N.J. Cooper and w.G. Breed Changes in glycoprotein composition of the plasma membraneof Brushtail possum spermatozoa, during epididymal migration.1030-1100 MORNING TEA-ATRIUM, CANBERRA ROOM, GALLERYTime Author TITlE: OOCYTE AND EMBRYO DEVELOPMENT(chair-Keith Harrison)0830-0845 Oral H. Jericho, E Nieto and D.H. Edgar Investigation of the effects of glucose and phosphate on earlycleavage stage embyros asibling oocyte stUdy0845-0900 Oral C. SjOblom, M. Wikland Glucose transport in murine blastocysts is stimulated byand SA Robertsongranulocyte-macrophage colony-stimulating factor (GM-CSF).0900-0907 Miniposter KM. Hartwich, J.S. Robinson Foetal outcome following the exposure of ovine embryos to highand S.K. Walkerammonium concentrations in vitro.0907-0915 Miniposter D. Sherrin, T. Breen, M. Hunter Problems associated with the detection of multinucleatedand K. Harrisonblastomeres in human embryos.0915-0930 Oral G. Abdelnour, S. Fleming, G. Coticchio Examination of the interaction of EGF, FSH and Activin a onand P. Illingworthmeiotic resumption in mouse oocytes0930-0937 Miniposter T.T. Peura, H.M. Hamilton Incubation of two hours in 6-DMAP is sufficient forand S.K. Walkerparthenogenetic activation of sheep oocytes.•

1lI_1II11l1111f11111l1111 (Continuod).....................•.......•..................•.................•............•.....................•............................................. (Conlinuod)••••••••••••••••••••••••• &••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••BALLROOM 2-FSAlASRB (Continued)0937--0945 Miniposter C.T. Murray and J. Catt0945--09520952-10001000-10151015-10301030-1100fEDERATION BALLROOM-fSAlASRBTIME1100-11401140-12201220-1330MiniposterMiniposterMiniposterOralS. Robson, M. Barry and R.J. NormanH.H. Hamilton, S.K. Walker, G.C. Webb,S. Maddocks and 11 PeuraPS. Duggal, 0.1 Armstrong,D.A. Magoffin and R.J. NormanMORNING TEA-ATRIUM, CANBERRA ROOM, GALLERYAUTHORDr Ryuzo YanagamachiDr Penny KaneLUNCH-ATRIUM, CANBERRA ROOM, GALLERYBALLROOM 1-fSAlASRBEntry into first cell cycle as a sensitive laboratory TIME AUTHORperformance measureAssessment of ovarian follicular vascularity by power doppler 1330-1345 Oral M. Zaitseva, P. Rogers and C. Gargettsonography during transvaginal oocyte retrieval in IVF cycles-A pilot study.Development of bovine-ovine interspecies clones: preliminary "1345-1400 Oral U. Manuelpillai, PAW. Rogersstudies.and B. VollenhovenTo what extent does feeding rumen-protected 3-omega fatty 1400-1407 Miniposter I. Mylonas, L. Winkler, J Makovitzky,acids alter the reproductive endocrinology of dairy cows.D-U. Richter, UJeschke, V. BrieseThe in vivo effect of leptin and feed restriction on rat ovarianand K. Frieseleukocytes.1407-1414 Miniposter C.T. Roberts, R.A. Earl, J.J.H.M. Erwich,J.S. Robinson and IV. KhongSPECIAL INVITED LECTURES(chairs-John Hearn and Lyn Hinds)ICSI and Oocyte ActivationReproductive Health Needs Worldwide; Constraints toFertility ControlTIME AUTHOR TITLE: OVARIAN DEVELOPMENT/ ASSISTEDREPRODUCTION· fEMALE(chairs-Jill Shaw and Bob Moor)1330-1345 Oral H. Newton and P. Illingworth In vitro growth of murine preantral follicles after isolation fromovarian tissue frozen/thawed in different cryoprotective agents1345-1352 Miniposter M. Snow, M. Cleary, S-L. Cox, J. Shaw, Viability of mouse ovarian tissue collected from deceasedM. Wolvekamp and G. Jenkin females.1352-1359 Miniposter M. Clearv, M. Snow, M. Wolvekamp, The effect of cooling rate and prolonged exposure to anJ.Shaw, S-L. Cox and G. Jenkin ischaemic environment in situ or in vitro on post thawovarian tissue viability.1400-1407 Miniposter o Mattiske, G. Shaw and J. Shaw Development of ovarian tissue from Macropus eugenii pouchyoung after grafting to SCID mice.1407-1415 Miniposter D. Gook, B. McCully, A.Nelson, Development of antral stage follicles in human cryopreservedC. Riley and J. McBain ovarian tissue following xenografting in SCID mice1415-1422 Miniposter J. Archer and D. Gook Isolation and culture of human follicles following thawing ofcryopreserved ovarian tissue1422-1430 Miniposter J.w. Catt Successful cryopreservation of biopsied embryos1430-1437 Miniposter P.R. Hurst, MW. Fisher and B.J. McLeod Follicle populations in prepUbertal, mature and aged red deer hinds.1437-1445 Miniposter O.L. Clow, P.R. Hurst and J. Fleming Studies on ovarian surface epithelial cell biology in the mouse.1445-1452 Miniposter KE Mate, K.S. Sidhu, EC. Molinia, Sperm binding and penetration of the zona pellucida in vitro butA.M. Glazier and J.C. Rodgernot sperm-egg fusion in an Australian marsupial, the brushtailpossum (Trichosurus vulpecula).1452-1500 Miniposter G.M. Magarey and K.E. Mate Intracytoplasmic sperm injection of Tammar wallaby oocytes.1500-1515 Oral D.S. Cram, B. Song, R. McLachlan Transmission of androgen receptor CAG repeat expansions inand A. Trounsonmen with spermatogenic disorders by intracytoplasmicsperm injection1515-1522 Miniposter N. Korfiatis, A. Trounson and Effects of donor cell cycle on in vitro development of BovineO. Lacham-Kaplan nuclear transfer embyros produced by piezo injection.1522-1530 Miniposter o Lacham-Kaplan, M. Diamente, Nuclear transfer by injection of mechanically andD. Pushett, V. Hall, I. Lewis chemically isolated fetal fibroblast nuclei in the bovine.and A. Trounson1530-1600 AfTERNOON TEA-ATRIUM, CANBERRA ROOM, GALLERY1415-1422 Miniposter P. Rogers, PAW. Plunkett and B. Affandi1422-14291430-14451445-14521452-14591500-15071507-15141515-15221522-15291530-1600TIME1600-16401640-17201900-lateMiniposterOralMiniposterMiniposterMiniposterMiniposterMiniposterMiniposterS. Robertson, A. O'Connelland A. RamsayE. Dimitriadis, L. Robband L.A. SaalamonsenS. O'Leary, 0.1 Armstrong, R. Kamaiand SA RobertsonW. Gu, M K Holland and P.J. KerrJ.G. Thompson, S. Cox, D. Berg,S. Meier and G. VerkerkA.M. Padula and K.L. MacmillanA.M. Padula and K.L. MacmillanF.C. Molinia, A.M. Glazier and J.V. MyersAfTERNOON TEA-ATRIUM, CANBERRA ROOM, GALLERYBALLROOM-fSA/ASRBAUTHORESHR Exchange lectureDr H.M. Picton, A. Mkabdkam,O. Salha, P. Wynn and R.G. GosdenBritish fertility Society ExchangeLecturer-Dr R.A. AndersonC.W. Martin, C.S. Riley, N.P. Groomeand D.IBairdCONfERENCE DINNERGREAT HALL, PARLIAMENT HOUSETiTlE: UTERUS(chairs-Peter Rogers and Sarah Robertson)Regulation of VEGF receptors by estrogen and progesterone inmicrovascular endothelial cells from human myometrium andfibroids.Effects of insulin like growth factors and estrogen on uterineleiomyoma and myometrial smooth muscle cell proliferationIsolation, purification and cultivation of normal humanendometrium cells. Immunhistochemical expressions ofdifferent cytokeratins and vimentin in freshly isolatedglandular epithelial cells.Endothelial activation precedes trophoblast invasion of themesometrial arteries in pregnant guinea pigs.Perivascular smooth muscle-Actin is reduced in theendometrium of women with progestin-only contraceptivebreakthrough bleedingThe effect of interleukin-6 deficiency on implantation, fetaldevelopment and parturition in mice.Expression of interleukin 11 and its receptor in the humanendometrium throughout the menstrual cycle.Intrauterine seminal plasma induces endometrial inflammatoryresponse in gilts.Immunocytes in the female rabbit reproductive tractEnergy substrate and ionic composition of day 7 bovineuterine fluid.Observations on oestrous suppression using hydroxyprogsteronehexanoate in oestrogen challenged ovariectomised cattle.Serial plasma progesterone concentrations in ovariectomisedholstein cows treated with new and reused CIDR devices.Superovulation of the brushtail possum during seasonalanoestrus with porcine FSH/LH.fSA PLENARY LECTURES(chair-Rob McLachlan)The initiation of human primordial follicle growth in vitro in ultrathin slices of ovarian cortex.Decline of inhibin Bin seminal plasma parallels suppressionof spematogenesis in a male contraceptive study.•

lIlJIlI(ConlinuGd)••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 00 ••••••••••• 0•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••OAK ROOM NORTH-ASRBTIME1645-17001700-17151715-17221722-17291730-17371900-IateOralOralMiniposterMiniposterMiniposterAUTHORT.C lavranos and RJ. RodgersR.B. Gilchrist, L.J. Ritter, D.B. Rowe,S. Fujii, RJ. Norman, S.A Roberstonand D.T. ArmstrongM.A. Abdo, and A.M. DharmarajanA.E. Drummond, M. Dysonand J.K. FindlayH.E Irving-Rodgers and R.J. RodgersCONFERENCE DINNERGREAT HALL, PARLIAMENT HOUSETITLE: OVARIAN fUNCTION AND FOLLICLE DEVELOPMENT(chairs-Jock Findlay and Tina Lavranos)Expression of the telomerase catalytic subunit (TERT) in bovineovarian follicles at different stages of follicle development.Differential expression of GM-CSF receptor mRNA subunits infollicular cells and the effect of GM-CSF deficiency on oocytemeiotic competence.Characterisation of caspase proteolytic cascade following tumournecrosis factor-alpha (TNFa) induced corpus luteum (Cl)apoptosis.Analysis of rat ovarian inhibin subunit mRNA expression by"Real Time" PCRlaminin and Type IV collagen composition of the follicularbasal lamina of bovine atretic follicles.TIME0830-09050905-09400940-10151030-11001100-11351135-121012.10-12451300-1400TIME1400-14151415-14301430-14451445-15001500-15151515-15301530-1545SPEAKERGayle JonesPeter KayeRobert DanielsMORNING TEA-ATRIUM, CANBERRA ROOM, GALLERYBob SeamarkSome results of IVF procedures in animalsAlan TrounsonCurrent research areas and future prospectsN.S. MacklonStimulation protocols and results-effects on egg and embryo quality,implantation rates and future implicationsLUNCH-CANBERRA ROOM, ATRIUM, GALLERYAUTHORW.R. Edirisinghe, J.E. Dowd and J.A. AllanJ.A. Allan, A. Catakovic and W.R EdirisingheN.E. Merry, M. Attard and D.H. EdgarN. Richings, H. Bourne and H.W.G. BakerL. Wilton, R. Williamson, H. Slaterand l. VoullaireL. Gras, G.M. Jones, A.P. Kauscheand A. TrounsonS.J. McArthur, K.A. De Boer, D. leigh,C.G. Robert, J.w. Catt, M.C. Bowman,J.A. Perrson, J.C. Andersonand R.P.S. Jansen1545-1615 AFTERNOON TEA-ATRIUM, CANBERRA ROOM, GALLERYFSA SYMPOSIUM ON EMBRYOLOGY(chair-David Edgar)Current knowledge of egg preparation for ovulation and fertilisationEmbryo metabolismMolecular basis of embryo development leading to implantationFSA FREE COMMUNICATIONS-SPERM AND EMBRYOS(chairs-David de Kretser and Harold Bourne)The effect of morphology and motility of testicular sperm on fertilisationand pregnancy ratesTesticular aspiration: Freezing seminiferous tubules and ICSI: A viablealternative to vasectomy reversalA comparison of four thaw protocols for frozen embryosAn assessment of factors associated with implantation in embryosgenerated from re-inseminationChromosome analysis of blastomeres from human embryos usingcomparative genomic hybridisationAnalysis of aneuploidy in embryos from 53 patients with poor prognosisof pregnancyExperience with the introduction of a comprehensive preimplantationdiagnostic service for serious genetic diseaseTIMEAUTHORFSA FREE COMMUNICATIONS-IVF CLINICAL(chair-Andrew Speirs)1615-1630D.C. Knight, J.P.P. Tyler and G.L. DriscollDietary-derived isoflavons are present in ovarian follicular fluid in womenundergoing transvaginal oocyte collection1630-1645G.L. Driscoll, J.P.P. Tyler, J.T. Hangan,P.R. Fisher, M.A. Birdsall and D.C. KnightA prospective randomised controlled dOUble-blind, double-dummy, comparisonof recombinant and urinary hCG for inducing oocyte maturation and follicularluteinization in controlled ovarian hyperstimulation1645-17001700-1715P. Benny, A. Gooreratne and S. HurdM. Rawashdeh, L. Arthur, H. SmithThe effect of lH priming prior to ovarian stimulation with FSH in GnRHatreated womenA retrospective study of luteal phase hormone concentrations in differentforms of luteal support for assisted reproduction•1715-1730C. Princehorn, P. O'Donnell and J. StangerFriday starts: A management advantage1730-17451745-1800R. Norman, C. Kirby and J. ThompsonG. Kovacs, A. Meeking and V. MaclachlanPregnancy following ICSI of the wrong sperm? 'De Novo' mutation orpUblic relations disaster?Why do people who commence IVF treatment stop

IInllll (Conlinuod)..•.•..••••...•.•..•....•..•..•.....•......•.....•........••..•.....•.•..••........•.......•..•.•.••...••.....••...•....•.BALLROOM 2-fSATIME SPEAKER fSA SYMPOSIUM ON REPRODUCTIVE SURGERY(chair-Ric Porter)(Conlinuod).............•...................•.................................................•........•.................•...•......•ALBERTTIME AUTHOR fSA fREE COMMUNICATIONS-COUNSElLING &PSYCHOSOCIAL(chairs-Kay Oke and Jenny Blood)0830-0855 Ossie Petruceo The place of surgery. selection of cases and results 0830-0845 A. Macdonald and RH. Cook Infertility and Assisted Reproductive Technology-Community understanding0855-0915 Ossie Petruceo Current trends in adhesion prevention. research areas and results ofanti-adhesion agents to date0845-0900 K. Bourne Pregnant again following the loss of an IVF pregnancy-"l feel like I havebeen pregnant forever"0915-0935 David Molloy Endoscopic surgery for repair of pelvic damage 0900-0915 K. Looi The welfare of the child-philosophy becomes a practice0935-0955 Andrew Speirs Microsurgery for repair of pelvic damage 0915-0930 L. O'Byrne The effect of first cycle follow up by counsellors0955-10151015-10351035-11001100-11201120-11401140-12001200-12201220-12401300-1400TIME1400-14151415-14301430-14451445-15001500-15151530-1600TIME1615-16301630-16451645-17001700-17151715-17301730-17451745-1800Bruce DowningOssie PetruccoMORNING TEA-ATRIUM, CANBERRA ROOM, GALLERYJohn KerinRobert WoolcottArnaud WattiezArnaud WattiezDavid MolloyLUNCH-ATRIUM, CANBERRA ROOM, GALLERYAUTHORL. Robinson, J. Crittenden and P. IllingworthE. Pearce, J. Crittenden and P. IllingworthPAL. Lancaster and T. HurstIL. Hurst and PAL. LancasterG.!. Leslie. C.A. McMahon, J. Cohen,F. Gibson, C. Tennant and D.M. SaundersAFTERNOON TEA-ATRIUM, CANBERRA ROOM, GAllERYAUTHORM.J. Davies. J.X. Wang and R.J. NormanJ.X. Wang. M.J. Davies and R.J. NormanJ. Marks. K. Stern, S. Quinn,R. Simmance and J. McBainJ. Dalrymple, ELeahy and A. TrounsonSA Robertson. D.J. Sharkey,K.P. Tremellen and K.G. DanielssonK. Tremellen, D. Valbuena, J. Landeras.A. Ballesteros. J. Martinez. S. Mendoza,SA Robertson, C. Simon and R.J. NormanN.D. Hussey, I Webb, J. Hall and Z. RudzkiEndoscopic tubal reanastomosis: results and costsMicrosurgery for tubal reanastomosis: results and costsFalloposcopy: Diagnostically useful but technically challengingSelective salpingography. tubal catherisation and transcervical tubalre-canalisationEndoscopic surgery vs Laparotomy for Endometriosis and FibroidsWhat to attempt and what not to attempt endoscopicallyWhere does IVF fit in?fSA FREE COMMUNICATIONS-IVf ADMINISTRATION AND OUTCOMES(chair-Doug Saunders)The use of a computerised database versus manual collection of data for theNational Perinatal Statistics Unit and the Reproductive TechnologyAccreditation CommitteeContribution of a nurses database to infertility practiceRising contribution of assisted conception to multiple births in AustraliaTrends and variations in perinatal death rates after assisted conceptionHealth and developmental outcomes of IVF children during the first five yearsfSA fREE COMMUNICATIONS-INFERTILITY (CLINICAL)(chair-Anne Clark)The influence of body mass on fecundity of women during AssistedReproduction TreatmentThe influence of body mass index (BMI) on pregnancy wastage followinginitial ART successAn effective group approach to lifestyle modification in infertile womenwith Polycystic Ovarian Syndrome (POS)Evaluation of aIifetstyle behaviour modification program for couplesexperiencing infertilityInsemination elicits an inflammatory response in the human cervixThe efLect of intercourse on pregnancy rates during assisted humanreproductionPreimplantation of genetic diagnostic of B-Thalassemia0930-09450945-10001000-10151015-10301030-11001100-11151115-11301130-11451145-12001200-12151215-12301230-12451245-1400TIME1400-14071407-14141415-1422M.J. Krust-McKayP. Hutchins, K. Oke. J. Craig and L. WiltonlB. Tolks, J. Ellis, R. Irwin and K. PetrieM.E. Evans MontroneMORNING TEA-ALBERT HALLS.L. De LaceyG. Phillipson. E. Baxter A. Gooneratne.K. Scrimshaw and I. SinK. Harrison. M. Condon and A. HaywardC.A. McMahon, F. Gibson. G.I. Cohen,J. Cohen. D.M. Saunders and C.C. TennantF. Gibson, C. McMahon, J. Cohen.G. Leslie and D. SaundersJ. Cohen. C. McMahon, F. Gibson.C.C. Tennant. D.M. Saunders and G.I. LeslieLUNCH-CANBERRA ROOM, ATRIUM, GALLERYAUTHORSelf-concept discrepancy theory: A model for understanding individualvariability in reactions to infertilityPGD-The role of the genetic counsellorA pilot study: The effect of written emotional disclosure on conception ratesin patients undergoing in vitro fertilization?The use of ajar metaphor in working with depressionBeing a non-mother: The politics of social conversationAssessment of staff attitudes to issues in Assisted Reproductive TechnologiesCharacterisation of semen donors accepting identity release and use bylesbians and single womenMothers of five year old IVF children: Acomparative study of psychosocialadjustment and parenting stressBehaviour at five years of age for children conceived through in vitrofertilisation (IVF): A prospective studyFive-year follow up of IVF fathers: Preliminary findings for parental adjustmentH. Wellsmore Sex selection for family balancing-the case againstS. Watson, S.M. Junk, J.M. Yovichand J.L. YovichI. Lalic and J.w. CattD.H. Edgar. H. Bourne and J. McBainFSA MINIPOSTER SESSION(chairs-Geoff Driscoll and Terry Thomas)The grading of embryos at one day and three days post inseminationare comparableSelection of 'early cleavage' embryos for embryo transfer: effect onpregnancy and implantation rateAn analysis of early pregnancy loss following the transfer ofcryopreserved embryosSingle embryo transfer-a pilot studyThe factors influencing couples to donate their excess frozen embryos1422-14291430-1437M. Bowman, J. Catt and M. LivingstoneB.A. Woodroffe, S.E. Watson. S.M. Junk.J.M. Yovich and J.L. Jovich1437-1444 A. Catakovic. W.R Edirisinghe, R Jemmott.J.E. Dowd. C.Kirby and J.A. AllanFrozen embryo pregnancy rates: benefits of pronuclear stage freezing1445-1452 E. Morris and S. StiegradVaginal ultrasound scanning practice of fertility nurses in Australia1452-1459 G. Kovacs, S. Breheny and V. MacLachlan What is the probability of conception for couples entering an IVF program?..1500-1507 A. Meeking, G. Kovacs and V. MacLachlan Why do couples who register for IVF not proceed with treatment?1507-15141515-1522P.M. Matthews and D.H. EdgarA. Lakmaker, J.D. StangerThe effect of high insemination concentration on IVF outcomeFertilisation rate and the duration of human sperm-egg interactionand K. Stevenson1522-1529 K. Harrison Indicators for male occupational infertility from national census data•

(ConlinUGd).....•••..•.•....•.•••...•..•.......••...•..•...•..••...•....•.•.••....••..•...•.•............•...•.....•...•...•...•...•.•••••••••••••••••ALBERT HALL (fSA) (Contioued)1530-1537 G.N. Clarke, D.Y. Liu, M. Martie,M. O'Bryan and HW.G. Baker1537-1544 S. Holden, P. Jackson, M. Frydenberg,D. De Kretser and R. McLachlan1544-15521552-15591600-16071607-16141615-1645J.w. Catt and M. HenmanS.J. McArthur, J.W. Catt and M.J. HenmanR. PerkinsJ. Vanderlelie, T. Perkins and K. BellAFTERNOON TEA-ATRIUM, CANBERRA ROOM, GALLERYSperm autoimmunity in a patient with severe asthenospermia and shortsperm tailsOutcome of open biopsy sperm retrieval in spermatogenic failure: Predictivefailure of testicular histologyContaminants in gas lines-big or little deal?Success in providing acomprehensive IVF laboratory service to regionalNew South WalesPerceptions of fecundity among women attending afertility clinicLuteal phase levels of estradiol may help predict pregnancy outcome inpatients undergoing in vitro fertilisation4 ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••BALLROOMTIME0830-09050905-09400940-10201030-11001100-11351135-12101210-12401300-1400SPEAKERRobert McLachlanDavid HandelsmanGail Risbridger, J. Schmitt,J. Fisher, P. ChoongFSA SYMPOSIUM ON THE MALE(rhair-Steve Steigrad)The ageing maleMORNING TEA-ATRIUM, CANBERRA ROOM, GALLERYDavid De KretserMark FrydenbergBronwyn StuckeyLUNCH-ATRIUM, CANBERRA ROOM, GALLERYAndrogen supplementation for the ageing maleCancer biologyTesticular cancer: Increasing frequency and emerging conceptsThe prostate cancer 'epidemic'The treatment of erectile dysfunction-from sex therapy to gene therapy- TIME0830-08500850-09100910-09350935-1005SPEAKERMartyn Stafford BellKim Riding(a patient) The patient's views(a surrogate) The surrogate's viewsFSA SYMPOSIUM ON SURROGACY (IT'S NOT AS EASY AS YOU THINK)(chair-John Leeton)Clinical indications, exclusions, techniques and resultsCounselling of participants in the ACT Surrogacy Program1030-1100 MORNING TEA-ATRIUM, CANBERRA ROOM, GALLERY1100-1140 Susan CooperAvoiding psychological and ethical pitfalls in gestational surrogacy1140-1220 Colin ThomsonLegal considerations, state and federally1300-1400 LUNCH-ATRIUM, CANBERRA ROOM, GALLERYTIME0830-08450845-09150915-10001000-10301030-11051105-11401140-12151215-13001300-1400SPEAKERFSA SYMPOSIUM ON POLYCYSTIC OVARY SYNDROME(chair-Rob Norman)Rick LegroThe difficulty of defining PCOsJohn EdenEnvironmental causesRick LegroGenetic causes-new genesMORNING TEA-ALBERT HALL(chair-Rick Legro)Anne ClarkLifestyle changesRob NormanInsulin modifying approachesGab KovacsOvarian drillingRick LegroDiscussion and closing remarksLUNCH-ATRIUM, CANBERRA ROOM, GALLERY•TIME1400-14451445-15451600-1700General Meeting of FSAFSA President's Debate'That the Units with the loudest trumpets are the best'FINAL COCKTAil PARTY AND PRIZES-ATRIUM, CANBERRA ROOM

SEASONALITY AND NEUROENDOCRINOLOGYSEASONALITY AND NEUROENDOCRINOLOGYAFfERNOON, NOT MORNING; EXOGENOUS MELATONIN PROVIDES A LONG NIGHTSIGNAL TO INITIATE OVARIAN ACTIVITY IN THE EWE.Michael Guerin, Jim Deed and Colin MatthewsDepartment ofObstetrics and Gynaecology, The University ofAdelaide, Adelaide South Australia 5000AIMSWe have previously shown in the seasonal Romney Marsh ewe that exogenous melatonin will only block the short night(priming) signal for timing seasonal oestrus when administered in the afternoon, not in the morning (1). This provided the fIrstconclusive evidence that the timing mechanism for seasonal breeding is based on the coincidence oflight or dark/melatoninsignals to the circadian system rather than on the duration ofmelatonin secretion. In this study we have examined whether thetiming of melatonin is important to the long night (inductive) signal that is required by the ewe todirectly initiate seasonalovarian activity.METHODSFive groups of 5 mature Romney Marsh ewes were placed in environment controlled rooms at the Waite Campus oftheUniversity ofAdelaide from 21 May 1999. All ewes were blood sampled twice a week for progesterone deteiminations tomonitor ovarian activity throughout. Initially all groups were subjected to eight weeks ofshort night photoperiod (10D: 14L).At the end of this period ewes were then subjected to the following treatments:Group 1 remained in short nights (lights off at 18:00 hr and on at 04:00hr)Group 2 short nights with daily melatonin infusion (to physiological levels) between 04:00 and 10:00 hrGroup 3 short nights with daily melatonin infusion (to physiological levels) between 12:00 and 18:00 hrGroup 4 long nights with lights offat 18:00 hr and on at 10:00 hr (16D:8L)Group 5 long nights with lights off at 12:00 hr and on at 04:00 hr (16D:8L)RESULTS(Results of ovarian activity are illustrated in Figure 1) There was no difference between the groups in the time they ceasedtheir initial activity in late July. Groups 3, 4 and 5 all exhibited renewed ovarian activity earlier than the control Group 1 (P

SEASONALITY AND NEUROENDOCRINOLOGYSEASONALITY AND NEUROENDOCRINOLOGYADVANCING A SHORT NIGHT SIGNAL DOES NOT NECESSARILY STOP OVARIANACTIVITY EARLIER IN THE EWE.Michael Guerin, Jim Deed and Colin MatthewsDepartment ofObstetrics and Gynaecology, The University ofAdelaide, Adelaide South Australia 5000AIMSWe have proposed that the signal for the cessation ofovarian activity in seasonal breeding ewes is light coincident with an.afternoon-located sensitive phase ofthe circadian pacemaker. In earlier studies when similar 8-week treatments. o~ short mghts(which advance the afternoon portion ofthe pacemaker into the light) have been utilised.we have rep~rted CO~ICtingresultson the timing ofoestrus offset (1 )(2). In this study we have provided 8 weeks ofshort mghts at tw~ different ~es, ~n~commencing four weeks earlier than the other, to examine for any effect on the timing ofthe cessanon ofovanan actiVIty.METHODSThree groups of6 mature Romney Marsh ewes were established at the Waite Campus ofthe ~niversi~ ofAdelaide in early1998. Group 1 remained outside under natural conditions throughout. Group 2 was br~ught mto envrronment c?ntrolledrooms and subjected to eight weeks ofshort night photoperiod (10D:14L) from 21 Apni. Group 3 was brou~ht m andsubjected to eight weeks ofshort night photoperiod (10D:14L) from 21 May. At the end ofeach 8-week penod Groups 2 & 3were subjected to long night photoperiods (14D:lOL) until 21 December 1998 when both groups were paddocked.All ewes were blood sampled twice a week for progesterone determinations to monitor ovarian activity throughout. ?n twooccasions during short night treatments and three during long night treatments all ewes were blood sampled over 2 mghts. Thefirst night under treatment conditions to monitor endogenous melatonin elevation and the second night under acutely extendeddarkness where melatonin acts as a marker for pacemaker activity.RESULTSThere were no differences between the groups in the timing ofthe mid-year cessation ofovarian activity (see Figure 1). Thetiming ofthe onsets and offsets ofinduced ovarian activity for Groups 2 &3 were not different. The measure~ents ofendogenous melatonin demonstrated that all animals in Groups 2 & 3 correctly perceived the short and long mght treatments.The marker ofpacemaker activity indicated that the short night treatments advanced the afternoon portion ofthe pacemakerinto the light.OutsideControlGroupNATURAL PHOTOPERIOD: 1 I•AIMSORAL MELATONIN ADMINISTERED TO CYCLING MARES IN AUTUMN DOES NOTADVANCE ANOESTRUS.METHODSM Guerin, J Deed, R Woodward*, D Washington* and C MatthewsDepartment ofObstetrics and Gynaecology, *Department ofAnimal ScienceThe University ofAdelaide, Adelaide South Australia 5000We have proposed that the timing for seasonal breeding is dependent on the correct sequence oflight anddark/melatonin signals to the endogenous circadian pacemaker. Studies in the ewe indicate there is an aftemoonlocatedsensitive phase ofthe pacemaker that sets up the reproductive system when light is coincident withitin theshort days ofspring/summer. The same sensitive phase then directly signals the onset ofoestrus when it iscoincident with darkness/melatonin in the lengthening days ofautumn (1). Exogenous melatonin in the afternoon,not the morning, will mimic long nights to maintain existing ovarian activity (2) and will initiate new ovarianactivity in ewes already primed with short nights (3).Exogenous melatonin, in the afternoon not the morning, has been shown to inhibit the effect ofadditional artificiallight that normally advances the time ofseasonal breeding in the horse mare (4). From studies ofmelatonin as amarker for the endogenous circadian pacemaker we have proposed that the mare may use a similar basicmechanism to time seasonal breeding as the ewe (5). Since the mare is considered to be a "long day" breeder, theadministration ofexogenous melatonin should be inhibitory to ovarian activity. In this study we administered oralmelatonin, in the afternoon or morning, to mares during the autumn to see ifeither treatment could mimic darknessand advance anoestrus.In late March 1999 three groups offive Standardbred mares were established at the Roseworthy Campus ofTheUniversity ofAdelaide (latitude 35°S). All mares were confirmed in oestrus, and ovulating, by ultrasound scanningand blood progesterones. Commencing on 30 March two groups were orally administered a daily 12 mgm dose ofmelatonin, in a 1 ml solution, given at either 30 mins prior to sunrise or 4 hrs prior to sunset. The dose given hadpreviously been shown to elevate plasma melatonin in the horse to normal nighttime levels, or greater, for at least 5hours. All mares were blood sampled twice weekly throughout and progesterone determinations were performed tomonitor ovarian activity. Anoestrus was taken as the date ofthe last confirmed ovulation for the season.; SHORT NIGHTS ~LONG NIGHTSNATURALRESULTSGrouplGroup 3: SHORT NIGHTS:: I I :LONG NIGHTSNATURAL____L~~ MA~_l!~ i JULY i AUG 1 SEPT I ocr I NOV I DEC I JAN !Figure 1. The ovarian activityfor the three groups during the course ofthe experiment. The shaded blocksillustrate the periods ofextended cyclic pattern as determined by twice-weekly plasma progesterone.CONCLUSIONSA short night signal (light on the afternoon portion ofthe pacemaker) four weeks earlier than natural oestrus cessation time didnot alter the timing, however it still provided the priming signal for induced ovarian activity in subsequent long nights. Thecircadian mechanism that times the offset ofovarian activity requires further investigation.Guerin MV & Matthews CD. (1998) 1. BioI. Rhytluns 13,60-69Guerin MV, Deed JR & Matthews CD. (1998) Proc. Aust. Soc. Reprod. BioI. 29,36(Acknowledgment. This research was supported by a Large ARC Grant to Matthews and Guerin)There were no differences in the timing ofanoestrus between the 3 groups. The Control Group ceased ovarianactivity on 18 May (2:19.5 days), the Morning Melatonin Group on 24 May (±19.6 days) and the AfternoonMelatonin Group on 15 June (± 38.4 days).CONCLUSIONSAlthough oral melatonin in the afternoon has been shown to block light advancing the onset ofoestrus (4), in thisstudy it has failed to act as darkness to advance anoestrus. It is possible that the signal to cease ovarian activity wasperceived before treatments began and the treatments were insufficient to alter the outcome. Alternatively, with themare's and the ewe's natural ovarian responses to increasing darkness different (the mare is normally anoestrousover winter and the ewe oestrous) it may be that melatonin has a different role in the timing events ofseasonaloestrus between the two species. .REFERENCES(1) Matthews CD, Guerin MV and Napier AJ (1995) 1. BioI. Rhythms 10,308-318(2) Guerin MV, Deed JR & Matthews CD. (1998) Proc. Aust. Soc. Reprod. BioI. 29, 36(3) Guerin MV, Deed JR & Matthews CD. (2000) Proc. Aust. Soc. Reprod. BioI. 31,(4) Palmer E and Guillaume D (1992) Anim. Reprod. Sci. 28, 21-30(5) Guerin MV, Deed JR, Kennaway DJ and Matthews CD (1995) 1. Pineal Res. 19,7-1526 27

SEASONALITY AND NEUROENDOCRINOLOGYSEASONALITY AND NEUROENDOCRINOLOGY4A SINGLE INTRACEREBROVENTRICULAR INJECTION OF OREXIN-ASTIMULATES PULSATILE LH SECRETION IN MATURE MALE SHEEPDominique Blache and Graeme B. MartinFaculty ofAgriculture (Animal Science), The University ofWestem Australia, Nedlands, WA 6907, AustraliaIntroductionIn mature Merino rams, the reproductive axis is stimulatedby nutrition (1) but the hormonal pathways involved arenot understood. We have been investigating potentialnutritional signals, such as leptin and insulin, withoutbeing able to imitate the full effect ofa feed supplement onLH pulse frequency (1). Recently, two novel hypothalamicneuropeptides, orexin-A and orexin-B, were shown to beinvolved in the control of feed intake in rats (2). Bothorexins increased feed intake when injected intracerebrallyin satiated male rats (3). Surprisingly, in ovariectomizedfemale rats, a single injection of orexin-A, but not orexin­B, reduced LH pulse frequency in the absence ofexogenous sex steroids (4) but increased LHconcentrations in animals pretreated with oestradiol andprogesterone (5). This dependence on the presence of sexsteroids is also seen in the responses of male sheep tochanges in nutrition (1). We therefore tested whetherinjection of orexin-A into the third ventricle of intactmature rams would increase LH pulse frequency.Materials & MethodsTen intact, mature, Merino rams were acclimatised toindividual indoor pens for 2 weeks. Cannulae wereimplanted in the third ventricle and the animals wereallowed 2 weeks to recover before being allocated into 2groups of same average bodyweight. Blood was sampledfor 36 h every 20 min starting at 07:00 h. The animals werefed a restricted diet of I kg wheaten chaff and 100 g lupinseed per day. They were fed 1 h before and 24 h after thestart of the sampling period. Orexin-A (Peptide InstituteInc., Osaka, Japan) was dissolved in water (6.4 nmoles in20 JlI) and injected intracerebroventricular (icv) using aHamilton syringe (n = 5). Controls (n = 5) were given thesame volume of vehicle. Blood plasma samples wereassayed for LH. Repeated measures ANOVA was used tocompare the number of LH pulses before and after icvinjection. The 36-h sampling period was divided into 4periods of 9 h (PI, P2, P3 and P4). LH pulse frequencieswithin periods were compared by t-tests.Resultsoiii i i 0Feed intake was not affected by orexin-A injection as theanimals were fed a restricted diet. During the period 9 to18 h after injection (P4), LH pulse frequency was 3-foldhigher in orexin-injected animals than in controls (Fig. 1).The same was seen when comparing values for orexininjectedrams before and after injection (PI to P3).Discussion and ConclusionOrexin-A increases the pulsatile secretion of LH (andpresumably GnRH) in intact, mature Merino rams.Importantly, nutritional supplements generally induce 3-4pulses per 9 h (l), so the size of the response to orexin-Asuggests that it is a key signal in the conununication ofmetabolic status to hypothalamic reproductive centres.The delay to the increase in LH pulse frequency (6.2 ± 0.8h) is surprising and suggests that neuronal pathways whichlink orexin neurons to GnRH neurons are complex. Thefact that we observed an increase in LH pulse frequency intestis-intact animals suggests that the interactions betweenorexin-A and sex steroids seen in females rats (3, 4) alsoexist in male sheep.References1. Blache, D. et al. (2000) 1. Reprod. Fert (in press)2. Sakurai, T. et al. (1998) Cell, 92,573-585.3. Haynes, A.C. et al. (1999) Peptides 20,1099-105.4. Tamura, T. et al. (1999) Biochem. Biophys. Res.Comm. 264, 759-762.5. Pu, S. et al (1998) Regulatory Peptides 78, 133-36Supported by the Australian Research Council.4 b)P < 0.05-18 -9 0 9 18 PI P2 P3 P4Time relative to injection (h)Time periodFigure 1: a) LH profile ofa ram injected icv with 6.4 nmoles orexin-A (t = 0); black circles indicate pulses. b) LHpulse frequency (mean ± sem; n = 5) in rams treated with orexin-A (closed bars) or vehicle (open bars). Timeperiods relative to icv injection are PI: -18 h to -9 h, P2: -9 h to 0 h, P3: 0 to 9 h, P4: 9 to 18 h.EFFECTS OF CENTRAL ADMINISTRATION OF RECOMBINANT BOVINE LEPTIN ONPULSATILE LH SECRETION IN MALE SHEEP UNDER DIFFERENT FEEDING REGIMESPietro Celi1, Graeme B. Martini, Dominique Blache1, Philip E. Vercoe1 and Ross L. Tellam 21) Faculty ofAgriculture (Animal Science), The University ofWestem Australia, Nedlands 6907, Australia;2) CSIRO Tropical Agriculture, Indooroopilly 4068, AustraliaIntroductionReproductive activity is affected through a number ofpathways, and several metabolic hormones, includinginsulin and leptin, have been implicated as nutritionalsignals to the gonadotrophic regulatory centres of thebrain (1,.2). However, in Merino rams, we haveobserved that central administration ofleptin reducesboth feed intake and LH secretion in Merino rams fedad libitum (3). To further investigate whether leptin isa metabolic signal to the reproductive system in themale sheep, we tested whether intracerebral leptinwould increase LH pulse frequency in rams fed longtermwith a diet that is poor in energy and protein. Wedeveloped this hypothesis from the fmding that leptinconcentrations in both plasma and CSF are decreasedin rams fed with LD for five days (4), and alsodecreased in the plasma of sheep fed with LD for 2months (5).MethodsTwenty mature Merino rams with a cannula in the thirdcerebral ventricle were acclimatised to a daily ration ofeither a Low Diet (LD; 500 g wheaten chaff + 50 glupin grain + 2% minerals) or a High Diet (lID; the LD+ 1 kg lupin·grain) for 5 weeks. Rams were stratifiedon the basis of body weight and body condition thenrandomly allocated to treatments: a) LD + artificialcerebrospinal fluid (aCSF); b) LD + 0.4 J.lg/ h bovineleptin in aCSF; c) lID + aCSF; d) IID + 0.4 J.lg/ hbovine leptin in aCSF. Infusions were continuous (icv)for 5 days. Blood plasma sampled every 20 min for 24h on Days 0 and 5 ofthe treatment period was assayedfor LH and insulin. Repeated measure analysis ofvariance was used to determine the effects oftreatments on data gathered.ResultsPlasma insulin concentrations were reduced during thefIrst two weeks of the Low Diet, but then returnedtowards levels seen in HD-fed rams after 5 weeks ofdietary treatment (data not shown). On Day 5 ofinfusion, plasma insulin levels were not significantlymodified by diet, aCSF or leptin (Fig 1).LH pulse frequency also did not significantly differbetween the groups after 5 weeks of feeding with aLow or High Diet. In this situation, five days of icvinfusion of leptin did not significantly affect thefrequency ofsecretion ofLH pulses (Fig 2).Discussion and ConclusionWe expected leptin to increase LH pulse frequency inthe rams fed with LD, yet we observed no change, sowe have to reject the hypothesis that leptin representsthe metabolic signal to the reproductive centers inmature rams fed on different dietary regimes. Theobservation that LH pulse frequency does not differbetween HD or LD after five weeks of feeding agreeswith previous fmdings in our laboratory. Thestimulatory effect of nutritional supplements is lostafter 3-4 weeks, perhaps reflecting the fact that, in bothlong-term dietary groups, the animals have reached anew homeostatic status (1). Plasma insulin levels, infact, were not different after 5 weeks of fee~g ..therams a LD or lID, and they were not affected by theinfusion ofvehicle or leptin. In this situation, ~eramsmay also not be responsive to leptin administration.Other metabolic and reproductive hormones need to bemeasured before reaching a defmitive conclusiQn.The lack of a stimulatory effect on LH secretion insheep, now observed under a range of nutritionalregimes, calls into question the role of this adiposesignal in reproduction in the mature animal, althoughwe have yet to test it during a fasting-induced decreaseofLH pulsatility in sheep.References1) Martin, G.B., Walkden-Brown, S.W. (1995). 1.Reprod. Fert. 49: 437-449; 2) Messinis, lE., Milingos,S.D. (1999). Human Reproduction Update 5: 52-63; 3)Celi, P., Martin, G.B., Blache, D., Vercoe. P.E., Dynes,R.A., Tellam, R. (1999). Proc. End. Soc. Aust. 42: 84;4) Blache, D., Chagas, 1., Blackberry, M.A., Vercoe,P.E. Martin, G.B. (2000) 1.Repr.Fert. (submitted)5)Blache, D., Tellam, R.L., Chagas, L., Blackberry,M.A., Vercoe, P.E. Martin, G.B. (2000) 1.Endocrinology. (in press)..5 100""3CIJ.5 -.. 75~-~ § 50oS::::t.0..- 25~~Low DietO l-'-L--..I---L-_......-aCSF LeptinLow DietaCSFHigh DietTLeptinFigure 1: Plasma insulin concentration in rams infused icvfor 5. days with bovine leptin (0.4 J1g124 h. Horizontal bar:mean± sem ofpre-leptin-treatment values.aCSF Leptin aCSF LeptinFigure 2: Effect of 5 days icv infusion of"bovine leptin (0.4J1g124 h) on LH pulse frequency in rams. Horizontal bar:pre-leptin-treatment values (mean ± sem).28 29

SEASONALITY AND NEUROENDOCRINOLOGY5a.-ANDROSTAN-3a., 17~-DIOL VIRILISES THE DEVELOPING FEMALE MARSUPIALGENITAL TRACTM.W. Leihyl, G. Shawl, J.D. Wilsonl. 2 & M.B. Ren~eelI Department ofZoology, Omverslty ofMelbourne, Victoria 3010, AustralIa;. 2 Department ofInternalMedicine, Division ofEndocrinology, University ofTexas, Southwestern Medical Centre at Dallas, 5323Harry Hines Boulevard, Dallas, Texas 75235-8857 USAIntroductionAndrogens are essential fordevelopment of the malereproductive tract. In the absenceof androgens, the undifferentiatedurogenital system will develop intoa female tract. Two androgens areknown to be active during male 0 ...L..I:.'

SEASONALITY AND NEUROENDOCRINOLOGYFOUNDER'S LECTUREMOLECULAR CLONING OF WALLABY GnRH RECEPTOR GENETimothy C. Cheung and John P. HearnDevelopmental Neurobiology and Endocrinology, Research School ofBiological Sciences, The Australian NationalUniversity, Canberra, ACT 2601, AustraliaIntroductionGonadotrophin-releasing hormone (GnRH) plays apivotal role in the endocrine control ofreproduction andembryo development 1 • To understand the molecularevents associated with these complex physiologicalfunctions via GnRH-mediated pathways, the knowledgeof GnRH receptor (GnRH-R) gene expression andregulation is crucial. We are currently isolating thewallaby GnRH-R gene and investigating itsdevelopmental expression. The overall goal of ourwork is to understand the molecular mechanism ofGnRH-R in endocrine regulation of reproduction,embryo development, and sexual differentiation. In thiscommunication, we report our recent progress on thecloning of the wallaby GnRH-R gene. We have alsoisolated a partial GnRH-R cDNA from the testis of awallaby pouch young.Materials and MethodscDNA synthesisTotal RNA was extracted from postnatal 79 day old(P79) wallaby (Macropus eugenii) testis using QiagenRNA purification columns (Qiagen, Hilden, Germany).cDNA was synthesised using Expand ReverseTranscritpase (Roche Diagnostics, Germany) accordingto the product instruction manual.Primers Sequences (5'-3 ')Fl 5'-AGAGACACAAGGCTTGAAGC-3'Rl 5'-AAATTTCCAGTTTGTCCAGA-3'F2 5'-CAATGCTAAGATCATCTTCA-3'R1 5'-GTGCCTTAGGCTGAAGGTAT-3'Table 1: Primer selection for PCR cloning.PCR CloningPCR cloning was carried out using wallaby cDNA astemplate. Primer selection was based on the GnRH-Rgenomic sequence that we obtained previously(unpublished data). PCR was performed using primersFI & Rl, F2 & R2 (Table 1) and Pfu DNA polymerase(Stratagene, La Jolla, USA). Amplification conditionswere an initial 4 minutes at 94°C, followed by 35 cyclesof 30 seconds at 94°C, 30 seconds at 55°C, and 2minutes at 72°C for 2 minutes. Then, a fmal extensionwas carried out at 72°C for 10 minutes,ResultsPCR cloning of wallaby GnRH-R cDNA using totalRNA isolated from P79 testis yielded two cDNA clones.Comparison with the human GnRH-R cDNA sequenceindicated that both of these clones contained wallabyGnRH-R cDNA fragments, and a 530 bp nucleotideHomology in Homology inDNA Level Protein LevelHuman 83% 83%Bovine 83% 83%Sheep 83% 83%Pig 81% 79%Rat 79% 81%Mouse 77% 83%Horse 77% 83%Table 2: Comparison ofsequence homology ofwallaby GnRH-R with various mammalian species.sequence was constructed from these two fragments. Thispartial sequence has 83% homology with the cDNAsequence that corresponded to the exon 2 and 3 of humanGnRH-R gene 2 • It contains an open reading frame thatencoded a peptide with 146 amino acid residues, and thispeptide shares around 80% amino acid sequence identitywith human as well as various mammalian GnRH-Rsequences (Table 2).Discussion/ConclusionDuring the course of the cloning of wallaby GnRH-Rgene, we isolated a 530 bp cDNA sequence from the testisof a P79 pouch young. It is likely that this transcript is apart of the full-length GnRH-R transcript. Interestingly,this cDNA contains a 5'-untranslated region as well as aputative AUG start codon that is in good context fortranslation initiation with reference to the Kozakconsensus sequence 3 • It suggests that the transcript byitself may be able to serve as an independent transLltionalunit. Additional experiments are needed to investigatehow this transcript was generated.Translation using the putative start-site yielded a peptidewith 149 amino acid residues. This peptide appears tohave around 80% sequence homology when comparingthe amino acid sequence with placental mammalianGnRH-R but displays 95% sequence homology with thebrush-tail possum (Trichosurus vulpecula) GnRH-R.This analysis unambigeously indicated that the wallabyGnRH-R gene is much more diverse from the placentalmammalian GnRH-R but appears to be highly conserveamong marsupials. In this respect, GnRH-R may playadditional roles in regulating the unique reproductivecycle ofmarsupials.References1) Raga, F.,Casan, E. M., Kruessel, L., et al. (1999),Endocrinology. 140,3705-122) Kakar, S.S. (1997). Eur. 1. Endocrinol. 137,183­192.3) Kozak, M. (1992). Annu. Rev. Cell BioI. 8, 197­225.THE FUTURE FERTllJTY OF MANKINDRoger ShortDepartment ofObstetrics, Royal Women's Hospital, Carlton, Victoria, 3053?-&O ~-:>~ ~'l~-f- r\~over-J D:gJfcvv~-=- .J~ uftJu pc~ ~) ~ ~(c 1 tTl~32

ASSISTED REPRODUCTIVE TECHNOLOGY-MALEASSISTED REPRODUCTIVE TECHNOLOGY-MALEMOTILITY PARAMETERS OF FRESH AND FROZEN .ABALONE (Haliotis iris) SPERM.1. F. Smith (1), R.M. McDonald (1), R.D. Roberts (2), S. Adams (3), P.A. Pugh (I).{IAgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton 2001, NZ, (2) Cawthron Institute, Nelson, NZ. (3) Dept.Marine Science, Otago University, Dunedin, NZINTRODUCTION:Cryopreservation affects the motility parameters ofmammalian sperm and measurement of these can assist inthe prediction of the fertility of that semen (1). Similarmeasurements are needed for the development of successfulcryopreservation methods for the sperm of shellfish species.The NZ Aquaculture industry has identified a need forefficient cryopreservation techniques of gametes, of anumber of shellfish species, to enhance the ability of yearround-spatproduction, selection and conservation ofgenotypes. As part of the development of such techniquesthe methods and equipment settings for computer assistedsperm analysis (CASA) ofabalone sperm were investigated.METHODS:Abalone were induced to spawn using hydrogen peroxidetreatment (2). Semen was collected in a concentrated formas it was expelled from the respiratory pores. Sperm densitywas measured and sperm were diluted into a seawater(SW)-based diluent with either DMSO or glycerol as thecryoprotectant and loaded into 0.25mI straws. Motilitymeasurements were made, over time, on unfrozen samplesheld at ambient temperature (l5°C-RT) or refrigerated(5°C) and also on frozen/thawed samples at 2 stages of thefreezing process (after holding at -30°C and after storage inliquid nitrogen -196°C). Sperm samples were diluted to 6xl0 6 /ml in 1 mI SW +1%BSA in a tissue culture plate andvideo recorded at 20X. The videotape segments weresubsequently analysed using a 'Hobson Sperm Tracker'after optimisation of settings .using the on screen trackimaging overlay facilities of the equipment (3). Fertilisingability of sperm was also tested using freshly collected eggssuspended on a fme mesh in 10ml seawater.RESULTS AND DISCUSSION:Mean sperm density was 6.0xl0 s sperm /mI and initialdilutions were at [1 sperm: 5 diluent] for a concentration instraws of 1.2x lOS/mI.The optimum 'Tracker' imaging parameters for abalonesperm were (threshold +9/-7; filter -3,4,2,0; brightness 99;contrast 71; search ratio 13.5; minimum track points 40 or0.8 minutes oftrack time).There was no significant difference between the twocryoprotectants in the motility of the unfrozen (held) orfrozen/thawed sperm. Hold temperature had no effect on theTable 1: Mean values at different evaluation times for selected motility parameters and fertility.unfrozen sperm but there was a trend for the ambienttemperature sperm to survive freezing better. There was noeffect of hold time up to 3h on motility parameters (Table1). However, freezing and stage of freezing reduced overallsperm velocity (VCL), straight line velocity (VSL), beatcross frequency (BCF), linearity (LIN) and Straightness(STR) . These effects were generally more pronounced atthe lower freezing temperature. Similar effects were seen inthe visual motility estimates with a very low proportion offrozen sperm motile. Sperm fertility was maintained for upto 60-min ofstorage of sperm post-collection. The presenceof cryoprotectants in the diluent tended to reducefertilisation rates and this was more marked with glycerolthan DMSO. Fertilization rates with frozen thawed spermwere very low or nil in this experiment. Abalone spermhave much lower velocity than that of ram or bull spermand also a much lower ratio of VSL to VCL as reflected inlower LIN and STR. This reflects a. more spiral or circularform of movement by the abalone sperm. Cryopreservationreduces these parameters and this effect is similar to thatseen in the ram (4). The effect of maintenance of motilitybut loss of fertility upon storage is also seen in both speciesalbeit on a different time scale with the loss being morerapid in abalone.REFERENCES:(1) Smith, J.F.; Parr, 1.; Murray, G.R.; Clarke, A.;McDonald, R.M and Duganzich, D.M. (1998) Proc. N. Z.Soc. Aniln. Prod. 58:181-185.(2) Morse, D.E., Duncan, H., Hooker, N., Morse, A. \ 1977)Science 196: 298-300.(3) Briggs, R. M., Smith J.F., Duganzich, D.M. 1996.. Proc.13th Int. Congo Anim. Reprod. (Sydney) :3:P24-28.(4) Briggs, R.M & Smith, J.F. (1996Proc. "Techniques forGamete Manipulation and Storage 11. Hamilton, NZ. p56.ACKNOWLEDGE:MENTS:Staffat the Glenhaven Aquaculture Centre for assistancewith the project.The project was funded by FRST contract CAW 801.Email: smithj@agresearch.crLnz,. • .Parameter VCL VSL BCF LIN STR Visual FertilityEvaluation time J..Lmlsec J..Lmlsec Hz % % % motile %Unfrozen 60min 106.1 24.4 7.8 24.7 56.2 61 30.0Unfrozen 180min 98.1 23.2 7.6 24.9 56.2 56 2.0Post-Thaw at -30°C 84.3 14.6 4.8 17.7 48.4 16 < 1.0Post-TIlaw at -196°C 92.9 6.6 2.6 7.6 28.0 5 0.0SHORT-TERM STORAGE OF CANE TOAD (BUFO MARINUS) SPERM WITH SUBSEQUENTCRYOPRESERVATION.R.K. Browne, 1. Clulow and M. Mahony.Department ofBiological Sciences, University ofNewcastle, NSW, Australia.IntroductionConservation programs for threatened amphibians requirea range of reproductive technologies (1). Current freezingprotocols for of amphibian sperm require carefully stagedcooling, with both Me2S0 and glycerol in sucrose provingsuccessful cryoprotectants (2). Storage of sperm or testeson ice with subsequent cryopreservation would allowcollection in localities remote from cryopreservationfacilities. This study compared the motility of Cane Toad(B. marinus) sperm after storage for six days in a range ofdiluents, and after freeze/thaw.Materials and methodsTestis were macerated (1 wt: 1vol) in diluents consisting of10% (w/v) sucrose (sucrose) or Simplified AmphibianRinger (SAR) alone, or these combined with 15 or 20%(v/v) Me2S0 or glycerol (Sigma). Suspensions were thenplaced in 1.5m! Eppendorf tubes and stored at OoC (on ice)for 6 days. Relative %motility was then estimated under alight microscope. (Relative %motility (motility) =(%motility/initial %motility) x 100). These spermsuspensions or stored (6d10oC) suspensions in SAR or 10%sucrose with 15% or 20% Me2SO or glycerol then added,were frozen/thawed using the protocol of Browne et al,1998. Relative %motility was again determined at 3, 10and 24 min. post activation (only data for 3min. shown inTable 1). All results were subjected to ANOVA - P valuesrelate to differences in mean values.Results: Table 1. (means ± SE).Short-term storage:There was no significant (P>0.05) difference in pre-freezemotility between SAR (82 ± 8%), and sucrose (73 ± 11%)alone or SAR and sucrose with 15% Me2S0 as diluents.With glycerol at 15% and 20% motilities weresignificantly higher (P< 0.01) with SAR (87 ± 4%, 29 ±8%) than sucrose (9 ± 5%, 1 ± 1%).20% concentrations ofMe2S0 proved toxic in sucrose (1 ± 1%) and greatlyreduced motility in SAR (17 ± 11%).Cryopreservation ofsuspensions:Only 15% Me2S0 as cryoprotectant in SAR (18 ± 8%) andsucrose (46 ± 7%,), and 15% glycerol in SAR (19 ±8)gave moderate motility recoveries. Post-store addition of15% Me2S0 in sucrose gave a low recovery (6.8 ± 2.9%).In contrast 15% Me2S0 in SAR added pre- or post- shorttermstorage gave the same pre- and post-freeze/thawrelative motility (63 ± 14%; 18 ± 8%) and (66 ± 13%; 19 ±9%) respectively. The addition of 15% (v/v) Me2S0 prestoragegave a significantly higher (P

ASSISTED REPRODUCTIVE TECHNOLOGY-MALEEFFECT OF DILUENT AND OVIDUCT CONDITIONED MEDIA ON TIlE MOTILITY ANDVIABILITY OF BRUSHTAa POSSUM (TRICHOSURUS VULPECULA) EPIDIDYMAL SPERM.J.F. Smi th, R. M . McDona ld, D. K. Berg, K.. S S 1'dh U(.), F•• C M0l' mla . (..).AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton 2001, New Zealand; Co-operative ResearchCentre for Conservation and Management ofMarsupials, (·~acquarieUniversity, NSW 2109 Australia and(")Landcare Research, PO Box 69,Lincoln 8152 New Zealand.ASSISTED REPRODUCTIVE TECHNOLOGY-MALENON-INVASIVE COLLECTION AND CRYOPRESERVATION OF AMPHIBIAN SPERMM. Pomering, R.K. Browne, 1. Clulow and M. MahonySchool ofBiological and Chemical Sciences, University ofNewcastle, NSW 2308INTRODUCTION:Establishment of in vitro fertilisation procedures forthe possum is needed for both species preservation andas a measure of the effectiveness of bio-controlsystems aimed at the reproductive process.Secretory proteins from oviductal explants have beenshown to bind to possum epididymal sperm and toinfluence the capacitation status (% thumbtack'T'shape)(l). Computer assisted sperm analysis(CASA) measurements of sperm motility parametersand viability staining were undertaken to quantitate thechanges in sperm function during incubation withoviductal secretions.MATERIAL AND METHODS:Sperm were obtained by back-flushing the caudalepididymis with either EMEM or RSD-l (2) , diluted to25xl0 6 /ml and held at 35°C in a 5% CO 2 atmosphere.Conditioned media from the isthmus segment of theoviduct (CMOI) was prepared beforehand (3) andstored frozen. CMOI was diluted 1: 1 with eitherEMEM or RSD-l prior to use and sperm were dilutedto 5x106/ml in this and incubated. Aliquots were takenat 0, 2 and 4 hours for measurement. CASA wasperformed using a 'Hobson Sperm Tracker' optimisedusing the on screen imaging display facilities. Spermwere further diluted to 0.5xl0 6 /ml and 50JlI placed intoIml of mineral oil in 24 well tissue culture plate andassessed under a 5% CO 2 atmosphere. Live/deadviability measurements were performed on a FACScanflow cytometer using SYBR-14 and PI (4).Table 1 Selected motility parameters and proportion ofthumbtack 'T'shaped sperm.Parameter VCL VSL MAD BCF STR T'sTreatment JlffiIsec Jlm/sec 0 Hz % %EMEMOh 178.2 71.6 64.9 13.8 65.6 6EMEM2h 132.2 57.6 62.1 7.8 55.7 37EMEM4h 141.7 80.7 49.3 12.7 69.4 68E+CMOIOh 163.3 26.3 95.6 6.7 48.1 6E+CMOI2h 155.8 41.8 75.9 6.8 49.0 41E+CMOI4h 115.3 48.3 59.4 6.3 48.1 56RSD-10h 170.7 35.4 85.0 9.4 53.3 5RSD-12h 126.1 24.1 87.3 5.0 42.9 16RSD-14h 103.7 37.1 68.7 4.9 46.2 31R+CMOIOh 160.2 48.2 78.5 9.3 53.4 5R+CMOI2h 163.3 27.6 92.6 6.6 47.2 20R+CMOI4h 128.7 41.8 76.1 5.9 45.7 35RESULTS:There were significant (P

ASSISTED REPRODUCTIVE TECHNOLOGY-MALE~ --'~~(~....:;~ASSISTED REPRODUCTIVE TECHNOLOGY-MALE./ j,..r--~ :"'-:....,....",I -" C" 'J' , ('s. ~ [or: /,- J(:J'" J S~) (> (( •/. " ..... y/.,j- -.~ - -i, .'~ ~ f' /,-,.,., ""4~:- e.. - ,'...... ~.7.SUCCESSFUL CRYOPRESERVATION OF NON-HUMAN PRIMATESPERMATOZOASue E Watson!, Stephen M Junk\ Sherri Huntress 2 , Terry P Fletchey.2, John L Yovich 11PIVET Medical Centre, Cambridge Street, Leederville; 2Perth Zoo, Labouchere Road, SouthPerth, Western AustraliaIntroductionHuman spermatozoa have successfully been cryopreserved since the 1950's using a glycerolbasedcryoprotectant (1). The post-thaw survival and motility reflects the fertilizing capacityofthe sperm (2). The cryopreservation ofspermatozoa in non-human primates would assist inbreeding programmes, particularly for endangered species. Presently, however, animals areusually transported for breeding purposes, with cryopreserved spermatozoa rarely being used.The aim in this preliminary study was to determine whether cryopreserving spermatozoa intwo non-human primates (silvery gibbon and orang utan) could be, performed using simpletechniques used in human cryopreservation.Materials and MethodsThe testes and epididymis were collected from a silvery gibbon (Hylobates moloch) during acastration procedure under general anaesthetic (GA). The operation was performed as theanimal was no longer required for breeding purposes. During a routine operation on an orangutan (Pongo pygmaeus) under GA, manual prostatic massage was performed resulting in anejaculated coagulum. The testicular and epididymal samples and the ejaculated coagulumwere dissected to release the spermatozoa and resuspended in culture medium (hepes bufferedT6 medium). A droplet of each suspension was observed at 400X magnification to assessmotility. All sperm suspensions were diluted 1:1 with a 15% glycerol-based cryoprotectantmedium. The dilutions were aspirated into 0.5mL sterile straws and placed in liquid nitrogenvapours for 30 minutes prior to plunging in liquid nitrogen for storage (3). A straw of eachwas subsequently thawed after 24 hours and the motility of the contents assessed under themicroscope.ResultsThe motility for each ofthe samples pre and post cryopreservation are in the table below:Species Origin ofSperm Motility Motility % Survival(pre-freeze) (post-thaw)Orang utan (n=l) Ejaculated 18% 13.6% 76%Gibbon (n=I) Testicular 89% 37% 42%Epididymis 95% 290/0ConclUSion31%Survival of retrieved and ejaculated spermatozoa in the human after cryopreservation isvariable, but at our centre approximates 50%. The survival of the samples in this studyranged from 31 % to 76% after cryopreservation. This preliminary study demonstrates thatcryopreservation may be possible for long-term storage in these non-human species.1. Brotherton, J. (1990) Arch. Androl., 23; 25-282. Nagy, Z. et al (1995) Ferti!. Steril. 63; 808-8153. Edirisinghe, W.R. et al (1996) Hum Reprod. 11; 2474-2476GLYCOLYTIC ENZYME ACTIVITY IN HYPOTONICALLY-TREATED BOAR~~,''\L c::,' i"', SPERMATOZOA"'~' 1t(\.

· ASSISTED REPRODUCTIVE TECHNOLOGY-MALE ASSISTED REPRODUCTIVE TECHNOLOGY~MALEROLE OF THE MALE REPRODUCTIVE DUCTS IN CONCENTRATING SPERM AND IgGANTIBODIES: SIGNIFICANCE FORIMMUNOCONTRACEPTIONIMMUNE RESPONSES IN FEMALE RABBIT REPRODUCTIVE TRACT TOINFLUENZA HA OR RABBIT ZPB DELIVERED BY RECOMBINANT MYXOMAYmUSESW Gu 1,3, PJ Kerr 2,3, MK Holland 1.2, R Jones 4, PA Janssens :3, RF Seamark 1IPest Animal Control CRC, 2CSIRO Wildlife and Ecology; 3Division ofBiochemistry and MolecularBiology, Australian National University, ~e Newcastle University.Introduction:The European rabbit is a major pest species inAustralia. Instead of using lethal methods toreduce the population we are exploring thedevelopment of a viral vectored immunocontraceptivevaccine. In this study we usedrecombinant myxoma viruses expressinginfluenza haemagglutinin (MV-HAil) or rabbitzona pellucida protein B (MY-ZPBi 2 ) toinvestigate the immune responses in the femalereproductive tract to antigens delivered by therecombinant virus.Results:1. Antibodies in reproductive tract andovarian follicular fluids followingimmunizations with MV-HAThirty-two female rabbits were immunised intradermally(Ld), intra-nasally (Ln), and intravaginally(Lva) with 10 6 pfu MV-HA. Ovulationwas induced by hCG treatment in Ld infectedrabbits. HA specific IgG and IgA were measuredin serum and the tract fluids obtained by flushingand micro-puncture (including uninfectedcontrols). High-level anti-HA IgG titer wasinduced in serum but was 100 fold lower in thereproductive tract fluids and the titer in flushingswas comparable to that in micro-puncturesamples (Figure 1). HA specific 19A wasdetected in trace amounts in a few fluids but onlyafter mucosal immunization. Induction ofovulation did not affect HA specific IgG or IgAtiters in serum or the tract fluids. However, whenHA specific IgG in ovarian follicular fluid wasmeasured at 15 days post-infection the titreswere identical to those in the serum (4.85± 0.17,mean±S.D).2. Immunological and histologicalresponses in ovary to immunization withMV-ZPBEight female rabbits were intra-dermallyimmunized with 10 3 pfu MV-ZPB. Ovaries werecollected at day 15 and 30 post-infection. IgG in1115 days 1130 daysSerum Oviduct UteIllS VlIgina Oviduct Uterus VaginaFlullllltlgMlc:ro-pUncweFigure 1. Antloody titers in serum and the tract fluids.ovary was detected by immunofluorescencespecifically bound to the zona pellucida ofoocytes in secondary and tertiary follicles(Figure 2). The number ofpre-ovulatory follicles(2.3±1.5, mean± S.D) were significantly reduced(P

ASSISTEB REPRODUCTIVE TECHNOLOGY-MALEJSA PRESENTATIONSTHE ROLES OF SOX GENES IN MARSUPIAL SEX DETERMINATIONTHE ENTRY OF IMMUNOGLOBULIN INTO THE MALE REPRODUCTIVETRACTH. Ecroyd\ K.W. Beagle/, N. Newcombe' and R. C. Jones'.lDepartment ofBiological Sciences, University ofNewcastle, NSW 2308 and2Discipline ofPathology, Faculty ofMedicine and Health Sciences, The University ofNewcastle, NSW 2308INTRODUCTIONThe male reproductive tract is often considered animmunologically privileged site due to the presence ofboth anatomical (1,2) and immunological barriers thatact to shield reproductive antigens in the duct lumen.Knowledge of the local immunity of the malereproductive system and its regulation (both locallyand systemically), is fundamental to studies in anumber of fields. This includes the development ofvaccines against male STD's and maleimmunocontraceptive vaccines. The success of suchvaccines is dependent upon the induced antibodiesaccessing the target antigen in sufficient titres as to beeffective. The aims of the present study were toestablish the site(s) of entry of specific 19A and IgGantibody into the duct lumen ofthe male reproductivetract and to determine immunisation regimes thatmaximise their delivery.MATERIALS AND METHODSMale Balb/c mice were immunised with tetanustoxoid (IT) by subcutaneous, oral, intranasal (IN),rectal or transdermal routes. Luminal reproductivefluids were collected from the rete testis (RT), caudaepididymidis (CE), seminal vesicles and ventralprostate (P) gland and specific anti-tetanus IgG andIgA antibody titres of these fluids and serum assayedby ELISA-spot assay.RESULTSSpecific IgG was induced in RT and CE fluids at lowlevels (0.3-1.5% and 0.25-2.3% of the blood serumtitres respectively) following each immunisationregime, except following transdermal immunisationwhich induced higher levels (8.60/0 -RT fluid and7.6% -CE fluid) compared to the serum titre (Fig. I).The concentration of IgG in prostatic fluid followingrectal immunisation was higher than that induced atthe RT and CE and independent of the serum IgGtitre. The entry of 19A into the male fluids was alsofound to be independent of the blood 19A titre (Fig.2). 19A was detected in prostatic fluid following eachimmunisation regime, the highest titre being inducedfollo:ving IN immunisation at levels 3-8 fold higherthan In serum. 19A was present at low levels in fluidfrom t?e RT. fo.llowing each immunisation exceptrectal ImmUnISatIOn and only detected in CE fluidfollowing systemic immunisation. Specific 19A wasnot detected in seminal vesicle fluid following eithersystemic or mucosal immunisation.DISCUSSIONThe results show that some IgG enters at the rete testis(1) and is concentrated within the epididymal lumen. Theluminal titres seem to be dependent on blood titres, butonly reach a small proportion of them. The entry ofspecific 19A into the male fluids is interpreted as beingthrough local production by resident lymphocytes andsubsequent SC-mediated transport into the duct lumen.Similarly, it is concluded that IgG in prostatic fluidfollowing rectal immunisation arises due to its localproduction. Thus mucosal immunisation(s) may proveuseful in order to elicit an appropriate immune responseto antigens in the male reproductive tract.25:i 20a 15eJOr-----------------..2.20/..S,2Y. ,.EllITtluidI!lCEtluid5lPfluid:3 w 100.9%"'YSlJb.Q IntranasalRectal Transcbmll195 ------._--- --.. Serum1)KI'1I1id----Figure 1: The concentration (EU/ml) of anti-IT IgG antibody inreproductive fluids of male mice following various immunisationregimes. Mean ± SEM for 5 animals per group. % - percentage ofserum IgG titre..mOlid1!5 POlidFigure 2: The concentration (EU/ml) of anti-IT 19A antibody inserum and reproductive fluids of male mice following variousimmunisation regimes. Mean ± SEM for 5-10 animals per group,*p

·\"W? ~A. 10J-Y\A)~Vr~~~~ -VEGF, INTRAVASCULAR NEtJr OPHILS AND HUMAN ENDOMETRIAL ANGIOGENESISJSA PRESENTATIONSCaroline E. Gargen, Fiona L. Lederman and Peter A.W. Rogers ..Monash University Dept. Ob$tetrics & Gynaecology, Monash Medical Centre, Clayton, Vlctona, 3168JSA PRESENTATIONSPROGESTERONE DOWNREGULATION OF MATRIX METALLOPROTEINASE-l (MMP-l) INTHE HUMAN ENDOMETRIUM: EVIDENCE FOR INDIRECT TRANSCRIPTIONALREGULATION.IntroductionAngiogenesis is the formation of new vessels from preexistingvessels. Angiogenesis occurs periodically in thehuman endometrium as part of the menstrual cycle,particularly in the functionalis (1). A complex subepithelialcapillary plexus also develops with each cycle.During angiogenesis endothelial cells (BC) proliferate asthey migrate into avascular tissues or within vessels asthey elongate or intussuscept. Vascular endothelial growthfactor (VEGF) is expressed in glands, stroma and focallyin microvessels of human endometrium (2), but thecellular source for endometrial angiogenesis has not beendetermined. The aims of this study were to elucidate therelationship between microvessel VEGF and endometrialangiogenesis in the subepithelial capillary plexus, thefunctionalis and basalis of human endometrium and toidentify the VEGF-expressing cells associated with thesemicrovessels.MethodsFull thickness endometrium from 18 hysterectomy samples(3 menstrual, 7 proliferative and 8 secretory) withoutendometrial pathology were examined by immunohistochemistry.The percentage of pr ·n= 2 3 3 6 7 75 8 8menstrual proliferative secretoryFigure 1 Microvessels expressingfocal VEGF in the (-)subepithelial ~lexus, (~ ) funetionalis & (0) basalis ofhumanendometrium P

JSA PRESENTATIONSJSA PRESENTATIONSNON-RANDOM ARRANGEMENT OF CHROMOSOMES IN Sminthopsiscrassicaudata AND Macropus eugenii SPERMLK. Greaves!, M. Svartman l *, M.J. Wakefield!, D. Taggarr & J.A.M. Graves llDepartment ofGenetics, La Trobe University, Bundoora, Victoria, 30832Department ofZoology, Melbourne University, Parkville, Victoria, 3052Chromosome arrangement in spenn has been studied over many years. The dataare contradictory. There is evidence for non-random chromosome arrangement inmonotremes (1) and planaria (2), but not in frogs or chickens (3). Whether chromosomearrangement is non-random is a particularly interesting question, as specific arrangementswithin sperm could set up specific arrangements in the fertilised egg that may affect genefunctioning in the embryo. Marsupials are an ideal model to study because the spennhead is large and asymmetric, and the chromosomes are few and large, making it an easymodel to use for determining chromosome positions.Fluorescence in situ hybridisation (FISH) was used in this study to explore thearrangement of chromosomes within testis sperm of Sminthopsis crassicaudata andMacropus eugenii. Specific chromosomes were isolated through microdissection or flowsorting and DNA was isolated and labelled with._,fluorescent dyes. These labelled probes were then '3 /~~~~""hybridised to the spenn of the species studied, f .t?.~.. \.4- ; #"MNlt1lii; iwhere they "painted" the ep.tire chromosome from,i '/',lll'i iwhich it originated. '5 iWe. demonstrated that there is a nonrandomchromosome arrangement within thespenn of both marsupial species. Eachchromosome occupied a discrete region whichwas consistant between sperm from the one 6species (see figure). The X and Y chromosomes .2occupy the same position in X-bearing and Y­bearing spenn.As an example ofthe importance ofchromosomepositioning for gene regulation we looked at theSminthopsiscrassicaudataMacropuseugeniiposition of the X chromosome, and asked whether it is important in paternal X­inactivation. The observation that the X chromosome in both species sat at the point ofcontact between the sperm and egg may be significant for paternal X-inactivation.1. Watson, J.M., Meyne, J. and Graves, J.A.M. (1996). Ordered tandem arrangement ofchromosomes in the sperm heads of monotreme mammals. Proc. Nat!. Acad. Sci. USA. 93,10200-10205.2. Joffe B.L, Solovei, LV. and Macgregor, H.C. (1998). Ordered arrangement andrearrangement of chromosomes during spermatogenesis in two species of planarians(Plathelminthes). Chromosoma. 107, 173-183.3. Solovei, LV., Joffe, B.I., Hon, T., Thomson, P., Mizuno, S. and Macgregor, H.C. Unorderedarrangement ofchromosomes in the nuclei ofchicken spermatozoa. Chromosoma. 107, 184­188.Supported by the Australian Research Council·Supported by Fundayao de Amparo 'a Pesquisa do Estado de Sao Paulo (FAPESP)PROSTAGLANDIN H SYNTHASE-2 GENE EXPRESSION IN THEENDOMETRIUM AND PLACENTA OF THE TAMMAR WALLABYL.T Sebasti~ G.Shaw, G.E. Rice l and LJ ParryDepartment ofZoology, The University ofMelbourne, Parkville, 3052Iperinatal Research Centre, Royal Women's Hospital, Carlton, 3053IntroductionThe onset and progression of labour is associated withincreased prostaglandin (pG) production. Activation ofenzymes involved in prostaglandin synthesis, such asProstaglandin H Synthase (pGHS)· may influence theonset ofparturition. PGHS mediates the conversion ofarachidonic acid into Prostaglandin H 2 and as such,could be a rate-limiting step in prostaglandin synthesis.There are two isoforms of this enzyme, PGHS-l andPGHS-2. In the eutherian placenta, PGHS-2 mRNAand protein levels increase at term (1).In the tammar, plasma PGF2a metabolite (pGFM)rises dramatically within minutes ofbirth. There is alsoan increase in the production of PGs by theendometrium and placenta in late gestation (2).Premature birth and the peri-partum surge in PGFMare both induced by cortisol injection (3). The purposeofthis study was to examine PGHS-2 gene expressionin the placenta and endometrium of the tammarthroughout pregnancy and ascertain whether or notcortisol influences the timing of parturition via the.prostaglandin synthesis pathway.Materials & MethodsPGHS-2 gene expression was examined in uterine andplacental tissues, obtained from pregnant tammarwallabies shot in the wild on Kangaroo Island.Tammars (n = 3) received two injections (Lm.) 12hours apart, of the cortisol analogue dexamethansone(4 mg/mL) on day 24 ofthe 26 day gestation. Tissueswere collected 24 hrs later on day 25. Total RNA wasextracted from tissues and used in either RT-PCRreactions or in Northern analysis. Heterologousprimers from highly conserved regions of knowneutherian PGHS-2 sequences were used to obtaininformation on the tammar PGHS-2 cDNA. Tammarspecific, radiolabelled oligonucleotide probes (40-mer)and riboprobes (400 bp) were used in subsequentSouthern and Northern analyses.ResultsA 470 bp fragment of the tammar PGHS-2 cDNAmolecule has been cloned and shows 80-90% aminoacid homology compared with several eutherianspecies. Tammar PGHS-2 is first expressed in theplacenta on day 19 of the 26-day gestation (Fig. 1).Southern blotting confirmed the specificity ofthe PCRproducts and showed also PGHS-2 gene expression inthe endometrium of the gravid uterus. No PCRproducts are seen in the nongravid endometrium ormyometrium.10 17 19 20 21 22 23 24 25 26Fig.I. RT-PCR of yolk sac placenta through gestation.PGHS-2 gene expression is highest at the end ofpregnancy onday 26.This was confirmed by Northern blotting; a 4.5 kbtranscript is expressed only in the endometrium ofthegravid uterus at term (Fig.2), and the placenta.. . . . . .23 - 23 - 25 - 25 - 26 - 26 -;~~~:~:fl&&~~~:~~~~~: .:r.~bE

JSA PRESENTATIONSSPECIAL INVITED LECTURESIDENTIFYING MARKERS OF THE MAMMALIAN MALE GERM LINE USINGSUBTRACTIVE HYBRIDISATIONRoman S.D., Wallace C, Sutton K.A.School ofBiological and Chemical Sciences, University ofNewcastle, Callaghan NSWREGULATION OF GENE EXPRESSION IN EARLY MAMMALIANDEVELOPMENTMarilyn MonkMolecular Embryology Unit, Institute ofChild Health,30 Guilford Street, London WCIN lEHIntroduction Primordial Germ Cells (PGC's) developearly in mouse embryogenesis. These cells subsequentlydevelop into the reproductive cells of the mature germ linevia complex maturational processes requiring both cellmigration and proliferation (reviewed in 1).PGC's differentiate along male, or female, linesaccording the sex specific signals they receive from theirsomatic surroundings. Sry activation in Sertoli cellprecursors, over the period from 10.5-12.5 days postcoitium (dpc) leads to the development of testes (2). In themale, PGC's interact with the somatic Sertoli cells in thetestis chords and differentiate into the precursors ofSpermatogonial Stem Cells (SSC's).After a period of growth and proliferation, the malegerm-line is induced into mitotic arrest at 13-14 dpc. Thesse precursors then relocate to the centre of theseminiferous chords where they await the signal for postnataldevelopment into spermatogonia (reviewed in 3).There is very little known about the genes controllingPGe development and maturation into SSC's. It is thoughtthat failures in the proper transition from PGC's to SSC's inmale individuals may lead to testicular cancer (4,5). .The main focus of this work is to identify andcharacterise genes that are involved in the transition of thegerm line from PGC to SSC.As preliminary work to further identifying suchgenes, a male specific subtractive library has beengenerated via subtractive hybridisation experiments.Experimental DesignMale 12.5 dpcgenital ridge RNAIDiscard IFemale 12.5 dpcgenital ridge RNASubtractive hybridisation experimentsAssessment for differential expression... Male SpecificGerm or somatic cell expression?~GermFunctional studiesMethodsGenital ridges were isolated from 12.5 dpc embryos.The sex of the embryos was ascertained by examing for thepresence or absence of sex chords. Subtractivehybridisation were performed utilising a commercial kit(Clontech).A portion of the library produced by subtractivehybridisation was screened for differential expression.Plasmids (210) were blotted in duplicate onto nylonmembranes and screened with male and female probes toassess for differential expression across a larger portion ofthe library. Probes were generated from total RNA isolatedfrom male and female genital ridges of 12.5 dpc embryosvia radio-labelled eDNA synthesisResultsBFemaleFigure 1: Identification of differential expression via bulk screening of 21 0plasmids with probes generated from total RNA ofa) male genital ridges andb) female genital ridges isolated from 12.5 dpc embryos.• 21 clones exhibited male specific expression.• ATP synthase ~-subunit (circled) shows strongexpression on both membranes.In parallel, inserts from 47 colonies were sequenced.Ofthese, 5 possessed minimal insert (less than 20 bp). Theother 42 inserts are a minimum of 250 bp in length. 22corresponded to genes with general house-keepingfunctions. II corresponded to genes present in theGenbank database but not previously implicated ingonadogenesis and 9 were novel fragments.Using this information, combined with the amount ofbacteria transformed and the quantity of library ligated, weestimate the size of the subtractive library to beapproximately 1475 clones. Of this, approximately 20%are novel sequences.ConclusionsThis process has identified 21 potential candidates asa starting point for further identification andcharacterisation into their role in the male germ lineReferences1. McLaren, A., (1999). Genes Dev. ll:373-3762. Koopman, P., Gubbay, J., Vivian, N., Goodfellow, P.& Lovell-Badge, R., (1991). Nature. 351:117-1213. Lin, H., (1997). Annu. Rev. Genet. ll:455-4914. Moller, H., (1993). Eur. Urol. 23:8-135. Oliver, R.T., (1998). Curro Op. One. lQ:266-272Over the past few decades, investigations into the molecular mechanisms regulatinggene expression, and their refinement to the sensitivity ofthe single cell, have led todiscoveries which have challenged long-held dogmas influencing our understandingofthe basic nature ofearly mammalian development.Consider these questions.• Is the germ line continuous and sacrosanct in its developmental potency?• Must the germ line be set aside very early from very few cells before anyrestrictions in developmental potency in the developing embryo?• Is fertilisation the beginning, the to, ofthe genetic programme ofdevelopment?• Are the gametes tabula rasa - the ground state in terms ofgeneticprogramming?• Is it possible that the germ line could be subject to epigenetic modification tocause transgenerational changes in inheritance?• Is adaptive genetic change in inheritance possible?Research in my laboratory over the years has surprised me many times withunexpected results that challenged these dogmas and have slowly caused aparadigm shift in terms ofour understanding oftnammalian embryonicdevelopment.In this presentation, I will summarise our work over the years which has broughtabout this paradigm shift - our research on X-inactivation and differentiation infemale embryonic development (and continuity ofthe germ line), on correlations inX-chromosome inactivation mosaicism between somatic and germ cell lineages (andthe origin ofthe germ line), on changes in methylation and genetic programming inearly embryos (and totipotency), and on imprinting (and transgenerational variationin inheritance). In the second half ofthis talk, I will present some ofour currentwork on the extension ofthese concepts to studies on gene expression in humanembryonic development.eO(1 ~Y&C':s- 0t~~sf----~ ~tfJ l~--:;> id~ -) -f-c.R.--- ShA, A-e48 49

SPECIAL INVITED LECTURESMALE REPRODUCTIVE TRACT/SPERM FUNCTIONWHY CHROMOSOMES SEPARATE AT MEIOTIC ~APHASEBob Moor and Yanfeng DaiLaboratory ofProtein FunctionThe pattern of chromosome segregation at anaphase determines whether the cell division will be equatorial(mitotic) or reductional (meiotic). During mitosis sister chromatids lose cohesion in early anaphase andsegregate to opposite poles ofthe dividing cell. By contrast, in anaphase I of meiosis sister chromatids remaintightly associated and homologous chromosomes move together to the same pole; sister chromatids onlyseparate at anaphase II. A variety of proteins, most notably the cohesions, are required to maintain the bindingbetween sister chromatids throughout the first meiotic division (MI). The sequential proteolysis of selectedclasses of the chromatid binding proteins and cyclins, mediated by the anaphase-promoting complex (APC),controls both the separation of homologous chromosomes and their ordered poleward movement duringanaphase I of meiosis. Before this stage suppression of APe function by proteins of the MAD and cdc20 genefamilies prevents premature chromatid separation and the meiotic errors that otherwise result in irreversible fetaldefects. We shall focus in our communication specifically on the intracellular signals that regulate APC functionand anaphase progression during meiosis in mammals.We have cloned and determined the expression patterns of MAD2 and cdc 20, changes in their spatialdistribution during meiosis and the consequences on meiosis of up- or down-regulating these proteins before orduring anaphase. Interactions between MAD2, cdc20 and upstream regulatory proteins have been analysedusing yeast two-hybrid and co-precipitation approached. Both MAD2 and cdc20 are stored in the oocytecytoplasm as inactive masked proteins. After activation, cdc20 translocates to the kinetochores while MAD2 islocalised most heavily around the spindle pole. Up-regulation ofMAD2 blocks oocytes in metaphase I while upregulationof cdc20 has no discernible effect on meiosis. By contrast the down-regulation of MAD2 inducespecific but dramatic spindle distortions and hastens entry into anaphase I while the down regulation of cdc 20blocks oocytes in metaphase I. These combined results emphasise the key role of MAD2 and cdc20 proteinsduring meiosis.Events in the upstream signalling pathway have been investigated by precipitation and translation studies. Ourresults demonstrate that MAD2 binds strongly to cdc20 which, in tum, activates the APe and inducesproteolysis of the cohesions and cyclins. Moreover, cdc20 binds the ERK proteins and is linked to the meioticcycle regulator mos via the Mos-MAPK pathway. In an extension of our studies of the anaphase signallingnetwork we next examined the regulation of Mos. Transcripts of the c-mos proto-oncogene are stored duringmeiosis as masked messengers and occur in three different forms which differ only in the 3' untranslated region(3 'UTR). Hybrid messages consisting of a luciferase coding region coupled to the mos 3'UTR have beeninjected into oocytes to study tlle mechanisms of mos mRNA translational control. We show that mostranslation is regulated not only by the length ofthe 3'UTR but also by specific V-rich and A-rich motifs which.in tUlR act in different ways on tile long and short form mos transcripts. We propose that transcript length actsas a rheostat to control both the level of translation and the stage during meiosis at which the messages aredegraded while the A-rich and V-rich motifs act as on-off switches. Although we have cloned some of the RNAbinding proteins we have yet to demonstrate how signals from the membrane interact with these bindingproteins to activate the 3' motifs in stored meiosis-related transcripts.IMPACT OF EPIDIDYMAL MATURATION ON THE REDOX REGULATED PATHWAYSCONTROLLING SPERM CAPACITATIONBeverley Lewis and John Aitken* . .*School ofBiological and Chemical Sciences, University ofNewcastle and MRC Reproductive BIOlogyUnit, Edinburgh.IntroductionAs spermatozoa progress through the c.Pididymisthey acquire the competence to capaCItate. Thecellular mechanisms regulating this centralelement of the maturation process are notunderstood. .Tyrosine phosphorylation is widely regarded asone of the keystones of capacitation (1). Incapacitating spermatozoa, tyrosinephosphorylation is controll~d by a re~oxregulated cAMP mediated SIgnal transductioncascade that appears to be unique to this celltype (2-4). Since the function~l comp~t~nce ofthis unusual pathway dunng epIdIdymalmaturation has not been examined, an analysisofthis system was undertaken using the rat as ananimal model.MethodscAMP was monitored using the Biomol, formatA cyclic AMP enzyme immunoassay kit,acetylated version (Biomol" PA, ~SA).Tyrosine phosphorylation was detected WIth thePY20 monoclonal antibody (Affiniti, Exeter,UK) using conventional Western blot protoc~ls.Immunolocalization of the sites of tyrosmephosphorylation was achieved using an alkalinephosphatase labeled second antibody.ResultsThe induction of reactive oxygen speciesgeneration with NADPH resulted in an increasein intracellular cAMP in both caput and caudalspermatozoa, providing a phosphodiesteraseinhibitor was present in the form ofpentoxifylline (Fig la). However only ir: maturecaudal epididymal spermatozoa did thIS redoxinduction of cAMP generation result in anincrease in tyrosine phosphorylation (Fig 1b).The functional development of this cAMPtyrosinephosphorylation path~ay d';1ringepididymal maturation was as.soc~ated WIth ashift in the cellular 10callzatlOn of thephosphorylation sites from the acrosome (Fig.1c) to the sperm tail (Fig. 1d)ConclusionsRedox regulation of cAMP generation is aconsistent feature of spenn biochemistrythroughout all stages of ~pididyrnalmaturation. However only In maturespennatozoa does cAMP induce thephosphorylation of a novel set of proteinsmany of which are located in the sJ?enn ~ail.In this location these phosphoprotelns mIghtplay a key role in controlling the motilitychanges (hyperactivation) asssociated with theattainment ofa capacitated state.These results contribute to our understanding ofthe cellular mechanisms responsible forepididymal sperm maturation. Spe.cifically,they focus attention on the mechamsms bywhich cAMP stimulates tyrosinephosphorylation, as the key event controlli~g thepotential ofmaturing spermatozoa to capaCItate.WGo).,ac..2.0(00x 1.5li')E~ 1.0a.~

MALE REPRODUCTIVE TRACT/SPERM FUNCTIONMALE REPRODUCTIVE TRACT/SPERM FUNCTIONMECHANISMS OF ACTIVATION OF MAMMALIAN SPERMM. A. Wade, R. J. Aitken, R. C. Jones and R. N. MurdochSchool ofBiological and Chemical Sciences, University ofNewcastle N.S.W. 2300 AustraliaIntroductionThe molecular mechanisms involved in motilityinitiation are unknown. We have previously shownthat caudal epididymal sperm (CES) are activatedby a 40-fold dilution with a physiological solutionbut not with l.OS-fold dilution. The low dilutionregime was used to test the effect ofphannacological reagents, and demonstrated theinvolvement of cyclic adenosine monophosphate(cAMP) in motility activation (1). Other studieshave indicated the importance of the bicarbonateion (2) and recently, reactive oxygen species (ROS)such as hydrogen peroxide and/or the superoxideanion in spenn activation (3). It has also beensuggested that in the rat, release from viscoelasticrestriction provided by 'immobilin' in caudal fluid,initiates motility (5). The current study examinedthe involvement of these factors in motilityactivation.Materials & MethodsCES were obtained by backflushing the luminalcontents from the cauda epididymidis. Samples of1J.11 of luminal fluid were placed under watersaturated paraffin oil at 37°C. Reagents in 50 111 of300mM sucrose were added to the 1 J.11 dropletsand mixed. Alternatively 4 J.11 BWW, a modifiedTyrodes medium (6) or caudal epididymal fluid(CEF) was added (5-fold dilution) to the droplet.Motility was rated on a subjective scale of 1-10using the whole-droplet technique (4).ResultsThe 5-fold dilution in CEF had no effect but BWWactivated full motility (Fig. 1) which was sustainedfor more than 4 hours. A l.05-fold dilution with300mM sucrose had little or no effect when usedalone or when HC0 3 or ROS was added. However,the addition of dibutyryl cAMP (16mM) initiatedlnotility but this declined to zero within 60minutes. The decline in motility was not due tolack of energy substrate, acidification of cells ordegradation of cAMP. After the decline, a 5-folddilution with BWW re-activated motility (motilityscore = 8-10) which was sustained in excess of 4hours. Motility which was dilution-activated couldnot be prevented by inhibitors or protein kinase A(staurosporine or H89) whereas motility inducedby dibutyryl cAMP was prevented by the inhibitors.Figure 1 Activation of rat CES in response to 5­fold dilution in BWW or CEF, or the addition ofdibutYrYl cAMP, HC0 3 or ROS in 50nl of 300-mMX-x-x-x-x-x-+--cAMP__.._HCOlROS-X-S·foldioBWW__ill__ S·fold io CEFII I·'~"'~' ,m m10 20 30 40 soTIme (Minutes)'------------------------_.-sucrose (means from at least 5 animals).DiscussionThese results indicate that caudal epididymalplasma contains an inhibitor of motility activation.Dilution in a simple, defined medium such asBWW is sufficient to remove this inhibition andactivate movement. The inhibitory factor cannotbe overcome by HC03 or ROS, which are knownto have central roles in capacitation (2 & 3).Moreover the inhibitory factor cannot beimmobilin because motility can be activated in theabsence of dilution by raising the intracellularconcentration of cAMP. On the basis of theseresults we hypothesize that CEF suppresses theactivation ofmotility by inhibiting the cAMPIPKAsignal transduction system.References1. Armstrong V.L. Clulow 1. Murdoch R.N. Jones R.C.(1994) Biology ofReproduction 38,77-842. Aitken, R.I. Harkiss,D. Konx, W. Paterson, M. Irvine S.(1998) Biology ofReproduction 58, 186-963. Aitken RJ. Harkiss,D. Konx, W. Paterson, M. Irvine S.(1998) Journal ofCell Science 111, 645-664. Pholpramool ,C. Zupp,J.L. Sechell, B.P. (1985) Journal ofReproduction and Fertility 75,413-205. Ussleman M.C. Cone R.A. (1983)Biology ofReproduction29. 1241-536. Biggers J.D. Whitten W.K.· Whittingham D.G. (1971)Methods in Mammalian Embryology, San Francisco,Freeman Press,pp 86-116Anti-actin monoclonal antibody inhibi~s hyperactivation and the zona pellnicdainduced acrosome reaction ofhuman spermLiu Dyl,2, Martic M 1 , Clarke GN 3 , Grkovic 1 4 , Garrett c 1 , Dunlop :ME 5 and Baker HWG 1 ,2,6JDepts. Obste. & Gynae., 4Anatomy & Cell Biology and 5 Medicine, University ofMelbourne, 2ReproductiveBiology Unit and 3 Andr?logy Lab., Royal Women's Hospital and 6 Melbourne IVF, Melbourne, AustraliaIntroduction: The human zona pellucida (ZPinduced acrosome reaction (AR) is critical forsperm penetration ofthe ZP. Hyperactivation (HA)is a marker of sperm capacitation, which occursbefore the AR. Our previous study found that actinplays important role in the both HA and the ZPinduced AR (1). Aim of this study was todetermine if anti-actin monoclonal antibody (Ab)can inhibit HA and the ZP induced AR of human.sperm.Materials and Method: Oocytes that had failedto fertilize in IVF were used. Sperm samples wereobtained from normozoospermic men with normalsperm-ZP binding. Motile sperm selected by swimuptechnique were incubated with 4 oocytes for 2 hin human tubal fluid supplemented with 0.5%BSA, with or without 1:100 actin Ab (leN) orcytochalasins B and D. The sperm bound to theZP were dislodged by repeatedly aspirating theoocytes with a small-bore pipett« and the AR wasdetermined by fluorescein la'Oelled 'Pisum Sativumagglutinin. Effect of the actin. Ab on sperm HAwas assessed by Hamilton-Thorn MotilityAnalyzer (2). Immunobeads (anti IgG Dynebeads)test and pre-embedded electron microscopy (EM)immunogold labeling on sperm pre-incubated withthe actin Ab were performed to detect ifthe actinAb could react with either surface or internal actinofcapacitated human sperm.Results: The actin Ab significantly inhibited boththe ZP-induced AR (Fig 1) and sperm HA (controlvs test, mean±SD, n=10, 13±5% vs 5±4%,P

MALE REPROD.UCTIVE TRACT/SPERM FUNCTIONMALE REPRODUCTIVE TRACT/SPERM FUNCTIONFINAL CONSTRUCTION OF SPERM ACROSOME MAY BE ANACTIN-MEDIATED PROCESSMiniie Lin, Chris Scarlett and R. John AitkenCooperative Research Centre for Conservation and Management ofMarsupialsSchool ofBiological and Chemical Sciences, The University ofNewcastle, NSW 2308, AustraliaIntroductionIt is now known that a number of most dynamicprotrusions at the somatic cell periphery are actinmicrofilament-containing structures, and theconstantly assembling and disassembling of themicrofilament (F-actin) within the cell protrusionsdetermine the structural reorganisation and functionsof the cell (1). Posttesticular change in the form ofthe acrosome as a concomitant of sperm passagethrough the .epididymis has been found in someeutherian mammals. The changes of the acrosomerange from a major reorganization expressed in theguinea pig and chinchilla to more modest changeseen in the rabbit, some primates (not human) andsome rodents (2). However, recently we confirmedthat some marsupial spermatozoa display the mostradical reorganization seen in any species so far (3).Therefore, the marsupial acrosome is an idealstructure to study the role of actin filaments on thefinal construction ofthe mammalian acrosome duringsperm maturation in the epididymis.Materials and MethodsSpermatozoa were freed from the testis and caput,corpus and cauda epididymis from 5 tammarwallabies for examinations. For EM, tissues werefixed in 2.5 % (v/v) glutaraldehyde and 2%paraformaldehYde in O.lm cacodylate buffer forovernight at 4°C, then treated with 1% osmiumtetroxide for 4 hr. After processing through thedehydration and critical point drying, the tissues werecoated with gold and examined in a JSM 840 SEM.For F- actin immunofluorescences, spermatozoa werefixed for 60 min in 4% formaldehyde in PBS at 4°C,and air dried on Poly-L-Iysine coated sliders;permeabilised with cold methanol and acetone (-20°C) for 10min each; stained with 50J.lg/ml Phalloidin­FITC conjugate solution in PBS for 60 min at RT;after 3 washes with PBS, F-actin fluorescence wasdetected under a Ziss Axiovert 10 fluorescentmicroscope.Results and DiscussionsIn the tammar wallaby, the acrosome is a sheet oftissue with elongated 'scoop' shaped protrusionswhen spermatozoa leave the testis. The acrosomalprotrusions condensed into a compact button-likeorganelle as the spermatozoa transit through theepididymis (Fig. 1, top row). The occurrence of actinfilaments within the acrosome was temporally andspatially associated to the process of the acrosomeshaping in the epididymis (Fig. 1, bottom row). Actinfilaments were assembled when acrosomalprotrusions started to fuse together in the caputepididymis, and disassembled after the acrosomecondensation was completed in the distal corpus andcauda epididymis. Future work will be directed atidentification and characterising sperm-specificproteins involved in the actin assembly anddisassembly during sperm epididymal maturation. Itis ~ticipated that the identification ofthose proteins,which may be exploited for the manipulation of malefertility, particularly in those mammalian species,such as in rabbits, mice and some marsupials, whoseacrosome requires a morphological maturation in theepididymis.Fig. 1. Fonnation ofthe wallaby acrosome (thetop row) and occurrences ofF-actin filamentsin the acrosome (the bottom row) as spermtransit from the testis to caput, corpus, andcauda epididymis (arranged from left to right inboth rows). A, acrosome., Reference:(1) Carraway et aI., (1998) In "Signalingand the Cytoskeleton", p 8-30, Springer.(2) Fawcett & Bedford (1979) In "TheSpermatozoon", P 11-19, Urban &Schwarzenberg.(3) Lin & Rodger (1999) 1. Anat. 194:223­232.DEVELOPMENT OF PSA-IO MONOCLONAL ANTIBODY REACTIVITY WITHBRUSHTAIL POSSUM (TRICHOSURUS VULPECULA) SPERMATOZOA DURINGEPIDIDYMAL MATURATIONM.S. Harris a and J.C. Rodger bCooperative Research Centre for the Conservation and Management ofMarsupialsaLandcare Research, Lincoln, NZ and bMacquarie University, NSW, AustraliaIntroductionThe PSA-10 monoclonal ant:J.body (mAb) binds to antigen(s) on the fibrous sheath and midpiece fibrenetwork (MFN) of mature tammar wallaby (Macropus eugenii) 'spermatozoa and the fibrous sheath.,midpiece fibre network and acrosome ofmature bmshtail possum (Trichosurus vulpecula) spermatozoa(1). This study examined the development ofPSA-l0 mAb immunoreactivity with the acrosome andmidpiece ofpossum spermatozoa during epididymal maturation.MethodsSpermatozoa were collected from the testes and proximal and distal regions ofthe head, body and tailof the epididymides of three possums after mincing the tissue in phosphate buffered saline (PBS)containing protease inlnbitors. Sperm were pooled by region, fixed in 4% paraformaldehyde andlabelled with PSA-I0 ascites fluid or myeloma ascites as described previously (1). The fluorescencepatterns on 100 randomly selected sperm were counted for each ofthree subsamples of sperm. pooledfrom different regions of the tract. Data was analyzed using a two-tailed Fisher exact test. Freshlydissected tissue from the possum and wallaby male reproductive tract was fixed in 4%paraformaldehyde for 5 h on ice before being processed for post-embedding immunogold labellingwith PSA-l0 ascites and myeloma ascites control (1).ResultsPSA-IO acrosomal immunor~activity was evident on most possum spennatozoa from the proximalhead ofthe epididymis but not those from the testis (Table 1). PSA-I0 immunoreactivity with restrictedregions of the midpiece was detected first on some possum sperm from the proximal·head of theepididymis (Table 1). The proportion of sperm showing midpiece immunoreactivity increased withepididymal transit, with the entire posterior midpiece ofall spermatozoa from the proximal body oftheepididymis being labelled. Immunogold labelling of the possum midpiece was associated first withgranular material surrounding the mitochondrial sheath, before the formation of the midpiece fibrenetwork. Labelling of the :MFN was evident after formation in the distal head of the epididymis.Immunogold labelling ofthe possum acrosome was associated with the outer acrosomaI membrane.Table 1. The percentage of fixed possum spermatozoa (n=3) from different regions of the malereproductive tract demonstrating acrosomal, midpiece and principal piece immunoreactivity with PSA­10 ascites fluid.Percentage ofpossum sperm demonstrating PSA-I0 mAbOrigin ofsperm inmale reproc Immunoreactivity with given structure (Mean % ±SE)Acrosome Midpiece Principal PieceTestis O±O O±O 100±0Proximal Head Epididymis 92.9±1.6* 11.9±2.0* 100±0Distal Head Epididymis 98.8±1.2* 96.0±2.1* 100±0ProximalBody Epididymis 100±0 100±0* 100±0* WIthin column, values are sIgmficantly different from preVIOUS (P

MALE REPRODUCTIVE TRACT/SPERM FUNCTIONMALE REPRODUCTIVE TRACT/SPERM FUNCTIONSNARE proteins, Syntaxin and Munc-18 are present in human spermMONOCYTE CHEMOATTRACTANT PROTEIN-l(MCP-l) ISUP-REGULATED IN THE TESTES OF LIPOPOLYSACCHARIDE-TREATEDADULT RATS: A POSSIBLE ROLE IN MONOCYTE RECRUITMENTOrapin Gerdprasen, Moira K. O'Bryan, David P. Nikolic-Paterson*, Kimberley L. Sebire,David M. de Kretser and Mark P. HedgerMonash Institute ofReproduction and Development, and *Department ofNephrology,Monash Medical Centre, Clayton, Victoria 3168IntroductionAt 12h after lipopolysaccharide (LPS)-administration to adult rats, there is a large increase of peripheral bloodmonocytes in the testicular interstitium (1). The biological trigger for this increase is unknown. Monocytechemoattractantprotein-I (MCP-I) is a memberofthe CC chemokine family and is themajorstimulus for monocyteinfiltration at other sites of inflammation (2). It is produced by a number of cell types, including monocytes,fibroblasts and endothelial cells in response to stimulation by bacterial endotoxins and inflammatory cytokines.These observations suggest that MCP-I may be involved in the recruitment ofmonocytes into the testis during LPSinducedinflammation.Materials & MethodsAdult male Sprague-Dawley rats were injected (Lp.)with saline alone or saline containing either a lowdose (0.1 mglkg body weight) or a high dose(5mglkg) ofLPS (from E. coli). Rats were killed atvarious time points (0-72h) after treatment. Testeswere fixed by whole-body perfusion with Bouin'sfixative. Testes and liver samples were collected forextraction oftota! RNA (3).Expression of rat MCP-I mRNA transcripts wasmeasured by Northern blot analysis using a 32p_labelled cDNA probe, and by in situ hybridisationusing a digoxigenin-Iabelled riboprobe. GAPDHwas used as loading control for the Northerns. Thesense cRNA was used as the negative control for insitu hydridisation. In order to assist with theidentification ofthe testicular cells producing MCP­1, double-labelling of in situ hydridisation-Iabelledsections was performed by immunohistochemistry,using the monocyte antibody, ED I, or the residentmacrophage antibody, ED2. Other testicularinterstitial cell types were identified by establishedmorphological criteriaResultsMCP-I mRNA was undetectable in saline-injectedcontrol testes. Expression ofMCP-I inthetestes wasup-regulated in a dose-dependent manner, 3-6h afterLPS-treatment(Fig. I).In situ hybridisation/immunohistochemical doublelabellingshowed intense labelling for MCP-ImRNA in several types ofinterstitial cells, includingmonocyte-like lIlacrophages, Leydig cells,peritubular and perivascular cells (data not shown).Resident testicular macrophages (ED2j wereunlabelled.ConclusionsMCP-I expression increases dramatically immediatelyprior to the increase in number ofmonocytes in the LPStreatedtestis, indicating a central role for this chemokine intesticular inflammation. The failure of resident testicularmacrophages to produce MCP-I in this model providesfurther evidence ofthe non-inflammatory phenotype ofthistesticular celltype.18S..MCP-IGAPDHLowdoseLPSo 3 6 12 18 24 72*o 3 a 12 11 24 nTmeHigh dose LPSo 3 6 12 18 24 72IfFig. 1: A Northern blot analysis ofMCP-l and GAPDH mRNA inthe rat testes following systemic LPS-injection. Histograms showMCP-l/GAPDH ration (values in the histograms are mean + scm,n=3 animals per group) (* p < 0.05 compared with saline-injectedcontrols).ReferencesI. Gcrdprascrtct al submitted for publicat~on.2. Gura T (1996) Sciencc272:954-63. O'Bryan MK, Schlatt S, Phillips DJ, de Kretser DM, Hedger MP (2000)Endocrinology 141:238-246.**TImeM. Martie\ D. Y. LiuI, M. E. Dunlop2, H. W.G. Baker l1 University ofMelbourne Departments ofObstetrics and Gynaecology Royal Women's Hospital and 2MedicineRoyal Melbourne Hospital, Melbourne, AustraliaIntroductionThe acrosome reaction (AR) is an importantexocytotic process during human fertilisation andinvolves fusion between the plasma and outeracrosomal membranes of sperm. In a variety ofsomatic cells exocytosis requires membrane proteinsknown as SNAP (soluble NSF attachment protein)receptors (SNAREs) (1). Syntaxin is acytoplasmically oriented plasma membrane SNAREwhich forms a complex with the SNAP 25 (25kdsynaptosomal-associated protein) and VAMP (thevesicle-associated membrane protein) prior tomembrane fusion. SNAREs have been identified insea urchin sperm and are possible regulators of theAR (1). Munc-I8 is a syntaxin binding protein withisoforms expressed in many tissues including thetestis. Phosphorylation with protein kinase C inhibitsMunc-18 association with syntaxin and allowsmembranes to fuse (3). In the present study we reportthe presence ofsyntaxin and Munc-I8 homologues inhuman sperm.MethodsMotile sperm from healthy volunteers were selectedby swim up technique, air dried on glass slides andfIXed in 95% ethanol. Slides were stained by indirectimmunofluorescence and immunoperoxidasetechniques using polyclonal syntaxin (Calbiochem,NSW, Australia) and monoclonal Munc-18(Transduction Laboratories, Ky, USA) antibodies, atthe concentration of 1:100 following standardprocedures. In some experiments sperm were furtherpermeabilised with 0.1% Triton-X-IOO (lCN, CA,USA). These sperm lost their plasmalemma and theouter acrosomal membrane To assess the effect oftheAR on syntaxin localisation, sperm were incubatedwith 3JlM A23187 and double stained with pisumsativum agglutinin labelled with rhodamine andsyntaxin antibodies labelled with fluorescein.Controls omitting the syntaxin and Munc-I8antibodies or substituting them with an irrelevantmonoclonal antibody showed no staining in all testsResultsIndirect immunofluorescence localised syntaxin tothe equatorial segment of human sperm. Acrosomereacted sperm lost staining ofthe equatorial segmentand showed different staining patterns: on the tail,postacrosomal area or complete loss of staining. Thevariation in syntaxin localisation after the ARsupports a possible role for this protein in regulatingthe AR. Indirect immunofluorescence andimmunoperoxidase staining localized Munc-18 onthe tail of human sperm only after the sperm hadbeen treated with 0.1%Triton X-IOOFigure 1. Immunofluorescence microscopy ofhumansperm treated with syntaxin polyclonal antibodies.Controls were negativeConclusionThis study reports the presence of syntaxin in theequatorial segment of human sperm with its loss ortranslocation after the AR suggesting it may beinvolved in the membrane fusion of the AR. Thenature ofthe anti-Munc-18 staining in the sperm tailis uncertain.References1) Sudhof, T. C. (1995) Nature 375: 645-532) Schulz, J. R., G. M. Wessel, G. M. et al. (1997)DevBioII9I(I): 80-73) Fujita, Y. Sasaki, T. et al. (1995) J BioI Chem270: 5857-635657

MALE REPRODUCTIVE TRACT/SPERM FUNCTIONMALE REPRODUCTIVE TRACT/SPERM FUNCTIONAZOOSPERMIA, HYPOSPERMATOGENESIS, LOW FSH, IDGH INHIBIN BAND POSSmLE INTRATESTICULAR GENITAL TRACT OBSTRUCTION.HWG Baker~ CJ Stem and DM RobertsonUniversity ofMelbourne Department ofObstetrics and Gynaecology~Melbourne IVF and Prince Rerny's Institute ofMedical Researc~ Monash Medical Centre~Melbourne~ Vic.Royal Women~s Rospita1~Background: isolated FSH deficiency is very rare. Most disorders ofspermatogenesisare caused by primary testicular abnormalities which tend to be associated with lowinhibin B and high basal and GnRH stimulated FSH levels. We present a case study ofaman with a spermatogenic defect and low FSH levels.Presentation: 12 months ofprimary infertility, two azoospermic semen tests and withnormal volume (2.0, 2.5mL) and orchiopexy for a right undescended testis ofthe age ofseven years. Puberty occurred unremarkably. He stopped growing around age 17 to 18years. His wife had good health and regular ovulatory cycles. Physical examinationshowed normal beard and body hair, long limbs (height 182cm, arm span 192cm, pubis tofloor 94cm), a scar in the right inguinal area and moderate testicular atrophy (volumes:right 10mL, left 14rnL).Investigations: karyotype, Yq microdeletion, serum LH, prolactin, testosterone,oestradiol, 17 hydroxyprogesterone, hCG, sperm antibodies, thyroid function andpituitary N1RI were normal. Serum FSH (0.5-0.7, N 1.0-7.0IUIL) was low and respondedpoorly to GnRH (max 1.0, N>3nJlL). Inhibin B levels were high (324, 383,N

MALE REPRODUCTIVE TRACT/SPERM FUNCTIONMALE REPRODUCTIVE TRACT/SPERM FUNCTIONWHOLE BODY HEAT-STRESS OF MALE MICE IMPAIRS SPERM-ZONA PENETRATIONS. Maddocks, B.P. Setchell and J. YaeramDepartment ofAnimal Science, University ofAdelaide, SA 5064DEVELOPMENT OF DNA FINGERPRINTING FOR APPLICATION IN ASSISTED REPRODUCTIVETECHNOLOGYIntroductionReduced fertility of domestic animals is common duringsummer months. This is believed to be a result ofheat stress.We have previously reported (1) that whole-body exposureofmale mice to a temperature of36°C for 12 h per day for 2days has a dramatic effect on paternal fertility, disruptingspermatogenesis, reducing sperm fertilising ability andreducing litter size when heated males are mated to normalfemale mice. In the present study, we have gone on toinvestigate with in-vitro fertilisation, sperm-egg binding andsperm-zona penetration using sperm collected from malemice exposed to whole-body heating.Materials and MethodsSix-to-eight-week old female and 10 to 12-week old maleC57/CBA F1 mice were used. The males were heated for12h per day for 2 consecutive days in a cabinet maintained at36°C and 65% relative humidity. Superovulated female micewere used to source eggs subjected to in-vitro fertilizationusing sperm taken from control and heated males at 7, 10and 14 days after heating. Swum-up sperm were subjected totriple-staining to assess acrosome integrity. Cumulus-freeoocytes were stained with Hoescht-33342 stain andincubated at 37°C with 2 x 10 6 swum-up sperm for 20 min.Eggs were then fIxed, washed and examined for spermbindingand sperm-zona penetration.ResultsSperm-egg bindingAll eggs successfully bound between 7 and 38 spermatozoaand the average numbers of sperm bound per egg weresimilar (p>O.OS) regardless of the treatment period postheatingofthe males (Table 1).Table1 Binding of spermatozoa collected from male micesubjected to heat at 36°C for 2 days (12h/day) to cumulusfreeoocytes obtained from normal female mice.Days post- No. No. eggs No. eggs No. sperm boundheating males incubated with sperm per eggAverage RangeControl 12 87 87 23±Oo4 10-38Day 7 4 86 86 22±0.S 10-32Day 10 4 82 82 22±0.5 7-32Day 14 4 90 90 23±0.9 7-33Acrosome statusMost live sperm were recovered acrosome intact, althoughsignificantly reduced numbers of live, acrosome-intactsperm were obtained from heat-exposed males (Table 2).Most dead sperm were acrosome reacted, with numberssignfIcantly increased in heat-exposed males (Table 2).Table2 Effect of heat stress on acrosome (AR) status andproportion oflive/dead spermatozoa.Percentages ofspermDays post- No. LiveAR- LiveAR- Dead DeadARheatingmales intact reacted AR- reactedintactControl 4 7S±3.9 a 8±0.9 3±0.9 14±Oo4aDay 7 4 6S±1.8 b 7±OA 4±0.9 24±1.S bDay 10 4 64±0.9 b 8±0.9 3±0.6 26±1.2 bcDay 14 4 60±1.1 b 8±1.0 3±0.5 30±2.0 cValues with different superscripts within a colunm differ significantly(p

MALE REPRODUCTIVE TRACT/SPERM FUNCTIONINDUCED TRIPLOIDY BY HEAT SHOCK IN NILEM (Osteochilus hasselti C.V.) FISHYulia Sistina, Rino Trijatmoko, Irwan Setiadi and Retno WidiastutiFakultas Biologi Universitas Jenderal Soedinnan Purwokerto, IndonesiaINTRODUCTIONA protocol to produce nilem triploid fish has beendeveloped from a fish gynogenesis protocol developedfrom other species (1). Nilem fish, a cyprinid, havepotential to be a popular commercial fish since theyhave a good taste, less bone than tawes and produce alot ofeggs for a 'caviar'. One ofthe disadvantages isthat they grow very slowly, compared to othercyprinids. Triploid fish have been known to havehigher growth rates due to sterility (2). In triploid fish,the third set ofchromosomes is from the second polarbody and the two other sets are from the male andfemale pronucleus. We report successful production oftriploid nilem fish by heat shock (HS) 400 C for 90seconds to protect second polar body extrusion. ThisHS has been developed from successful production ofnilem gynogenesis.MATERIALS AND METHODSEggs and spermatozoa were collected from matureparents by stripping. Prior to stripping, standardhypofisation procedures have been used, using carpfish as a donor, to ensure the maturation of gametesused. Milt was diluted in Ringer solution (1:100).Gynogenesis procedure was carried out at the optimumtime for polar body extrusion after sperm and eggmixing (fertilisation). Gynogenesis was performed asbefore (1). Briefly, diluted milt was IN-irradiated for5 min and then used to fertilise eggs. HS treatmentwas performed to produce diploid gynogenesis.Triploidy was achieved using normal (untreated)diluted sperm with eggs then followed by HS 40> C for90 seconds at three different times, 1 min, 3 min or 5min after sperm-egg mixing (fertilisation time). Theassessment included fertilisation rate, egg hatching,larvae and fry survival rate, fry size and diameter ofthered blood cells. Percentages of triploids weredetermined from the diameter ofred blood cells. Thecontrol was sperm and eggs without heat shock (normalfertilisation).RESULTS AND DISCUSSIONFrom the three different times of heat shock afterfertilisation, different percentages of triploids wereobtained compared to the control group which were alldiploid (Table 1). Morphologically there were nodifferences between diploid and triploid larvae or fry,which were very different between haploid and diploidfrom the gynogenesis study.Table 1. Percentage fertilisation, hatching, frysurvival, and triploids (mean ±SD at 21 days.Initial % % % %HS Fertility Hatch Survive TriploidominControl88±6 99±.5 97±1 01 min 46±24 84±9 87±3 70**3 min 44±7 91±1 86±3 80**5 min 38±8 46±11 44±14 50** ** **** = p

MALE REPRODUCTIVE TRACT/SPERM FUNCTIONCHANGES IN GLYCOPROTEIN COMPOSITION OF THE PLASMA MEMBRANE OFBRUSHTAIL POSSUM SPERMATOZOA, DURING EPIDIDYMAL MIGRATIONN. J. Cooper, and W. G. BreedDepartment ofAnatomical Sciences, The University ofAdelaide, South Australia 5005.INTRODUCTIONIn eutherian mammals, there is both processingand post-translational modification of sperm(glyco)proteins during their migration along theepididymis and, in addition, other moleculesbecome bound to the sperm plasma membrane(PM) that are sYnthesised and secreted by thelining epithelial cells (1,2).There have been few reports on the proteincomposition of the sperm PM of marsupials.Previously, in our laboratory, we extracted sixproteins from caput spermatozoa that were notpresent in caudal sperm extracts, while 8 proteinswere extracted from caudal sperm (3). Whetherthese proteins were glycosylated was not, however,determined.In a preliminary study carried out in this lab, it wasfound that the glycoprotein composition of thepossum spermatozoon PM changes duringepididymal maturation (4). The aim of this studyhas been to characterise the glycoproteins presenton the possum sperm PM. For this, lectins wereused to detect glycoproteins in sperm extracts afterSDS-PAGE and western blotting.METHODSCaput and caudal epididymal spermatozoa,obtained from adult male brushtail possums, werewashed with phosphate buffered saline (PBS), andincubated in 0.05% NP-40/PBS plus proteaseinhibitors, for 10min on ice. Sperm were thencentrifuged, and the supernatant removed andstored at -200 e. A sample of sperm from eachstep was removed, fixed and, processed fortransmission electron microscopy.Protein extracts were run on a 10-15% SDS-PAGE(16x20xO.75cm), using denaturing and reducingconditions. The proteins were thenelectrophoretically transferred to nitrocellulose(n/c) sheets using Towbins transfer buffer. Thelectin staining method of Srivistava and Olson(1991)(5) was then used to stain foroligosaccharides. The n/c was washed, incubatedwith biotinylated-lectin (5ug/ml) in Tris bufferedsaline (TBS) containing 2mg/ml haemoglobin, andthe appropriate sugars (0.2M) were used ascontrols. Binding was detected with streptavidin­HRP (5ug/ml), and DAB.Lectins used were Soybean Agglutinin (SBA),which is specific for N-acetyl-d-galactosamine(NAcGal), and Concanavalin A (ConA), which isspecific for glucose and mannose.RESULTSNo positive staining was observed with SBA onthe n/c, but ConA resulted in positive staining ofbands of MW 17, 26, 39, 56.5 and 61 kDa fromcaput sperm, and MW of 27 and 41.5kDa fromcaudal sperm. The ConA+sugar control wasnegative, indicating that the sugar inhibitedbinding oflectins to proteins on the n/c.CONCLUSIONSResults from this preliminary study show that, (1)NAcGal may not be present in the PM of possumsperm, and (2) glucose and mannose are present onboth caput and caudal sperm, but the bands thatstain positively with this lectin differ for the tworegions.REFERENCES1. Orgebin-Crist MC, Danzobo J, Davies J (1975).In Handbook of Physiology: Endocrinology, Vol.5. Eds RO Greep and DW Hamilton. AmericanPhysiological Society, Washington.2. Cooper, TG (1999). Journal of Reproductionand Fertility, Supplement, 53: 119-136.3. Lamont AE, Clarke H, Cooper NJ, HollandMK, Breed WG (1998). Journal of Reproductionand Fertility, 114: 169-177.4. Cooper NJ, Holland MK, Breed WG (1998).Reproduction and Fertility, Supplement, 53: 221-226.5. Srivistava A, Olson GE (1991). MolecularReproduction and Development, 29: 357-364OOCYTE AND EMBRYO DEVELOPMENTINVESTIGATION OF THE EFFECTS OF GLUCOSE AND PHOSPHATE ON EARLYCLEAVAGE STAGE EMBRYOS: A SmLING OOCYTE STUDY.Helena Jericho\ Felix Niet0 2 and David.H.Edgar 1 ,2IMelbourne IVF, Freemason's Medical Centre, 320 Victoria Pde, East Melbourne 3002, Victoria.2Reproductive Biology Unit, Royal Women's Hospital, Carlton 3053, Victoria.INTRODUCTION: Cleavage stage embryos have a substantial endogenous energy source. Thisinternal reserve means that embryos are very adaptable, and can cope with culture conditionswhich are probably less than ideal. Recent studies have shown that conventional embryo culturemedia such as HTF may actually be detrimental to embryo development and implantation due tothe presence ofglucose and phosphate, which may interfere with embryonic metabolism. This hasled to the development of a modified HTF (P 1), which does not contain glucose or· inorganicphosphate. The aim ofthis study was to compare the effects of culturing embryos in HTF and PIon blastomere number and morphology, whilst eliminating patient differences by. randomisingembryos from each patient to the two different media. Implantation rates were also investigated,and were based on embryo transfers where the embryos from each group were not mixed, thusmaking it possible to determine unequivocally from which group the pregnancy resulted.MATERIALS AND METHODS: In order to eliminate possible effects ofglucose free media onsperm function, only patients being treated by ICSI were included in the trial. After oocytecollection, the oocytes were denuded and cultured in 30 JlI droplets ofeither RTF or PI under oil.The media were supplemented with 4mg/ml RSA (Albumex). After ICSI, the injected oocyteswere washed and transferred to the appropriate culture drops. The assignment of oocytes toculture media was random, with each patient having approximately 50% ofinjected eggs culturedin HTF, and the remainder in Pl. Embryos were transferred back to the uterus at approximately44 hours post insemination.RESULTS: A total of 533 HTF cultured eggs were subjected to ICSI, with 327 fertilisingnormally (61.4%). Of these, 5.5% (18/327) arrested before the first cleavage. Within the ·Plculture group, 66.10/0 (319/483) fertilised normally and 4.7% (15/319) arrested. There is nosignificant difference between these results. At approximately 40-44 hours post leSI, theembryos were assessed for blastomere number. Within the RTF group, 30.7% (95/309) had 2blastomeres, 13.0% (40/309) had 3 blastomeres, and 56.3% (174/309) had at least 4 blastomeres.The cleavage rate of embryos within the PI group was not significantly different, with 30.3%(92/304) having 2 blastomeres, 9.9% (30/304) with 3 blastomeres, and 59.9% (182/304)developing to 4 or more cells. The degree offragmentation ofall embryos was also assessed. Thetwo groups produced embryos of similar quality, with 82% of embryos in each group having lessthan 10% cytoplasmic fragmentation. Implantation rates derived from embryo transfers whichinvolved only HTF or only PI cultured embryos were 10.6% (5/47) and 12.5% (8/64)respectively. This result is not statistically different.CONCLUSION: Embryo culture medium which does not contain glucose or phosphate did notappear to have an effect on embryo quality or implantation rate when compared to mediumincorporating these components.6465

OOCYTE AND EMBRYO DEVELOPMENTGLUCOSE TRANSPORT IN MURINE BLASTOCYSTS IS STIMULATED BYGRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF)Sjoblom, C. l , Wikland, M. l and Robertson, S.A?lFertilitetscentrum, Carlandersplaten 1 40229, Goteborg, Sweden, and 2Department ofObstetrics &Gynaecology and Reproductive Medicine Unit, The University ofAdelaide, South Australia 5005.INTRODUCTION _ 6Glucose is a major energy substrate in compacted .s~5pre-implantation embryos, and growth factorsfI)ftIincluding insulin and IGF appear to promote m4anabolic activity, survival and proliferation in C) 3blastomeres through increasing the activity of ~facilitative glucose transporters (1). The cytokine ~ 2GM-CSF has been shown to stimulate the growth ~ '0 1and development ofboth mouse and human pre-Eimplantation embryos through binding to the 0.- Q. 0chain ofthe GM-CSF receptor (2,3). In other cell 0,08 0,4 2,0 10 Con (ng/ml)lineages, including Xenopus oocytes and melanoma Fig. I Effect ofdose ofGM-CSF on 3-H* OMG uptakecells, ligation ofthis receptor can stimulate glucose in murine blastocysts (mean±SD pmoles 3-H* OMG /uptake through a kinase-independent pathway (4). blastocyst) *p< 0.005The aim ofthis study was to investigate the effect of~l\GM-CSF on glucose transport in mouse pre- . ~.rJ' ~implantation embryos. .', C,~ '{"('" (30) (21) (8) (8) (10) (10) (17)MA TERIALS AND METHODS i..,.r/~. g 5Blastocysts (n=369 in total) recovered from 'isuperovulated 4 week old Balb/c x C57BV6 female j5 4mice at 97 h post hCG after mating with CBA x ~ 3C57BV6 males were allocated into HTF media with 0addition ofrecombinant murine (rm)GM-CSF (0.08, ~ 20.4, 2.0, 10 ng/ml) or various other cytokines ~including IGF, macrophage-CSF (M-CSF),interleukin (IL)-2, IL-IO and IL-3 (concentrationsECo 0shown in Fig. 2). After 1 hour incubationblastocysts were transferred for 4 min into mediawith non-metabolisable analogue 3-H* ortho-methylglucose (3-H* OMG, 25 mM), then washed inglucose-free media and assessed by betascintillation counting.RESULTS3-H* OMG uptake into blastocysts was significantlyincreased by incubation in rmGM-CSF (Fig 1). Theresponse to GM-CSF was dose-dependent with amaximum effect (50% increase) at 2 ng/ml. Theextent ofthis effect was comparable to that ofIGFused at a dose previously found to have optimaleffects (1). None ofthe other cytokines tested hadan effect on glucose uptake (Fig 2).REFERENCES1. Pantaleon M et at. (1996) Mot.Reprod.Dev. 44:71-762. Sjoblom C, et al. (1999) Hum. Reprod. 14:3069-3076.4. Robertson S et al., (1998) Proc. ASRB Abstract 62.3. Ding DX et at (1994) PNAS USA. 91:2537-41.GM·CSF2nglml(36)(46)(66)IGF-1 M-CSF IL-213 Ps:IIml 200IUlml 200IU/ml(47)IL·10200IU/ml(51)IL·3200IUlmlConFig 2 Effect ofcytoldnes on 3-H*OMG uptake in murineblastocysts (mean±SD pmoles 3-H* OMG / blastocyst)*p< 0.005CONCLUSIONThese data show that GM-CSF can promote glucoseuptake in murine blastocysts, presumably throughbinding to trophectoderm cells via the a-chain ofthe GM-CSF receptor. Improved glucoseavailability might thus provide a mechanismwhereby GM-CSF can improve blastomere survivalin human and murine pre-implantation embryos (2).Conversely, blastocyst culture in cytokine deficientmedia might be associated with blastomereapoptosis due to suboptimal glucose availability.Further experiments are required to delineate theeffect of GM-CSF on specific glucose transportermolecules in blastocysts.Materials and methodsThe NH4+ concentration of media (mOdified~synthetic oviduct fluid (SOF) supplemented witheither 20% HS or amino acids) was determinedover 5 days using a clinical bichromatic ratetechnique (Dimension, Dade Behring, USA))Assays were kindly conducted by staff at th~Dept. of Clinical Chemistry, QEH, Woodvill

OOCYTE AND EMBRYO DEVELOPMENTOOCYTE AND EMBRYO DEVELOPMENTPROBLEMS ASSOCIATED WITH THE DETECTION OF ~LTINUCLEATEDBLASTOMERES IN HUMAN EMBRYOSIntroduction:D. A. Sherrin, T. M. Breen, M. R. Hunter and K. L. HarrisonQueensland Fertility Group, 225 Wickham Tce, Brisbane, QLD, 4000Multinucleation of cleavage stage embryosdisplaying 2 pronuclei and 2 polar bodies atfertilization check has been recorded as beingpresent in between 8%1 and 74%2 of humanembryo cohorts. Several studies haveconfirmed that embryos with multinucleatedblastomeres (MNB) are developmentallyincompetent due to chromosomalabnormalities and recommend that theyshould be excluded from routine embryotransfer unless there are insufficient numbersofnormal embryos for transfer I.2,3.Materials and Methods:Prior to the embryo transfer, embryos wereobserved on a warmed stage at 200xmagnification using an Olympus IMT-2inverted microscope to detect multinucleatedblastomeres and assess morphology. If therewere sufficient normal embryos, the embryoscontaining MNB were rejected for embryotransfer, regardless of their morphologicalstatus.Results:Over a four-month period, embryos from 73cases were assessed for multinucleation ofblastomeres and 37 (50.7%) were found tocontain at least one embryo with one or moreMNB. Of the embryos assessed, 12.2%(61/498) contained one or more MNB, and18.0% (11161) of these displayedmultinucleation in all of the blastomeres.Multinucleated embryos. were transferred in 5patients where there were insufficient normalembryos and none ofthe patients in this groupbecame pregnant.The majority of embryos (95%) in whichMNB were detected were at the 2-cell or 3­cell stage when the multinucleation wasobserved.Conclusions:Multinucleation of normally fertilizedembryos was discerned more often at the 2­cell and 3-cell stages than at the 4-cell or laterstages. Difficulties were experienced inobserving all ofthe nuclei at the 4-cell or laterstage as any degree of embryo fragmentationcan obscure one or more nuclei. The invertedmicroscope currently used to assess embryosfor multinucleation has a short workingdistance, not allowing the operator access toroll the embryos so that all ofthe blastomerescan be comprehensively observed. As well asthis, cell nuclei are only visible duringinterphase so that blastomeres which havebegun mitosis are unable to be assessed. Atleast 50% of our transferred embryos are atthe 4-cell stage or later, and it was thoughtthat we may be missing some multinucleationdue to the problems outlined above.The embryos are now assessed very early onDay 2 so that we can observe more embryosat the 2-cell stage, and again just beforetransfer when many have cleaved to the 4-cellstage. This practise does not appear to haveaffected the pregnancy rate, but our concernshave prompted the purchase of a HD IVFchamber and a powerful stereo zoommicroscope so that more comprehensiveobservations, which will include rolling theembryos, can be performed in a controlledenvironment.References: (1) Pelinck, M. J., et. al. Human Reprod., 13:4: 960-963, 1998(2) Balakier, Hand Cadesky, K. Human Reprod., 12:4: 800-804, 1997(3) Jackson, K. et .al. Fertil. and Steril., 70: 60-66, 1998~~'v\ L~EXAMINATION OF THE INTERACTION OF EGF, FSH AND ACTIVIN A ONMEIOTIC RESUMPTION IN MOUSE OOCYTESGina Abdelnour, Steven Fleming, Giovanni Coticchio and Peter Illingworth.Department ofReproductive Endocrinology and Infertility, University ofSydney, NSW 2145Introduction: A variety of ovarian autocrine and paracrine factors may modulatefolliculogenesis, and hence oocyte maturation, The oocyte developmental programme thatleads to its meiotic resumption is governed by a precise quantitative and temporal pattern ofsignal transduction that triggers germinal vesicle break down (GVBD) proceeding to the firstmetaphase (MI) and second metaphase (MIl). FSH plays an essential role in this process. It isknown that a variety of growth factors can modulate FSH action by autocrine and paracrinemechanisms. The purpose ofthis study was to examine the effect ofEGF, activin A and FSHon cumulus-enclosed oocytes maintained in their meiotic arrest in vitro. The presence of adirect physiological role was also investigated by using oocytes denuded from their cumuluscells.Materials and Methods: Three week old female Quackenbush mice were sacrificed 48 h postPMSG (5 IV/ml) injection. Ovaries were dissected and large antral follicles were punctured torelease cumulus enclosed oocytes (CEO). CEO and denuded oocytes (DO) were cultured in aMEM medium containing 0.3% BSA and 4 mM hypoxanthine, to maintain their meioticarrest. Medium was supplemented with EGF (0.01 - 1 ng/ml), FSH (0.5 - 75 mIU/ml) andactivin A (50 - 200 ng/ml) either alone or in combination. Oocytes with no treatment wereused as controls. Percentages ofGVBD (MI + MIl) were assessed after 17-28 h.Results: The results show that EGF and FSH alone, were able to override the inhibitory effectofhypoxanthine and induce GVBD in a dose related manner. A maximal stimulatory effect ofEGF (Ing/ml) and FSH (75 mID/ml) was observed in CEO (73% and 79% GVBDrespectively) but was minimised in DO (11 % and 20% GVBD respectively). Percentages ofGVBD did not increase when they were combined at half-maximal concentrations, howeverthe progression from MI to MIl was significantly improved; 39% when combined versus 24%in EGF only, and 26% in FSH only (P< 0.05). Activin A (100 ng/ml) did not affect meioticresumption when used alone (7% GVBD). Furthermore it had no additive effect on EGF andFSH at half-maximal doses. However when combined with high concentrations of FSH (75mIV/ml), percentages ofGVBD declined significantly by 12% (P

OOCYTE AND EMBRYO DEVELOPMENTOOCYTE AND EMBRYO DEVELOPMENTINCUBATION OF TWO HOURS IN 6-DMAP IS SUFFICIENT FOR PARTHENOGENETICACTIVATION OF SHEEP OOCYTEST.T. Peura, H.M. Hamilton and S.K.WalkerSouth Australian Research and Development Institute, Turretfield Research Centre, Rosedale,SA 5350, AustraliaIntroduction and aimsActivation of oocytes in the absence offertilising spermatozoa after micromanipulationsuch as nuclear transfer, is aprerequisite for subsequent embryonicdevelopment. Chemical activation by Ca++­Ionophore and 6-dimethylaminopurine (6­DMAP) is a commonly used activation methodin sheep and cattle. The length of 6-DMAPincubation varies from 4 to 5 h in cattle and 3to 4 h in sheep. The purpose of this study wasto evaluate ifparthenogenetic activation in thesheep is influenced by (i) a shorter (2h vs 3h)incubation period in 6-DMAP and (ii) theconcentration ofCa++-Ionophore (10 J.lM vs 20J.lM).Materials and MethodsIn vitro-matured sheep oocytes were freedfrom cumulus cells 24 h after maturation andexposed to either 10 or 20 JlM Ca++-Ionophore(Sigma) in synthetic oviduct fluid (SOF)­Hepes medium for 10 min followed byincubation for either 2 or 3 h in SOF-mediumcontaining 2 roM 6-DMAP (Sigma). Someoocytes from each treatmen~ group were fixedin Hoechst 33342-Glycerol at 2 and 3 h afterCa++-Ionophore incubation to determine theprogression of nuclear events. The remainingoocytes were subsequently cultured inSOFaaBSA-medium for 7 days, after whichmorulae and blastocysts were fixed in Hoechst33342-Glycerol for determination of cellnumbers. A total of seven replicates wereperformed and the development rates and cellnumbers were analysed with GraphPad Instatstatistical program using unpaired t-test afterarcsin transformation ofthe percentage data.ResultsData for the Ca++-Ionophore treatments werecombined as there were no statisticallysignificant differences. Evaluation of nuclearkinetics after 6-DMAP treatment revealed nodifferences between the two treatment groupsin respect of chromosome decondensation andformation of early pronucleus. Likewise, nostatistically significant differences wereobserved between the groups in eitherembryonic development rates (Table 1) or incell numbers in either compact morulae (CM),blastocysts (B), expanded (XB) and hatchedblastocysts (HB) (Table 2),Table 1. Development rates ofparthenogeneticsheep embryos after 2 or 3 h incubation in 6­DMAP.Group N Cleavetfl Morulao Blastocysf% % %2h 397 88.4 11.8 27.03h 399 86.5 15.5 32.3aDlfferences withm columns not statIstIcallysignificant (p>0.05)Table 2. Mean cell numbers (±SEM) ofparthenogenetic sheep embryos after 2 or 3 hincubation in 6-DMAP.Group CM Ir XB" HIr2h 41±3.3 78±5.7 119±4.l 153±16.33h 46±3.0 78±3.1 117±4.5 169±9.4aDlfferences withm columns not statIstIcallysignificant (p>0.05)Discussion and conclusionsIt is concluded that the progression of nuclearevents in sheep parthenogenetic developmentis so rapid that the incubation time in 6-DMAPcan be shortened to only two hours withoutcompromising activation. This finding haspractical benefits in experimental design ofnuclear transfer procedures. Our subsequentexperiments in sheep somatic cell cloningusing this modified activation protocol haveyielded good embryonic development ratesand pregnancies that have also resulted in theproduction ofoffspring.Acknowledgments: This research wassupported by the CRC for Premium QualityWool.ENTRY INTO FIRST CELL CYCLE AS A SENSITIVE LABORATORY PERFORMANCE, MEASUREMurray CT. and Catt 1.Sydney IVF, NSW 2000, Australia.Introduction: Laboratory performance measures(LPM) are routinely used in our IVF laboratory as partof a quality assurance (QA) programme. Control andwarning limits are set according to historical data andused to detect significant variation in specificlaboratory procedures (1,2). However, whilst the mostfrequently used LPMs such as fertilisation, cleavage,embryo utilisation, pregnancy and implantation ratesgive a good indication if something serious is amiss,they are not sensitive enough to detect the early signsof any problems. Some of our previous studies (3)indicate that the time for zygotes to enter into the firstcell cycle may be sensitive to changes in conditions,including oocyte!embryo abuse and mediacomposition. We have therefore extended our currentQA system to investigate whether the proportion ofembryos entering the first cell cycle at 24 hours postinsemination could be used to establish a LPM andthen to assess the sensitivity ofthe LPM.Materials and Methods: The LPM values for entryinto the first cell cycle at 24 hours post insemination(hpi) were calculated for a 2 month period and thecontrol ranges were set at ± 3 sd and the warninglimits at - 2sd from the mean values. IVF and ICSIcases have been kept separate, since IeSI has provento be more sensitive in the past. Prior to revision ofthe M91 culture media formulation it was observed (3)that ICSI embryos entered first cleavage significantlylater than IVF embryos. However, following thefonnulation revision they observed a reverse in theseresults (3). Analysis of the procedures separatelyshould confirm these findings.Results: The Control Range and Warning Limitsderived from 52 cycles of IVF and 26 cycles of ICSIare shown in Table 1. There were 352 IVF zygotesand 160 ICSI zygotes used to derive the IVF and ICSIvalues. This LPM will be :further refined over time.Table 1: 24 Hours Post InsemmatlOn Cleavage RateControl RangeWarning LimitsIVF I ICSI IVF I ICSI56.75-61.72 I 57.34-72.44 57.58 I 59.86The data to date is presented in Graphs 1 & 2 as thepercentage of zygotes that have cleaved at 24hpi ofthe total observed zygotes.Initial observations of the cleavage rate at 24hpiseparately for IVF and IeSI indicate that there is nosignificant difference between the cleavage rates at24hpi ofICSI embryos compared to IVF embryos (X 2test).References:Graph 1: 24 Hours Post InseminationIVF Cleavage Rate80..,------------70 -- :~ ~ :::: ~:::=cJ

OOCYTE AND EMBRYO DEVELOPMENTOOCYTE AND EMBRYO DEVELOPMENTASSESSMENT OF OVARIAN FOLLICULAR VASCULARITY BY POWERDOPPLER SONOGRAPHY DURING TRANSVAGINAL OOCYTERETRIEVAL IN IVF CYCLES - A PILOT STUDY.Steve Robson, Michael Barry and Robert J Norman.Reproductive Medicine Unit, The University ofAdelaide, Queen Elizabeth Hospital,Woodville, South Australia, 5011.Introduction Previous studies employing power Doppler sonography prior to oocyteretrieval suggest that oocytes from follicles ofhigh vascularity yield embryos that aremore likely to lead to ongoing pregnancy. Assessment offollicular vascularity at thetime of oocyte retrieval might hold certain advantages: the potential to misidentifyfollicles ofpre-determined vascularity is reduced, and inconvenience to thesonographer during routine cycle monitoring is eliminated.Methods A total of26 women were recruited to the study. Transvaginal oocyteretrieval was performed using an Aloka SSD-2000 ultrasound machine with a UST­980-5 angled probe and standard needle guide in the usual manner. The vascularity ofeach individual follicle was scored using standard criteria 1 from 1 (no vascularity) to4 (total circumferential vascularity) prior to aspiration. Follicles were aspiratedindividually, and the embryologic performance ofeach oocyte was followed. Apartfrom Doppler assessment, every other aspect ofembryologic and clinical managementwas conducted according to normal unit protocol. In the first 12 cases, the procedurewas videotaped to validate the reproducibility ofgrading scores. Fluid from follicleswas assayed for oestradiol (E 2 ), progesterone (P) and testosterone (T).Results A total of275 follicles were studied in 26 women. 52 (19%) weregrade 1; 136 (49%) grade 2; 84 (31%) grade 3; and 3 (1%) grade 4. Yield ofoocytesper follicle was similar for all grades: 58% in grade 1; 71 % in grade 2; 730/0 in grade3; and, 67% in grade 4. There were no differences in follicular fluid concentration ofeither E2, P or T between grades. Fertilisation rates were similar across grades inboth IVF and IeSI [IVF - 11/22 (50%) grade 1; 32/43 (74%) grade 2; 13/20 (65%)grade 3 {no grade 4 oocytes by IVF}. IeSI - 8/9 (89%) grade 1; 36/53 (68%) grade2; 27/38 (71 %) grade 3; 2/2 (100)% grade 4]. A total of 50 fresh embryos weretransferred. Since embryo transfer cohorts were made up ofembryos derived fromvarying grade oocytes, implantation rates could not be calculated for each grade.Where pregnancy ensued, the mean grade ofoocyte/embryos in the transfer cohortwas 2.44 in IVF and 2.83 in IeSI (overall mean 2.60), compared to mean grades of1.95 in IVF and 2.05 in IeSI (overall mean 2.00) where no pregnancy resulted. Whenthe cohort contained at least one embryo from a grade 3 or 4 follicle, the pregnancyrate was 50%. Ifthe cohort ofembryos transferred was derived from oocytes fromfollicles ofgrade 1 or 2 only, the clinical pregnancy rate was 15%. The mean age ofwomen with clinical pregnancy was 30.1±4.38, compared to 34.5±5.99 in nonpregnantwomen.Conclusions Power Doppler assessment offollicular vascularity at oocyte retrievalis not difficult. Pregnancy rates were increased when at least one embryo from ahigh-grade (3 or 4) follicle is included in the transfer cohort.1. Bhal P, Pugh N, Chui D et al. The use oftransvaginal power Doppler ultrasonography to evaluatethe relationship between perifollicular vascularity and outcome in in-vitro fertilization treatment cycles.Hum Reprod 1999; 14: 939-45. 0,./\DEVELOPMENT OF BOVINE-OVINE INTERSPECIES CLONES: PRELIMINARYSTUDIESH.H. Hamiltonab, S.K. Walkera, G.C. Webb b , S. Maddocksband T.T. Peura aaSARDI, Turretfield Research Centre, Rosedale, SA 5350,bUniversity ofAdelaide, SA 5005, AustraliaIntroduction and AimsIt has been recently shown that the oocYtecytoplasm from one species is capable ofdirecting early embryonic development ofanother species (1). The purpose of this studywas to determine if ovine oocyte cytoplasm cansupport early bovine embryonic developmentafter the transfer of somatic nuclei from bovinefoetal fibroblasts into enucleated ovine oocytes.Materials and MethodsThe cloning procedure used was adapted fromCampbell et aI. (1996). In vitro matured sheepoocytes were enucleated by aspiration 20 to 24 hafter maturation, followed by fusion with bovinefoetal fibroblast cells. Successfully fusedcouplets were activated in 10JlM Ca 2 +-ionophorebefore a 2 h incubation in synthetic oviduct fluid(SOF) medium containing 2mM 6-DMAP.Cattle foetal fibroblasts between passages 3 and6 were used as donor cells following serumstarvation for 9 to 10 days. In 2 replicates theresulting clones were karyotyped (metaphasearrested with 20JlM Nocozadole, fixed withethanol+acetic acid and stained with Giemsa).The bovine fibroblast cell line was alsokaryotyped. In 5 replicates the fused coupletswere cultured in SOFaaBSA-medium for 8 days,at which time the embryos were scored visually,then fixed in Hoechst 33342-glycerol todetermine the number of cell nuclei. As thecontrols, 3 replicates were also carried out usingovine foetal fibroblasts as donor cells.ResultsThe bovine fibroblast karyotyping revealed thecorrect number of chromosomes (60XY),although one spread out of the 30 had anabnormal chromosome fragment. Twelve out of31 karyotyped. bovine-ovine interspecies clonesgave readable metaphase spreads, and all werederived from the bovine fibroblasts (as indicated.by the presence of clearly identifiable bovinechromosomes). As Table I shows, a largerproportion of interspecies clones arrested beforethe 16-cell stage than intraspecies clones. Therewere also a number of cleaved clones with onlyone nuclei (Table 1). The overall developmentof sheep-derived clones was better than theclones derived from cattle cells.Discussion and conclusionsThese preliminary results suggest that bovinenuclei obtained from foetal fibroblast cells caninitiate early pre-implantation embryodevelopment with the support of ovine oocytecytoplasm. The degree of nuclearreprogramming will be evaluated in furtherstudies.References(1.) Dominko T, et al. (1999) Bioi. Reprod.60:1496-1502·(2.) Campbell KHS, et al. (1996). Nature380:64.Table 1: Final developmental stages of cloned embryos derived from transfer of bovine and ovinedonor cells into ovine cytoplasts. Percentages of developmental stages calculated from cleavedclones.DonorFibroblastNCleaved(%)5-8 nuclei(%)73

OOCYTE AND EMBRYO DEVELOPMENTTO WHAT EXTENT DOES FEEDING RUMEN-PROTECTED 3-0MEGA FATTY ACIDSALTER THE REPRODUCTIVE ENDOCRINOLOGY OF DAIRY COWSS. MeierDairying Research Corporation, Private Bag 3123, Hamilton, New ZealandINTRODUCTIONThe importance of fatty acids in controllingendocrine function has, until recently, beenunderestimated. Fats and specific fatty acidsare precursors to hormones that controlreproduction. The importance of progesteroneand prostaglandins is well recognised in thereproductive processes both are SYnthesisedform fats or fatty acids. It is therefore possiblethat, through the manipulation of fats and fattyacids, both progesterone and prostaglandinscan be altered so that reproductive function isenhanced. The manipulation of reproductivefunction using fatty acids has previously beendemonstrated (1,2,3).MATERIAL & METHODSTwenty 2-year old Holstein-Friesian dairy cowswere allocated to this experiment. From day 26­33 postpartum all animals were SYnchronisedusing an intravaginal progesterone insert(CIDR-B, InterAg Ltd., Hamilton, NewZealand) and oestradiol benzoate (1 mg i.m.,Intervet Pty Ltd., Castle Hill, NSW, Australia).Ten cows were allocated to receiveapproximately 220g ofrumen-protected fish oil,contained approximately 18 g ofeicosapentaenoic (EPA; C20:5ro3) anddocosahexaenoic acid (DHA; C22:6ro3).Weekly blood samples were taken from calvingby coccygeal vessels and analysed for plasmacholesterol. Plasma progesterone (P4)concentrations were also measured 3 times aweek throughout this period. On day 16 of thesecond oestrous cycle oxytocin (aT; 100 LU.,Butocin, BOMAC Ltd., New Zealand) wasadministered. Frequent blood sampling via ajugular vein catheter was carried out. Theseplasma samples were then stored and analysed13,14-dihydro-15-keto prostaglandin F2et(PGFM).RESULTSAfter 6 weeks of treatment plasma cholesterolwas significantly elevated in the supplementedgroup compared to that of the control group(6.53±0.61 vs. 4.53±O.26 mmol/l, respectively;p

SPECIAL INVITED LECTURES"'\.~ ISPECIAL INVITED LECTURESICSI and OOCYTE ACL> nONR. YanagimachiInstitute for Biogenesis Research, University ofHawaii School ofMedicine, Honolulu,Hawaii 96822 USAUnder normal conditions, a mature oocyte is activated upon its fusion with a fertilizatingspermat.ozoon. There have been two major theories of oocyte activation: (1) the G protein-IP 3~roduc~on theory and (2) the sperm factor infusion theory. The former requires ligand-receptormteractions b:tween the oocyte's and the spenn's plasma membranes, while the latter requiresmembrane fusIon between two gametes. Although successful oocyte activation by rcsr seems tofavour the .l~tte~ view, two mechanisms may be working simultaneously or consecutively duringnormal fertilIzation. At least part of the sperm-borne oocyte-activating factor (SOAF) is intracellularbe~aus~ the spermatozoon from which its plasma membrane is completely removed is still capable ofactivating an oocyte by rCSI. The intracellular SOAF is associated with the perinuclear theca whichhas been ~ought to be a purely mechanical structure. SOAF may be present partly in a soluble forman~ partly. m a les~ s~luble fonn. SOAF becomes biologically active during spermiogenesis. It lacksstnct specIes-specificIty as ~viden~ed, for ex~ple, by full activation of mouse oocytes after injectionof the sperma~ozoa of a WIde vanety of speCIes (e.g., the mouse, human, pig, fish and sea urchin).resr may frol when the spennatozoa do not have enough SOAF (e.g. round-headed human~p~nnatozoa) or th~ oocytes are not. sensetive enough to SOAP. In such cases, treatment of spennillJectedoocytes With parthenogenetic agents (e.g., calcium ionophore and electric shock) may solvethe problem.As of today, m~ian species in which normal live offspring were obtained by rcsr include:~~an, ~ouse, rabbIt, cat, sheep, cattle, horse, pig and monkey. Although a live spermatozoon can beillJected Into an oocyte, s~erm ".immobilization" (damaging spenn plasma membrane) or removingspe~ plasma mem?rane llumediately before resr is recommended for successful fertilization. Thep~rsistence of the .mtact ~perm plas~ membrane around the head of the injected spennatozoonhinders/retards the mteractlOn ofoocyte s cytoplasm with sperm nucleus and SOAF.REPRODUCTIVE HEALTH NEEDS WORLDWIDE: CONSTRAJNTS TOFERTILITY CONTROLPenny KaneAssociate Professor, Office for Gender and Health, University ofMelboumeThe scope ofthe term 'reproductive health' as defined in the 1994 UN conference onPopulation and Development, is outlined.and some further aspects of reproductivehealth as it affects both women and men are identified.An attempt is made to quantify the impact, in terms of mortality and morbidity, ofthese various components of male and female reproductive health. .Use is made ofsurvey data and the estimates of deaths and the total disease burden from each wasundertaken by the WorId Health Organisation in collaboration with the WorId .Bankand Harvard School ofPublic Health.These data suggest that maternal causes are the greatest contributors to the diseaseburden amongst women aged 15-45. In sub-Saharan Africia at least one quarter andin the Middle East and in India one fifth, of all ill-health affecting women in thoseages is related to childbearing. Many of the pregnancies are unintended and indeveloping countries a quarter to a half of those who want to delay or avoid furtherpregnancy are not using contraception. Induced abortion accounts for 61,000pregnancy-related deaths, and sexually transmitted diseases and other illnesses alsoresult from unsafe sexual practices.At least 1 million deaths each year are due to unsafe sex. These deaths - and anoverall disease burden of 50 million disability-adjusted life-years - are entirelypreventable. Yet half ofall couples worldwide are either not using contraceptives, oronly use traditional methods.Some of the constraints to contraceptive use are outlined. These range from lack ofinternational commitment to improving reproductive health, to social and economicfactors - in particular those which affect women's status and ability to make their owndecisions. They include lack of biomedical; research into new methods and poorsocial science research, as well as inadequate knowledge amongst sections ofthemedical professions as well as the public.The paper concludes that men and women have the right to demand better servicesand the knowledge and conditions in which to use them. Those in the moredeveloped countries have the responsibility of ensuring adequate financial andtechnical support to make reproductive health possible everywhere.7677

OVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALEIN VITRO GROWTH OF MURINE PREANTRAL FOLLICLES AFTER ISOLATIONFROM OVARIAN TISSUE FROZENffHAWED IN DIFFERENT CRYOPROTECTIVEAGENTS .Helen Newton and Peter IllingworthDepartment ofReproductive Medicine, Westmead Hospital, Sydney, NSW 2045.Introduction: Ovarian tissue cryopreservation is potentially a new technique for preserving thefecundity of cancer patients. One strategy for harvesting mature oocytes from frozen/thawed tissueis to isolate follicles for in vitro growth to antral sizes, followed by in vitro maturation andfertilisation ofthe oocytes. The aim ofthis study was to examine the growth and endocrine functionof murine preantral follicles isolated from ovarian tissue after; (i) freeze/thawing in differentcryoprotective agents using a slow freeze protocol, (ii) vitrification.Materials and Methods: Ovaries from 21 day old Balb/c mice were bisected and cryopreserved ineither 1.5 M solutions of dimethyl sulphoxide (DMSO), glycerol (GLY) or propylene glycol(PROH) using a slow freeze/rapid thaw protocol, or 5 M solution of DMSOIPROH using a rapidfreeze/rapid thaw protocol (vitrification). Preantral follicles 100-135 J.lm in diameter were isolatedfrom fresh and frozen/thawed tissue by manual dissection and cultured individually for 8 days in200 J.l111EM-a supplemented with 50 IU/ml penicillin, 50 J.lg/ml streptomycin, 5.5 J.lg/ml sodiumpyruvate, 3 roM L-Glutamine, 10 J..Lg/ml transferrin, 5 J..Lg/ml insulin, 5 ng/ml sodium selenite, 10%FCS and 90 mIU/mIFSH. Follicular diameter was measured on days 1, 4 and 8 and spent mediawas assayed for inhibin A and B and oestradiol. On day 8 1.5 ill/ml hCG and 5 ng/ml EGF wasadded to the culture media and sixteen hours later oocyte-cumulus complexes released from thefollicles were stripped and assessed for maturation.Results: At the time of isolation follicles recovered from frozen/thawed tissue appearedmorphologically normal and the number of follicles collected from frozen/thawed and fresh tissuewas comparable. Non-viable follicles began to degenerate within 72 hours of culture and thisprocess was typically characterised by either oocyte exclusion or follicular collapse. In the freshcontrol group 79±3% of follicles (n= 72) survived for 8 days of in vitro growth and this wassignificantly higher (p

OVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALETHE EFFECT OF COOLING RATE AND PROLONGED EXPOSURE TO AN ISCHAEMICENVIRONMENT INSITUORIN VITRO ON POST THAW OVARIAN TISSUE VIABILITY.M. Clearyl,2, M. Snow l ,2, M. Wolvekamp2, J. Shaw 2 , S-L. Cox l ,2 and G. Jenkin l ,2lDepartment ofPhysiology and 2Monash Institute ofReproduction and Development, MonashUniversity, Clayton 3168, AustraliaIntroductionOvarian tissue cryopreservation andtransplantation has been predominantlyperfolTIled using freshly obtained material. Theeffect of obtaining ovarian tissue where a timedelay exists between the death of the animal andcollection/cryopreservation of the tissue has notbeen established. This situation frequently ariseswhen obtaining material opportunistically fromroad kills or a distant location. In these casestissue will be subjected to prolonged periods ofischaemia before collection and/or while it isshipped to the laboratory .The time delay could be reduced by freezing thetissue in the field. There are commerciallyavailable portable freezers which do not requiremains power and are suitable for use in the field,but these give a faster cooling rate (I°C/min)than is conventionally used for ovarian tissue(0.3°C/min). This study looked at the effects ofthe cooling rate on post thaw ovarian structureand function. It also investigated whether tissueswhich experienced prolonged exposure to anischaemic environment (in situ or in vitro) couldbe cryopreservedMaterials and MethodsWhole ovaries were collected from 3-4 week oldfemale inbred virgin Balb/c mice. These werecryopreserved using a commercially availablepassive cooling device (Nalgene 5100-0001,Sigma). We verified that this gives a cooling rateof .....1°C/minute when placed in a -84°C freezeror on dry ice. Control tissue was cryopreservedby conventional slow cooling (0.3°C/minute) in aprogrammable biological freezer (Cryologic) (1)Tissue ischaemia was simulated by leaving theovaries for 0, 3, 6, 12 and 24 hours after deatheither in situ in the body or in vitro in PBSsupplemented with Penicillin and Streptomycinstored at room temperature. Tissue viability wasassessed by grafting the ovarian tissue to thekidney capsule of ovariectomised female Balblcrecipients. Two weeks after grafting the tissuewas collected and the morphology assessed byhistology.ResultsThere was no significant difference in follicleviability between tissues cryopreserved at lOCIminute in a portable freezer, or O.3°CI minute ina programmable freezer. The viability fell as thelength of exposure to an ischaemic environment(in situ or in vitro) increased, with no healthygrowing follicles being present in tissuerecovered, and cryopreserved, 24 hours afterdeath. However follicles at all stages ofdevelopment and little tissue destruction wasseen in tissue cryopreserved up to 12 hours afterdeath.DiscussionThis study showed that the use of a passivecooling device surrounded by dry ice or in a ­84°C freezer is a suitable alternative toconventional slow cooling. This means thatovarian tissue can be cryopreserved in the field,or in other situations where there is no access topower or a programmable freezing machine.This study also showed that tissue viability doesdecline as the interval between death andcryopreservation/ grafting increases. Howeverthe fall in viability was initially relatively slow.Thus even ovaries collected 12 hr after the deathof the donor animal contained viable germ cells.While there may be species differences thisindicates that viable germ cells should bl§ presentin ovaries collected opportunistically from recentroad kills and in material shipped interstate.The finding that ovarian tissue can becryopreserved with low technologycryopreservation equipment up to 12 hr after thedeath of the donor, or in PBS at roomtemperature, has practical implications, inparticular for groups working with rare andendangered animals.Reference(1) Shaw et a11999, Chapter 17. In: Handbook ofin vitro fertilization. CRC press. EditorsTrounson A.O. & Gardner D. 2nd editionOVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALEDEVELOPMENT OF OVARIAN TISSUE FROMMACROPUS EUGENIIPOUCH YOUNG AFTER GRAFTING TO SCID MICED. Mattiske l , G. Shawl and J. Sha~Department ofZoologyl, University ofMelbourne, 3010 and Institute ofReproduction & Development 2 , Monash University, Clayton, 3168.INTRODUCTIONOvarian grafting is a valuable tool which can be manipulated to investigate fund~ental bio!ogical q~estions suchas the control of follicular recruitment and the maturation of oocytes at ovulatIon. Ovanan grafting to a hostspecies is also a novel approach used in many eutherian species to preserve female. genn line~, ~ there is.3:Pincreasing need to maintain genetic diversity to preserve rare and endangered speCIes. In prehmmary s~dIes,ovarian tissue from taromar pouch young grafted to scm mice (mice with severe combined immune defiCIency)survived grafting and contained primordial and developing follicles (1). The ?urpose ofthis s.tudy was to determinewhether these follicles were capable of developing through to maturation and formation of embryos afterfertilisation.MATERIALS AND METHODSOvaries were collected from 2 to 10 week old tammar pouch young and randomly divided into two groups. Group·1 were grafted to the kidney capsule of adult, ovariectomised scm mice. GrouP. 2 was a control with tissue fixedat the time of collection. Graft recipients were killed 2-20 weeks after grafting and the grafts processed forhistology. The development ofthe ovarian tissue was compared to ovaries from normal pouch young ofthe sameage. Ovarian tissue that had been grafted for longer than ~6 weeks was che~ked for ~tral fol1i~les, wh~ch werecarefully punctured with a 30 G needle. Any oocytes found m the grafts were Incubated In maturatIon medIa (9.875ml EMEM 0.100 ml FCS, 0.030 g BSA and 0.025 ml FSH) at 37°C for 24-72 hrs or until polar bodies wer~observed. Spenn was collected from mature taromar males by electroejacula~ion.as described in (2). Oocytes.thathad extruded polar bodies were directly injected through the zona pellucIda mto the egg cytoplasm by mtracytoplasmic sperm injection (ICSI) and cultured at 37°C as in (3). Circulating levels of follicle stimulatinghormone (FSH) were measured in female recipient mice by RIA (4).RESULTS AND DISCUSSIONOvarian tissue from 20 day old pouch young (n=7) survived the grafting process and contained healthy genn cells.However, no follicles developed up to 120 days after grafting to scm mice. Ti~sue from pouch youn~ great~ ~an50 days old (n=7) survived the grafting process and developed to a stage wher.e It appeared morpholog.Ica1l?, snnI~arto nonnal adult ovaries. Follicles of all developmental stages were present m the transplanted ovanes, Inclu,(imgcorpora lutea. There was a significant difference between the circ~lating ~evels ofFSH ~ intact and oVariectomi~edcontrol animals (t = 12.7, p=0.001), and between grafted and ovanectormsed control anImals (t =-6.46, p =0.003),indicating that the wallaby tissue had a negative feedback effect on the mouse pituitary. O~ 25 oocytes collectedfrom grafts after 16 weeks and cultured, 14 were injected with spenn. Two cleaved, producmg a 4-cell and an 8­cell embryo.CONCLUSIONThese results demonstrate that ovarian tissue from marsupials retains developmental potential and is capable ofdevelopment into mature oocytes when grafted into a eutherian recipient. Furthermore, ICSI was successful and thefertilised oocytes developed into cleavage stage embryos.REFERENCE(1) Shaw, J., Mattiske, D., Connell, B. and Shaw, G. (1997) Pro~. ASRB 28:75. .(2) Taggart, D., Leigh, C., Steele, V., Breed, W., Temple-SmIth, P. and Phelan, J. (1996) Reprod. Fertll. Dev.8:673-680.(3) Renfree, M. and Lewis, A. (1996) Reprod. Fertil. Dev. 8:725-742.(4) Wreford, N., O'Connor, A. and de Kretser, D. (1994) BioI. Reprod. 50(5): 1066-7.We thank Dr Orly Lacham-Kaplan for perfonning the ICSI, Dr David Taggart for the tammar sperm, and Anne0'Connor and Sue Hayward for running the FSH assays.8081

OVARIAN DEVE~OPMENT/ASSISTED REPRODUCTION-FEMALEOVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALEDEVELOPMENT OF ANTRAL STAGE FOLLICLES IN HUMAN· CRYOPRESERVEDOVARIAN TISSUE FOLLOWING XENOGRAFTING IN SCID MICE.1 Debra Gook, IBrian McCully, lAngela Nelson, 2 Clyde Riley and 1,3 John McBain.lReproductive Biology Unit & 2Anatomical Pathology, Royal Womens Hospital and 3Melboume IVF,Melbourne, Victoria.Introduction: Despite the recent increase in ovarian tissuebanking, there has been little progress in establishingwhether follicles within this tissue are viable and capableof .function following cryopreservation. Although, ourprevious studyI has indicated that the propanediolcryopreservation method preserves follicle morphology, itgives no indication of functional preservation. Follicleculture has indicated some viability followingcryopreservation, but function and development arelimited and difficult to assess. The growth of a folliclefollowing autologous transplantation of cryopreservedtissue 2 indicates that this may have more potential. Theaim. of this study is to assess functional preservation offollicles within cryopreserved ovarian tissue xenograftedinto immunodeficient mice.Methods: Ovarian tissue was donated from 2 patients atrisk of loss of fertility: a 29 year old with breast cancer(control fresh tissue), and an 18 year old with acutemyeloid leukemia (frozen tissue). Thin slices of ovariancortex (4x3xlmm) were dehydrated in 15M propanedioland O.IM sucrose and cryopreserved using the slow freezeprotocol previously reported l . On thawing, the tissue wascut into smaller pieces (lx1xO.5mm) and a single pieceplaced under the renal capsule in immunodeficient (SCID)mice. Tissue was grafted to both kidneys and the micebilaterally oophorectomized. Non-frozen tissue was alsografted. Sites were examined at >24 weeks postoperativelyand fixed for histology.Results: Grafts were clearly distinguishable from renaltissue after 24 weeks on 9/10 sites with non-frozen tissue(5 mice) and 616 with cryopreserved tissue (3 mice). Boththe cryopreserved and non-frozen tissue contained at leastone antral follicle (2-3 mm in diameter) and a few smallfollicles (0.6 -lmm diameter). Increased circulation to thelarger follicles had also developed. Histologicalexamination verified these as normal antral follicles with alarge antrum, enclosed by granulosa and theca cells andcontaining germinal vesicle stage oocytes. Smaller antralfollicles and follicles at earlier stages were also presentwithin the tissue.Conclusion: Follicles within tissue cryopreserved usingthe propanediol procedure are viabile and have retainedfunction as indicated by their growth followingxenografting.1Gook, D.A. et aI., (1999) Hum. Reprod. 14,2061-2068.20ktay, K., (2000) Mol Cell Endrocin (in press).Ftgltte 1. An antralfollicle (--.. 5mm.)whicl1~opedfollowing xenografting 'of cryopreserved ·,hmnan ovariantissue under the renal capsule ina'scm mo'Use.Figure 2. Granulosa cell pedicle containing a GVoocytewithin a antral follicle (-3mm) which had developedfollowing xenografting of ayopreserved· human· ovariantissue.Figure 3. A section showing the tenal! ovarian interfaceand an early antral follicle within cryopreserved ovariantissue following xenografting.IS6:bATION AND CULTURE OF HUMAN FOLLICLES FOLLOWING THAWING OFCRYOPRESERVED OVARIAN TISSUEJanell Archer and Debra GookReproductive Biology Unit, Royal Womens Hospital, Melbourne, AustraliaIntroduction: Cryopreservation of ovarian tissuemay maintain future fertility for patients at risk oflosing ovarian function. Histological examinationhas shown that high levels of follicle survival arepossible following cryopreservation (Gook et al1999). However, the developmental competenceof these primordial follicles, which account for themajority of follicles in the human ovary, and ~etriggers which initiate growth and maturatIonremain unknown. Autotransplantation at a laterdate is an option for re establishing folliculargrowth but for oncology patients the risk of retransmission of cancers is unacceptable. Thereforein vitro growth and maturation may be preferable.The aim of this study was to isolate and culturefollicles from cryopreserved human ovarian tissue.Methods: Ovarian tissue donated by patients was slicedinto pieces approximately 4x3xlmm which weredehydrated in 1.5 M PROH+O.l M sucrose for 90 minsand then frozen as previously described (Gook et al1999). Following rapid thawing the tissue was digestedenzymatically with 125 - 250 m/mI collagenase IV and 5ID/ml DNAse in serum free Hepes buffered Hams FlOmedium with gentle agitation for 30-60 mins at 37°C.When sufficient dissociation had occurred, larger tissuepieces were removed and the remaining suspensioncentrifuged gently (800rpm) for 2 mins. The pellet andtissue pieces were rinsed in Hepes buffered Hams FlO+10% FCS +1OOmIU/ml FSH and follicle isolation wascarried out in this medium. Individual follicles withvarying levels of attached stroma were retrieved, rinsedand cultured on matrigel or fibronectin coated inserts inHams F12+10%FCS+100mIU/ml FSH. Follicles werecultured for between 2 and 14 days and morphology andsize were observed via light microscopy. A proportionwere also stained for mitochondrial metabolism(MitoTracker) and DNA content with Hoechst 33258.Results: Collagen bundle disruption in the stroma is aconsequence ofour current freezing protocol and enableslower enzyme concentrations and exposures to be usedthan for fresh tissue. With collagenase at 125 IU/ml thetissue remained fibrous making follicle dissectiondifficult. At 250 IU/ml for 60 mins excessive damageoccurred resulting in low numbers of intact follicles.The optimum conditions for retrieving intact freefollicles and allowing relatively easy dissection fromtissue was collagenase (200 IU/ml) and DNAse (5 IU/ml)for 30 mins although some inter patient variability exists.On the day ofisolation average follicle size was 44Jlm(n=83) with the majority (>95%) offollicles having 1to 2 granulosa layers. A number of naked oocytes wereobserved in the enzyme digestion suggestingcryopreservation and/or enzyme induced damage. Asignificant proportion (58%) of follicles which were intactat time of isolation extruded their oocytes in culture (4h­14days). Preliminary staining results show some mitoticactivity after 14 days in culture and also evidence ofmitochondrial metabolism.Figure 1 Human primoidial folliclesFigure 2 Human primoidial follicle which is extruding theoocyteConclusions: It is possible to retrieve good numbers offollicles from cryopreserved tissue with a combinationof enzymatic and manual dissection. However thelarge proportion of follicles extruding ooeytesduring follicle isolation and culture suggests a loss ofthe integrity of follicular structure.Reference: Gook, D.A. et al (1999) Hum. Reprod. 14,2061-20688283

OVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALEOVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALEBiopsiedControlSUCCESSFUL CRYOPRESERVATION OF BIOPSIED EMBRYOS404033(82%) 21734(85%) 251Experiment 2# ofembryos # Survived # HatchedBiopsiedControl24252019CattJ.W.Sydney IVF, Sydney, NSW 2000Introduction:Most centres offering PGn produce more 'nonnal' embryos than can be safely transferred. There istherefore a need to cryopreserve the excess embryos. However, cryopreservation ofbiopsiedembryos is compromised ifthe freezing is conducted on the day ofbiopsy(I,2). This may be due tothe effects ofacid TYrode's solution on the remaining blastomeres, the biopsy medium, or thephysical trauma ofblastomere removal. We have used an infra-red laser to breach the zona ofsupernumerary or abnonnally fertilised embryos and frozen them after biopsy. We have alsocultured embryos for an additional 48 hours post biopsy and then cryopreserved them.Materials and methods:Embryos were biopsied on day3 after insemination. An infra-red laser was used to breach the zona.A twenty micron hole was made and the zona thinned on either side ofthis breach. A biopsy pipette(30 J-lm Cook) was used to aspirate two blastomeres. The biopsied embryos were returned to culturefor either 2- 4 hours (Experiment 1), or two days (Experiment 2). Experiment 1 was controlled forby comparing with 40 day 3 embryos thawed for clinical purposes. Experiment 2 was controlled forby comparing with 25 day 5 embryos thawed for clinical purposes.Embryos were cryopreserved in experiment 1 using propanediol or, using glycerol, in experiment 2.The same freeze programme was used regardless ofcryoprotectant. Thawing was conventionalrapid thaws using propanediol/sucrose for experiment 1 or sucrose alone for experiment 2. Afterthawing, the embryos were immediately assessed to determine blastomere survival and assessedagain after 18 hours, to determine development. Embryo survival was defined as 50% or more ofthe blastomeres surviving.Results:Experiment 1Embryos Survived Blastomeres Blastomeres survived17145 (67%)211 (84%)Although the same percentage ofembryos survived, there were fewer blastomeres that survivedafter biopsy (p = 0.001 X 2 ).all transferred - 3 pregnancies (10 ET)Discussion:Embryos are susceptible to cryopreservation damage after biopsy. This would seem, at least in part,to be a function ofthe biopsy medium rather than the biopsy procedure. No embryonic material waslost through the hole in the zona during cryopreservation.A large hole in the zona had no effect on cryopreservation and embryo survival.Culturing embryos for 48 hours after biopsy gave good embryonic survival with only a fewembryos and blastomeres not surviving. Our current clinical protocol allows biopsy on day 3 andcryopreservation on day 5.FOLLICLE POPULATIONS IN PREPUBERTAL, MATURE AND AGED RED DEER HINDS.Peter R Hurst, Mark W. Fisher* and Bernie J. McLeod*.Department ofAnatomy and Structural Biology, School ofMedical Sciences, U~versity ofOtago,Dunedin, New Zealand and * AgResearch, Invermay Agncultural Centre, Mosglel, New Zealand.Introduction. A previous longevity study in a group ofhinds has shown that the success ofweaning calveswas above 60% until aged 16 years and then declined rapidly to only one calfweaned at age 19 and 20 (1).Further data on ovaries has been obtained from (n = 7) prepubertal «1 yr), mature (7 yr) and old (20 yr)animals. A stereological analysis was undertaken on 2 ovaries at each age to determine the numbers ofsmall, preantral follicles.Methods. Ovaries were weighed and all follicles> 2mm were dissected, counted and measured. For thestereological study ovaries were embedded in TechnovitTM: resin and serially sectioned at 40J.lm thickness.Every 20th section was optically sectioned with 100x and lANA objective to estimate.the number of .primordial and primary follicles by.the dis~~r proced~e. A. follicle ~ sco!ed only ifthe nucleus ofItsconstituent oocyte was detected entirely Wlthin the 3-dimenslOnal counting disector frame.Results. The descriptive characteristics (means ±S.D.) ofthe ovaries at 1 yr, 7yrand 20 yr: are shown inthe Table:arac erlS cvary weI t gm.No. >2mm folliclesDiameter oflargestfollicle, mm 7.25 ± 1.9 8.12 ± 0.77 5.24 ± 1.29o. nmor p :us , ,primary follicles 133,846 6,917Prepubertal and 7 yr. hinds had similar numbers ofantral follicles ofequivalent sizes bu~ the 7 yr..grouphad less primordial/primary follicles. At 21 years the deer had few, ifany detec~ble fo.llicles.. Duringoptical sectioning ofthe 7 year ovaries it was observed that 6% and 8% ofthe pnmordial follicles hadmorphological features indicative ofatresia. Most notable w~ the presence .ofat l~ast 1 ~~entedgranulosa cell nucleus (Figure). No such features were seen m any ofthe pnmordial follicles m theprepubertal animals.Figure showing 2 images(A & B) ofthe samefollicle separated by 3J.lm.In B the fragmentednuclear material ofagranulosa cell is indicatedby the arrow. Thearrowheads point to theoocyte nucleus.Discussion. This study has shown, predictably, that during ageing the population ofovarian fo~clesdiminishes to the point where old hinds have virtually no follicles present The chance observation ofgranulosa nuclear fragmentation in the 7yr. group is indicative ofgranulosa atresia an~ s:ugge~ts that as ~eovary ages the primordial follicle population is subject to increasing rates ofdegeneration. This hypotheslSis the focus offuture studies in the ovaries ofmice and humans.Reference. .1. Fisher, MW, McLeod, BJ, Mockett, BG, Moore, GH & Drew, K.R. (1996) Reproductive senescence maged red deer hinds. Proc. N.Z. Soc. Anim. Production. 56,344-346.References(1) Magli et al1999 Human Reprod 14 770(2) loris et al1999 Human Reprod 14 283384 85

OVARIAN DEVELO~MENT/ASSISTED REPRODUCTION-FEMALEOVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALESTUDIES ON OVARIAN SURFACE EPITHELIAL CELL BIOLOGY IN THE MOUSEOlivia L Clow, Peter R Hurst and Jean S FlemingDepartment ofAnatomy & Structural Biology, School ofMedical Sciences, University ofOtago,PO Box 913, Dunedin 9001, New ZealandMost human ovarian cancers arise from the singlelayer of cells, the ovarian surface epithelium(OSE), which surrounds the ovary.Epidemiological studies suggest human epithelialovarian cancer risk reductions are associated withboth pregnancy and oral contraceptive use (1), butlittle is known about the basic biology ofthe OSEcells. Weare studying epithelial rupture and repairassociated with ovulation, in young and old mice,to characterise mouse OSE cell morphology and toidentify patterns of cell proliferation and geneexpression associated with ovulation woundhealing.Methods: Ovaries from 20 2-4 month old micewere collected using a no-touch technique, atvarious time points from around the time ofovulation to 72hours after ovulation. The bursalmembrane was removed, before fixation of theovary in Karnovsky's fixative. Scanning electronmicroscopy was used to characterise mouse OSEcells and observe ovulation wounds at the apex ofovulatory follicles.Results: Mouse OSE has two distinct cell types,similar to the human (Fig 1: ref 2). Rounded,cuboidal epithelial cells lie in the areas betweendeveloping follicles, whereas the apex ofthe largerfollicles is covered with squamous cells, whichmay show flattened extensions, suggestingmigratory behaviour.Figure 1. Bar = 20 Jlrn.Cuboidal OSE cells lying between two follicularprotrusions. The more rounded cells appear tohave a lower density ofmicrovilli on their surface,compared with the flatter epithelial cells to theright ofthe picture.~,~I'Figure 2. Bar = 100 Jlm. Cuboidal cells appear toform invaginations into the ovarian stroma.I IFigure 3. Bar = 50 Jlm.Round structures present on the apex of largefollicles in newly mated mice, may be ovulationsites. Structure diameters were 150-200 Jlm at 2-4h, 60-100 /lm at 5-8 h and 30-40 J.l.m at 35 h postovulation.Establishing a mouse model ofovulation woundhealing will allow us to study the normalphysiology of gene products shown by geneticlinkage analysis to be associated with epithelialovarian cancer. An understanding ofthe basic cellbiology ofthe OSE will lead to new ways to detectgrowth abnonnalities in the OSE and thus newmethods for early diagnosis.1. Whittemore AS. Cancer 71: 558-65, 1993.2. Gillett WR, et al. Human Reprod 6: 645-650, 1991.SPERM BINDING AND PENETRATION OF THE ZONA PELLUCIDA IN VITRO BUT NOTSPERM-EGG FUSION IN AN AUSTRALIAN MARSUPIAL, THE BRUSHTAIL POSSUM(TRICHOSUR.US VULPECULA)K. E. Mate\ K. S. Sidhu l , F.C. Molinia 2 , A. M. Glazier & lC. Rodger lCooperative Research Centre for Conservation and Management ofMarsupials1 Department ofBiological Sciences, Macquarie University, Australia 2109 & 2 Landcare Research,PO Box 69, Lincoln 8152, New ZealandIntroductionCapacitation of sperm from any Australianmarsupial has not been achieved in vitro andattempts at in vitro fertilization (IVF) havepreviously been characterised by a complete lack ofsperm-zona pellucida (ZP) binding. Recently, cocultureof brushtail possum sperm with oviductepithelial cell (OEC) monolayers or with OECexplant-conditioned media has been shown toprolong motility, as well as induce capacitationassociatedchanges such as transformation to T­shape orientation (1). In this study we haveexamined the ability of these in vitro produced T­shaped sperm to bind to and penetrate the ZP ofsuperovulated possum eggs.Materials and MethodsFemale possums were superovulated with 15i.u.pregnant mare serum gonadotrophin (PMSG)followed 78-81 h later by 4 mg porcine luteinisinghormone (pLH). Eggs were collected from largefollicles (>2mm) 22-34 h after pLH. Oviducteptheial cell (OEC) monolayers and explants wereprepared from PMSGILH primed females asdescribed previously (1). Epididymal sperm werepreincubated for 2h with OEC monolayers orexplant-conditioned media prior to addition of eggs.In vivo derived T-shaped sperm flushed from theoviduct of artificially inseminated possums servedas a positive control. Eggs were examined after 4hincubation with sperm.Results & DiscussionThe ZP of approximately 25 % of folliclular eggswas penetrated by sperm that had been flushed fromthe oviducts of superovulated possums 4.5 to 6 hafter uterine insemination (Table 1). Spermpenetration was obvious as a large hole was createdin the ZP by the penetrating sperm resulting inextrusion of cytoplasm. Sperm capacitated in vitroby incubation with conditioned media from OECexplants or co-culture with GEC monolayers boundto and penetrated the ZP of possum eggs to thesame extent as in vivo capacitated sperm (Table 1).The timing of the superovulation hormonetreatments for the egg' donors had profound effectson the receptivity and/or penetrability of the ZP.When the interval between PMSG and pLH wasless than 76 h, sperm failed to bind or penetrate theZP. Despite this, eggs collected from donors treatedwith pLH 78-81 h after PMSG were penetrated byspermatozoa regardless of their nuclear maturationstatus (data not shown).This study provides preliminary evidence for thenecessity of sperm-oviduct epithelial cellinteractions for capacitation in Australianmarsupials and lends further support to thesuggestion that the T-shape head orientation isindicative of sperm capacitation. Despite theoccurrence of sperm-ZP binding and penetration,sperm-egg membrane fusion and egg activationwere not observed. The factor(s) responsible for thelack of sperm-egg membrane fusion have not yetbeen identified.Table 1: Zona pellucida penetration of eggs in vitroby sperm capacitated in vivo or in vitro in oviductepithelial cell (OEC) conditioned media or on OECmonolayersSperm Treatment No ofReplicates*In vivo 14 (103)OEC condo media 11 (62)OEC monolayers 9 (54)* Total number ofeggs in brackets%ZPPenetrated25.4±7.324.2±10.749.1±12.0References(1) Sidhu et al (1999) BioI. Reprod. 61: 1356-1361.86 87

OVARIAN DEVELqPMENT/ASSISTED REPRODUCTION-FEMALEOVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALEINTRACYTOPLASMIC SPERM INJECTION OF TAMMAR WALLABY OaCYTESGenevieve M. Magarey and Karen E. MateMarsupial CRC, Dept. ofBiological Sciences, Macquarie University, NSW 2109 Australia.IntroductionThe development of methods to attain fertilizationin vitro in Australian marsupials will be importantfor assisted breeding strategies to conserveendangered species and will also contribute to theunderstanding of marsupial gamete biology. In vitrofertilization (IVF) has not been achieved in anyAustralian marsupial so far using conventionaltechniques possibly due to unsuccessful spermcapacitation in vitro (1). Intracytoplasmic sperminjection is an IVF technique used successfully inhumans especially for cases of male infertility andhas also been successful in a number of domesticeutherian species (2). In this study the response oftammar wallaby oocytes to ICSI was assessed.Materials and MethodsSuperovulation of female tammar wallabies wasachieved using 8 x 12 hourly intramuscularinjections of 6mg porcine follicle stimulatinghormone (FSH) (FoIItropin V: Vetrapharm)followed 12 h later by a single intramuscularinjection of 4mg porcine luteinizing hormone (LH)(Lutropin V: Vetrapharm). Approximately 26-30 hafter LH, mature oocytes were collected fromfollicles greater than 2mm diameter by flushing withheparinised Dulbecco's Modification of EaglesMedium (DMEM: Trace Biosciences) containing25mM HEPES. Granulosa cells were stripped fromthe oocytes. Sperm were collected from malewallabies by electroejaculation.Oocytes were assigned to one of three treatmentgroups; Control, Sham and ICSL Control oocyteswere not subjected to injection, but otherwise werecultured· and assessed in an identical manner to theother groups. Injection was performed using aninverted microscope with modulation contrastoptics, heated stage, and left and rightmicromanipulators to operate holding and injectionpipettes respectively. Sham injected oocytes werepierced with the injection pipette but no sperm wasinjected. ICSI oocytes were injected with a singlespermatozoa using conventional ICSI techniques(3).Following treatment, all oocytes were incubated at36°C in 5% C02 in air for 17-19 h in DMEM.Assessment was performed using modulationcontrast and fluorescence microscopy after stainingwith Hoechst 33342 (Sigma Chemical Co. USA) orwith light microscopy after staining with 1%lacmoid (Sigma-Aldrich) in 45% acetic acid.Results and DiscussionNormal fertilization, as assessed by presence of 2pronuclei (2PN) (and disappearance of sperm headand tail), was achieved in 39% (39/100) of thesperm injected eggs (Figure 1). Significantly lesseggs formed 2PN in the sham or control groups(p=3PNSHAM~lPNt:JunknownB2PNIC SIfE;I degenerateFigure 1 Response of tammar wallaby oocytes to controltreatment, sham injection, or ICSI (sperm injection).Some eggs in both control and sham groups wereactivated, forming either 1 PN (15% (16/7 1) ofsham eggs and 23% (11/75) of control eggs) or 2PN(13% (9/71) of sham eggs and 7% (5/75) of controleggs). Significantly more of the sham injected eggswere activated compared to the controls (p

OVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALEOVARIAN DEVELOPMENT/ASSISTED REPRODUCTION-FEMALEEFFECTS OF DONOR CELL CYCLE PHASE ON IN VITRO DEVELOPMENT OF BOVINE NUCLEARTRANSFER EMBRYOS PRODUCED BY PIEZO INJECTIONNatasha Korfiatis, Alan Trounson & Orly Lacham-Kaplan.Centre for Early Human Development, Monash Institute ofReproduction & Development, Clayton, VIC, 3168.IntroductionDifferentiated somatic cells can be used to produceembryos and viable offspring following nuclear transfer(NT) (1,2). However, reconstituted embryos have asubstantially reduced developmental competence, asreflected by low live birth rates. The cell cycle phase ofthe donor nucleus is one of many factors which mayaffect the success of NT, and the ability of adifferentiated nucleus to be reprogrammed. Studiessuggest that Gl and quiescent donor nuclei (GO phase)are most compatible with reprogramming and embryonicdevelopment, therefore serum starvation of donor cellshas become a frequently used technique.The aim of the present study is to examine in vitrodevelopment ofbovine NT embryos in relation to the cellcycleMaterials & MethodCell CultureThe following treatment regimes were used tosynchronise/arrest bovine fetal fibroblasts. 1) Serumstarvation (S.S) using 0.5% FCS for 6 days to inducequiescence (GO), 2) conditioned medium (C.MED) for 7days, also to induce quiescence, 3) 38JlM Nocodazole(NOe) (Sigma) for 10 hours to induce metaphase arrest,and 4) actively dividing (A.DIV) cells (control). Todetermine cell cycle characteristics, cells were stained forDNA content with Propidium iodide (PI) andproliferation-associated nuclear antigen (Ki-67) whichstains positive in late G1, S, G2 and M phase nuclei.Using FACS analysis the percentage of cell nuclei inGO+G1 as well as GO+early G1 was determined.NT experimentsIn vitro matured oocytes were enucleated and injectedwith cells using the Piezo-Drill (Burleigh). Activation ofNT oocytes with calcium ionophore/DMAP wasperformed 3 hours after injection, except for the NOCgroup, where oocytes were activated first and injected 3hours later. Reconstituted oocytes were cultured in amodular incubator chamber at 39°C, 5% CO 2 , 7% O 2 inair.ResultsTreatment of bovine fibroblasts by serum starvation andconditioned medium significantly (p < 0.001) increasedthe proportion ofcell nuclei in GO/early G1 (88% & 85%respectively) compared to actively dividing cells (34%).Actively dividing cells showed a higher proportion ofcells in late G1 compared to the 8.S and C.MED treatedcells. Treatment of cell cultures with Nocodazolereduced the proportion ofcells in GO/G1 (37%).Following NT, higher proportions of embryos developedto blastocysts when donor cells were not treated (A.DIY)or were serum starved (table 1). However, a significantdifference was identified between the A.DIY group andthe group of oocytes injected with cells treated with· C.MED (p

UTERUSREGULATION OF VEGF RECEPTORS BY ESTROGEN AND PROGESTERONE INMICROVASCULAR ENDOTHELIAL CELLS FROM HUMAN MYOMETRIUM ANDFIBROIDSMarina Zaitseva, Peter Rogers and Caroline GargettMonash University Department ofObstetrics and Gynaecology, Monash Medical Centre, Clayton, Vic.,Australia, 3168IntroductionVascular endothelial growth factor (VEGF) is a majorregulator of normal and pathological angiogenesis. VEGFacts through two receptors, VEGFR-l/flt-l and VEGFR­2/KDR, which are expressed almost exclusively onvascular endothelium. Estrogen (E) and progesterone (P)have been shown to modulate endothelial cell proliferation(1,2). E and P also play an important role inpathophysiology of fibroids (benign tumours ofmyometrial SMC) (3).HypothesisE and/or P modulate the expression ofVEGF receptors onmicrovascular endothelial cell isolated from humanmyometrium and fibroids.AimTo study the effects of E and P on VEGF receptorexpression on MEC isolated from normal myometrium andMEC isolated from fibroidsMethodsPrimary MEC were isolated from normal myometrium andfibroids obtained from hysterectomy samples and culturedas previously described (4). MEC (up to passage 3) weregrown to confluence, made quiescent and stimulated withE (10 and 100 nM) and/or P (100 nM) for 18 hours inserum-free medium in the presence of 0.5% BSA and 10ng/ml VEGF 165 • Cells were harvested, washed, resuspendedin buffer and incubated with 860 ng/ml rhVEGF-biotin (R&Dsystems, USA) for 60 min at 4° C, followed by avidin-FITC(R&D Systems) for 30 min at 4° C. Double staining with anti­CD31 (mouse anti-human, Dako, UK), followed by PEconjugatedsheep anti-mouse IgG F(ab'h (Selenus, Australia)was performed on some samples to identify MEC in SMCcontaminated cultures. VEGF binding was analysed by flowcytometric analysis using MoFIo flow cytometer. Meanfluorescent intensity (MFI) was obtained for each sample. Forstatistical analysis data were log transformed and analysedusing parametric tests.ResultsVEGF receptor expression was determined by measuringthe level ofVEGF binding to MEC. Figure 1 shows that E(10 and 100nM) caused significant upregulation of VEGFbinding to myometrial MEC (P4020o*Figure 1. Effect of E and P on VEGF binding tomyometrial MEC. Estrogen at 10 & 100 nM but notprogesterone increased VEGF binding (MFI) to myometrial MEC(*P < 0.05 compared to control by Dunnet's test).-u::200~ 150OJc:c 100 c:su. 50C)w> 0~~o\ ~~~ ~~~ ~~~ R\~~ R\~~CP~ ~A\ ~\(;i "!l\(;i \~'t ~~'t~ ~A\Myometrial*FibroidFigure 2. Basal VEGF-binding of quiescent cells MEC.Fibroid MEC showed significantly greater basal levels ofVEGF binding (MFI) compared to myometrial MEC. (*P< 0.05 by unpaired student's t-test)ConclusionsThe present study shows that E upregulated VEGF receptorson myometrial MEC but had a variable effect in fibroid MEC.P had no effect on VEGFR expression in both myometrialand fibroid MEC. Fibroid MEC expressed higher basal levelsof VEGF receptors compared to normal myometrial MEC.Upregulation of VEGF receptors by E may be importantduring pregnancy and fibroid growth.References.1. Kirn-Shulze S., McGowan K., Hubchak S., Cid M., Martin M.,Kleinman H., Greene G., Schnaper W. (1996). Circulation, 94:1402-14072. Vazquez F., Rodrigues-Manzaneque J., Lydon 1., Edwards D.,O'Malley B., and lruela-Arispe M. (1999). J. BioI. Chern., 274:2185-21923. Andersen 1. (1998). From: VoUenhoven B. Baillieres Clin. Obstet.& Gynaecol., 12: 225-2434. Gargett C., Bucak K., and Rogers P. (2000). Human Reprod. (inpress).Hypotheses:• IGF-I and IGF-IT stimulate proliferation offibroid SMC in vitro• E2 enhances the proliferative effects ofIGF-I and IGF-TI on fibroid SMC in vitroProliferation assay readings after 96h in culture (figures shown are optical density units - [ODU])IGF-I (50ng/ml) IGF-IT (lOOng/ml) IGF-I+E2 IGF-IT+E2T C T C T C T Cfibroid 0.25* 0.2 0.24* 0.19 0.33* 0.19 0.32* 0.21myometrium 0.23 0.25 0.27 0.25 0.31 0.27 0.30 0.27ODU is proportional to cell number, T=treated, C=control. *significant compared with controls (p~O.05)UTERUSEFFECTS OF INSULIN LIKE GROWTH FACTORS AND ESTROGEN ON UTERINELEIOMYOMA AND MYOMETRIAL SMOOTH MUSCLE CELL PROLIFERATIONUrsula Manuelpillai, P.A.W. Rogers and Beverley VollenhovenDepartment ofObstetrics & Gynaecology;> Monash University, Clayton, Victoria 3168.Introduction:Uterine leiomyomas (fibroids) are benign tumours ofthe smooth muscle cells (SMC) ofthe uterusthat cause significant gynaecological problems and occur in up to 50% of women during theirreproductive years. The insulin like growth factors I, IT (IGF-I, IT) may play crucial roles instimulating fibroid SMC proliferation. Estrogen enhances IGF-I synthesis in the rat myometriumand may also stimulate IGF driven SMC proliferation. Therefore the effects of IGF-I or IGF-ITalone and in combination with estradiol (£2) on fibroid and myometrial SMC proliferation wereinvestigated.Materials and methods:Fibroid and adjacent normal myometrial tissue were obtained from pre-menopausal womenundergoing hysterectomy (n=5). Primary SMC cultures were treated with IGF-I, IGF-ll (10, 50,100nglml), IGF's and E2 (50nglml growth factor + 10nglrnl E2). Cells were grown for 24, 48, 96,144h and SMC cell proliferation was measured by a routine assay. Differences between treated andcontrol cultures were assessed by the Student's t test (p~0.05 considered significant).Results: i) IGF-I and IGF-ll significantly enhanced proliferation offibroid SMC. ii) proliferationoffibroid SMC treated with IGF-I+E2 and IGF-TI+E2 were also significantly elevated. E2 exertedsynergistic effects on IGF-I (48, 96h) and IGF-ll (96, 144h). iii) myometrial SMC did not respondsignificantly to any ofthe treatments except IGF-ll and IGF-ll+E2 at 144h.Discussion and conclusions:IGF-I and IGF-ll selectively enhanced proliferation offibroid SMC compared with myometrialSMC. In fibroid SMC, E2 appeared to exert synergistic effects on IGF-I earlier in culturecompared with IGF-ll. IGF-I alone or in combination with E2 did not have a significant effect onmyometrial SMC whereas IGF-ll alone or in combination was effective only after long periods inculture. Therefore fibroid and myometrial SMC appear to respond differently to the IGF's and E2.92 93

UTERUSENDOTHELIAL ACTIVATION PRECEDES TROPHOBLAST INVASION OF THEMESOMETRIAL ARTERIES IN PREGNANT GUINEA PIGSUTERUSISOLATION, PURIFICATION AND CULTIVATION OF NORMAL HUMAN ENDOMETRIUMCELLS. IMMUNHISTOCHEMICAL EXPRESSION OF DIFFERENT CYTOKERATINS ANDVIMENTIN IN FRESHLY ISOLATED GLANDULAR EPITHELIAL CELLSIoannis Mylonas*, Lubina Winkler, JosefMakovitzky, Dagmar-U. Richter, Udo Jeschke, Volker Briese,Klaus FrieseWomen's Hospital, Medical Faculty, University Rostock, Doberanerstr. 142, 18057 Rostock, Germany*Current address: Brown University, Providence, Rhode Island, USAIntroduction: The aims ofthis study were to (a) develop a simple, efficient and high reproducible methodfor the isolation, purification and cultivation ofhuman endometrial cells, and (b) characterize the differentcytokeratins (CK) and vimentin expression and (c) assess the purity of isolated glandular epithelial cells byimmunhistochemical methods.Materials and Methods: Normal human endometrium was obtained from 15 premenopausal, nonpregnantand nonnal menstruating patients. Endometrium samples were classified as proliferative (n=6) orsecretory (n=9) endometria. After a first collagenase digestion, stromal and epithelial cells were separatedby filtration. Glandular epithelial cells were further purified with two collagenase digestion steps,filtration, differential sedimentations at unity gravity and a Ficoll Gradient centrifugation. Glands werefurther suspended in small cell clumps with trypsin-EDTA. Stromal cells were purified with the use oferylyse-buffer, filtration and differential sedimentations at unity gravity. Morphological characterisationwas performed using immunhistochemistry for vimentin and CK 7, 8, 18 and 19.Results: Viability of glandular cells was 63-85% (n mean =76%) and of stromal cells >85%. The efficiencyof establishing a culture was 100% with a mean survival period of glandular cells of 4-6 weeks(proliferative phase) and 3-4 weeks (secretory phase). Subculturing attempts of glands were 33%successful. Stromal cells survived longer then 6 weeks and could be subcultivated more then 3 times.Glandular cells were polyhedral and grew as islands in a whorl-like wavy pattern around glandularfragments (Fig 1) confirming previous results [1-6]. Stromal cells were elongated and fibroblastic-like (Fig2). A small contamination «5%) ofstromal cells in glandular cell cultures was noted by microscopical andimmunhistochemical observations. The CK 7, 8, 18 and 19 produced clear labeling of cytoskeleton ofepithelial cells (Fig 3) from both proliferative and secretory phase, while stromal cells showed no CKexpression. Stromal and epithelial cells from both menstrual phases stained positively for vimentin (Fig 4).Conclusion: A simple and highly reproducible method which gave high cell viability, high cultureefficiency and minor different cell contamination was developed. The method allowed longer cultureperiods without addition of growth factors or use of extracellular matrix than previously reported [1-6].Since vimentin was, in contrast to previous reports [3,5,6], also expressed in glands at the time ofplating,an estimation ofcell culture purity should not be made using vimentin expression alone, but in combinationwith CK expression and morphological evaluation ofcultured cells. This method allows the propagation ofseparate endometrium cell types in vitro, which can be used to study endometrial function and implantationmechanisms.REFERENCES:1. Kirk D. King RJB, Heyes J, Peachey L, Hirsch PJ. Taylor RWT (1978) In Vitro;14: 651-6622. Satyaswaroop PG. Bressler RS, de la Pena MM. Gurpide E (1979) J ClinEndocrinolM~;48:639-6413. Osteen KG, Hill GA, Hargrove IT. Gorstein F (1989) Fertil Steril; 52(6): 965-9724. Matthews 0, Redfern CPF. Hirst BH, Tomas EJ (1992) Fertil Steril; 57: 990-9975. Vigano p. Di Blasio AM, De1I'Antonio G, Vignali M (1993) Acta Obstet GynecolScand; 72: 87-926. Ryan!P. Schriock ED and Taylor RN (1994) J Clin Endocrinol Metab; 78: 642-649ACKNOWLEDGEMENTWe like to thank the medical staff and nursesat the Women's Health Hospital of theUniversity Rostoek for supplying theendometrial biopsies. We also like to thankMrs. B. Fichtner or the excellent technicalsupport and Mr. S. Tomczak, MD andProfessor E. Koepke. MD -Director 0 theWomen's Health Clinic of KlinikumSuedstadt - RostoekFig 1-4: After plating the cells in culture dishes. glandularepithelial cells were polyhedral and grew as isllll1(l~ in awhorl-like wavy pattern around glandular fragment'.. (Fig2, x200). Stromal cells were elongated and fibroblasticlike(Fig 3). CK 8.18 and19 were strongly expressed inisolated glandular epithelial cells (Fig 4. x250) with astaining reaction more intense in the perinuclear region.Epithelial cells that form a glandular structure are stainedpositive for vimentin. The pattern of cellular stainingdiffers from that of cytokeratin antibodies. beinglocalized in the periphery ofcells (Fig 6. x250).Roberts CT 1 , Earl RA I, Erwich JJHM 1 , Robinson JS I and Khong TY 1 ,2IDept. ofObstetrics and Gynaecology University ofAdelaide Adelaide 5005, 2Dept. ofHistopathology Women's andChildren's Hospital North Adelaide 5006Backgro~nd The u~eroplacentalarteries d.uring pregnancy in guinea pigs are invaded by trophoblast and becomelarge calIbre complIant vessels as occurs m women(I). However, the precise cellular and molecular mechanismsinvolved are unknown. Impaired transformation of the utero-placental vessels in women has been associated withmi~car?age(2), preeclampsia and ~ntra~te~negrowth :estriction(3). Recer~tly, it has been suggested that endothelialactIVatIon precedes trophoblast mIgratIOn Into the deCIdual arterioles in women(4).Aim We .investigated the. chan~es ~hich take place in the mesometrial arteries in pregnant guinea pigs to test thehypotheSIS that endothelIal actIvatIOn precedes trophoblast invasion and to determine the extent of trophoblastinvasion ofthe mesometrial arteries.Me.thods .Seven guinea pigs,. were fed ad lib~tum,omated and sacrificed at day 60 post coitum (tenn = 67 days).GUInea pIgS were trans-cardlally perfused WIth 4 ro paraformaldehyde following an intraperitoneal overdose of~odium.pentobar~itone.Mesometri.al vessels were divided into four segments from the uterus to the arcade artery,ImmerSIOn fixed m the same fixatIve, and embedded such that the placental end of each was cross sectioned. In~nimals which sustained pregnancy in only one uterine horn, the non-pregnant horn was also processed and treated~n the sa~e way (~=3) as were both utenne h.oms of one animal which did not become pregnant. Sections wereImmunohlstochemIcally labelled for cytokeratm to label fetal trophoblast cells and examined by two observers todetermine the extent of trophoblast invasion distal to the placenta and the grade of vessel change on a scale of 0-6from no ?hange to completely ch~nged and line~ by endovascular trophoblast, respectively. The morphology oftheendothelIum formed a part of thIS vessel gradmg. Mesometrial vessels from each placental site were examinedseparately.Results Using the principle that the first changes that occur in the vessel will be seen distal to and the most recentproximal to, the placenta, the first sign of vessel change was endothelial 'ruffling', ie. the endothelium wascuboidal and protruded into the vessel lumen, and this occurred distal to trophoblast invasion. Interstitialtrophoblast around.t~e ve~sel wall was seen prior to the arrival of endovascular trophoblast. Finally, endovasculartrophoblast was VISIble In the vessel lumen and replaced the endothelium by which time the arteries werecompletely remodelled, lacking smooth muscle and were of large calibre. Trophoblast invaded to a mean ofsegment ~.6, about 2/3 of the way alo~g the vessels to the arcade artery. In the non-pregnant horn of animalspregnant m only one hom the endothelIum of the uteroplacental vessels was also 'ruffled' while that in the nonpregnantanimal was squamous.Discussion 'Ruffling' of the endothelium is a morphological indicator of endothelial activation.Immunohistochemical la?ell~ng for V-CAM expression (the molecular indicator of endothelial activation) iscurrently unde:way. l?urmg mflamm~tory response,s, endo~helial activation is induced by cytokines secreted byleukocytes dunng whIch the endothehum becomes ruffled and upregulates a variety of cell adhesion moleculesincluding VCAM-1 (5). This facilitates the further recruitment and adhesion of leukocytes to the endothelium. Wesuggest that a similar process ,occurs in the uteroplacental arteries during pregnancy which allows endovasculartrophoblast migration and adhesion to the endothelium. Gene ablation studies in mice have found that when uterinenatural killer cells (uNK.) are present in large numbers the decidual arterioles are tortuous anddilated(6) while theabsence of uNK. cells leads to atherosclerosis of these vessels and fetal loss after day 10 of pregnancy(7). Thesedata suggest that uNK. cells may be involved in arterial remodelling. Our future studies will aim to identify thecytokines which may be involved in this process.References(l)Hees H, Moll W, Wrobel K-H & Hees I (1987) Placenta 8, 609-626.(2)K.hong TY, Liddell HS & Robertson WE (1987) Br. J. Obstet. Gynaecol. 94,649-655.(3)K.hong TY, de WolfF, Robertson WE & Brosens 1(1986) Br. J. Obstet. Gynaecol. 93, 1049-1059.(4)Craven C, Morgan T & Ward K (1998) Placenta 19,241-252.(5)Abbas AK, Lichtman AH & Pober JS (1997) Cellular and Molecular Immunology 3 rd ed. WE SaundersPhiladelphia.'(6)Croy BA,Ashkar AA, Foster RA, DiSAnto JP, Magram J, Carson D, Gendler SJ, Grusby MJ, Wagner N,Muller W & Guimond M-J (1997) J. Reprod.Immunol. 35, 111-133.(7)GuimondM-J, Luross JA, Wang B, Terhorst C, Danial and Croy BA (1997) BioI. Reprod. 56, 169-179.94 95

UTERUSPERNASCULAR SMOOTH MUSCLE a.-ACTIN IS REDUCED IN THEENDOMETRIUM OF WOMEN WITH PROGESTIN-ONLY CONTRACEPTIVEBREAKTHROUGH BLEEDINGPeter A. W. Rogers, Debbie Plunkett and Biran Affandi*Monash University Department ofObstetrics and Gynaecology, Monash Medical Centre, 246Clayton Road, Clayton, Victoria, 3168, Australia, and *Department ofObstetrics an?Gynaecology, Faculty ofMedicine, University ofIndonesia, Jakarta, 10430, IndoneSIa.It has been shown that the endometrium ofwomen using progestin-only contraceptives hasincreased vascular fragility, although the structural basis for this weakness is unknown, as is itsrole in breakthrough bleeding. Perivascular cells such as pericytes and vascular smooth musclecells surround capillaries during the maturation process following angiogenesis, and act tostrengthen and stabilise the vessels. The aim ofthe present study was to quantify endometrialperivascular alpha smooth muscle actin expression in women with and without breakthroughbleeding problems using the subdermallevonorgestrel releasing contraceptive implantNorplant®, and compare it to normal menstrual cycle controls.Endometrial biopsies were collected from 15 normal cycle controls, and 37 women usingNorplant®, 20 ofwhom had significant breakthrough bleeding (BTB) problems prior to biopsyand 17 ofwhom had minimal BTB. Standard immunohistochemical doublestaining techniquesusing antibodies to CD34 and alpha smooth muscle actin to identify endothelial cells andsmooth muscle cells respectively allowed identification ofall endometrial microvessels.Microvessels were classified as level 0, 1 or 2 depending on whether perivascular alpha smoothmuscle actin was absent, discontinuous or continuous. For each control biopsy 40 vessels werescored in each of3 different zones: the sub-epithelial plexus, the mid-functionalis and thebasalis. In atrophic endometrium from Norplant® users, only 1 zone was distinguishable.In 15 controls the sub-epithelial plexus had significantly more level 0 v.essels than either thefunctionalis or basalis (61±4 versus 31±6 and 37±4%, P = 0.0006 and P = 0.0007 respectively).In contrast the functionalis and basalis had significantly more level 2 vessels than the subepithelialplexus (20±3 and 23±2 compared to 4±1 %, P = 0.0005 and P = 0.000 respectively).The major finding ofthe study was that in Norplant® users, where the relatively atrophicendometrium cannot be divided into different regions, women with breakthrough bleedingproblems (n=20) had significantly more level a vessels than those with reduced bleeding (n=17)(60±4 versus 46±4%, P = 0.0302). Norplant® users with breakthrough bleeding problems alsohad a non-significant reduction in level 2 vessels compared to women without bleedingproblems (4±2 versus 11±4%, P = 0.0667).These results demonstrate that perivascular alpha smooth muscle actin is reduced around theendometrial vessels ofNorplant® users with breakthrough bleeding compared to those with nobleeding problems, and strongly support the concept that reduced vascular structural integrityplays a key role in endometrial breakthrough bleeding.THE EFFECT OF INTERLEUKIN-6 DEFICIENCY ON IMPLANTATION, FETALDEVELOPMENT AND PARTURITION IN MICE.Sarah Robertson!, Aishling O'ConnelP and Alistair Ramsay2IDept ofObstetrics & Gynaecology and The Reproductive Medicine Unit, University ofAdelaide, SAand 2Dept ofImmunology and Microbiology, The University ofNewcastle, Newcastle NSW.INTRODUCTION: Interleukin-6 (1L-6) is a 26 kD rp.ultifunctionalcytokine that regulates various aspects oftheimmune response, acute-phase reaction and haematopoiesis and has functional redundancy with LIF and IL-lldue to shared use ofa common signal-transducing receptor (gpI30). lL-6 is synthesised in the uterus,particularly during the pre-implantation period ofpregnancy and at the time ofparturition (1,2). Myeloidleukocytes, endothelial cells and the conceptus express receptors for lL-6 but the physiological importance ofthiscytokine in female reproductive function is not known. Mice with a null mutation in the lL-6 gene (3) haveimpaired immune and inflammatory responses and a severely compromised acute phase reaction. The aim ofthese studies was to employ lL-6 null mice to investigate the role ofthis cytokine as a determinant ofsuccessfulpregnancy outcome.MATERIALS AND METHODS: Adult female cytokine deficient (IL6-1-) mice with a disruption in the firstcoding exon ofthe IL-6 gene were generated by conventional gene targeting techniques (3). Oestrous cycleswere examined in lL6-1- and cytokine replete (1L6+1+) mice (both C57Bl/6 background) by daily examination ofvaginal cell smears for two weeks. Mice were then mated with males ofthe same or opposite genotype andsacrificed at day 17 ofpregnancy. The number ofviable and resorbing implantation sites, and fetal and placentalweights in viable implantation sites were measured. The fetal:placental weight ratio was calculated as an index ofplacental function. In a second experiment, lL6-1- and lL6+1+ females were mated with males ofthe samegenotype and pregnancies were allowed to proceed to term, when the duration ofgestation was determined. Datawere compared by ANOVA and Bonferroni t-test.RESULTS: The length ofthe estrous cycle and the interval between placing with males and sighting ofa vaginalplug was not influenced by lL-6 genotype. There was a significant effect offetal and maternal IL-6 deficiency onparameters measured at day 17 ofpregnancy, when the mean number oftotal and viable implantation sites werereduced by 23% and 48% respectively, and resorption sites were increased by 140% (Table 1). Fetal andplacental weight, and the fetal:placental weight ratio in viable sites were comparable in lL6-1- and IL6+1+ mice.Fetal resorptions were also more frequent in lL6-1- females mated with IL6+/+ males. All parameters werenormal in IL6+1+ female mice mated with IL6-1- males. Parturition was delayed in lL6-1- mice (meaI!!SD lengthofgestation was 19.8.±0.5 and 20.62:0.6 in lL6+1+ and lL6-1- mice respectively, P

UTERUSUTERUSEXPRESSION OF INTERLEULIN 11 AND ITS RECEPTOR IN THE HUMANENDOMETRIUM THROUGHOUT THE MENSTRUAL CYCLE.Evdokia Dimitriadis, Lorraine Robb l , Lois A SalamonsenPrince Henry's Institute ofMedical Research, P.O. Box 5152, Clayton, 3168,Victoria, Australia'Walter and Eliza Hall Institute ofMedical Research, Parkville, 3050, Victoria, AustraliaIntroductionInterleukin 11 (lL-II) is a cytokine with pleitropicactivities on a range of cell types (1). IL-IIsignals via a homodimeric receptor complexcomposed of a specific binding chain, IL-llreceptor alpha (lL-IIRD) and the signallingcomponent gp 130. IL-ll plays a crucial role infemale fertility in the mouse (2). Female micewith a null mutation of the IL-l1RD chain wereinfertile due to a defective decidualizationresponse of the uterine stroma to the implantingblastocyst. In the human female, decidualizationof the stromal cells occurs in the secretory phaseof the menstrual cycle and involves biochemicaland morphological changes. If implantationoccurs, stromal cell decidualization continuesthroughout pregnancy. Therefore factors involvedin· decidualization are important for implantationand subsequent placentation. As a first step inelucidating the role of IL-llin human fertility theaim of this study was to examine the localizationand expression pattern of IL-l1 in the humanendometrium throughout the menstrual cycle.MethodsEndometrial biopsies were obtained from 30women who showed no apparent endometrialdysfunction. Biopsy sections were stained byimmunohistochemistry to immunolocalise IL-ll.Menstrual cycle sections were divided into 6phases with the following day ranges:Menstrual (ME) d1-5, early-proliferative (EP) d5­8, late-proliferative (LP) d9-13, early-(ES) d 14-18mid-eMS) d19-23 and late-(LS) d24-28 secretory,consisting of4 samples per phase.Antisera specific for human recombinant IL-l 1(R&D Systems) was used forimmunohistochemistry. Semi-quantitative scoringof staining intensity and distribution wasperformed. Results are expressed as means. Theexpression of IL-l1 transcripts in late secretoryphase endometrium was determined by in situhybridisation and IL-llR[ : mRNA expressionacross the menstrual cycle by reversetranscriptase-polymerase chain reaction (RT­PCR).ResultsImmunoreactive IL-ll was found associated withall cellular compartments. In the ME, and LPphases glandular epithelial and stromal cellsstained positively (Figure 1). In contrast, insecretory phase tissue, staining was seen in allcellular compartments (Figure 1). In the ES phasemoderate immunoreactivity was found in theluminal (LE) and glandular epithelial (GE) cells.More intense staining was seen in the endothelial(endo) and vascular smooth muscle (VSM) cellsin .the MS phase. However the most intensestaining was seen in the decidual cells of the LSphase, when they were present, with the nondecidualizedstromal cell staining intensityvarying little in the MS and LS phases. Theseresults were verified by in situ hybridisation. TheIL-II Ra was also found to be present throughoutthe cycle.Figure 1LEGEslromaendaVSM[REP-I"ICLPI DEs!111.151j ll:lLS I~decidualConclusionsImmunoreactive IL-ll is found in the humanendometrium in all the major cell types.IL-ll Ra mRNA was present throughout the cycle.In the proliferative phase moderate IL-Il stainingoccured in the glandular epithelial and stromalcells.Late secretory phase endometrial tissue showedthe most intense staining for IL-II which wasverified by in situ hybridisation.The most intense staining observed occurred inthe decidual cells.The presence of in vivo IL-II in a normalphysiological situation in secretory phaseendometrium implies that IL-II may be involvedin decidualization and the preparation of areceptive endometrium for implantation.ReferencesDu et al (1997) Blood 89: 3897-3908Robb et al (1998) Nature Med 4: 303-308INTRAUTERINE SEMINAL PLASMA INDUCES ENDOMETRIALINFLAMMATORY RESPONSE IN GILTSS. O'Leary, D.T. Annstrong, R Kamai and S.A. RobertsonDepartment ofObstetrics and Gynaecology, The University ofAdelaide, Adelaide, SA 5005IntroductionSeminal plasma (SP) has previously been thought tofunction simply as a transport and swvival mediumfor spermatozoa traversing the female reproductivetract However, emerging data in rodents nowindicates that factors in SP, most notablytransfonning growth factor beta (TGF~), act toevoke an inflammatory response in the uterineendometrium after insemination (1). This responseapPearS to improve implantation through inducingembryotrophic cytokine. expression and promotingimmunological tolerance to paternal transplantationantigens expressed on the semi-allogeneic conceptus.In pigs, exposure to seminal constituents has beenshown to improve litter size (2) and to influenceovarian function (3,4), but the physiologicalmechanisms mediating this effect are unknown. Theaim of this study was to begin to investigate themolecular basis of the interaction between SP andthe female reproductive tract in pigs. Specifically,we have measured the acute effect of SP onleukocyte recruitment and other inflammatoryparameters in the uteri ofgilts.Materials and MethodsAt the onset ofgonadotrophin-induced oestrus, eightLarge White X Landrace gilts were administeredeither seminal plasma (SP; n=4) or saline (pBS~ n=4)by transcervical infusion~ Reproductive tracts wereretrieved at autopsy 36 hours after treatment whenthe weight, luminal fluid content and vascular indexof uteri were scored. Endometrial tissue biopsieswere frozen in ocr and the abundance and locationof resident leukocytes were assessed byimmunohistochemical analysis of ethanol-fixedfrozen sections using monoclvnal antibodies specificfor CD45 (MCAI222), :MIlC class II (MSA3), andmacrophages (HB142). Antibody reactivity (%positivity) was quantified by video image analysis.Fig. 1. The. effect of intrauterine SP infusion onleukocyte populations in the endometrium of gilts-'-- .80 -A~70f 60t1 50l:lot'$. 4090..,--------------A'1-. .-1- At II30 T • • PBS20 •A SP10it = P

UTERUSUTERUSIMMUNOCYTES IN THE FEMALE RABBIT REPRODUCTIVE TRACTW GU 1 ,3, MK Holland 1,2, PJ Kerr l ,3lpest Animal Control CRC, 2CSIRO Wildlife and Ecology, 3Division ofBiochemistryand Molecular Biology, Australian National UniversityIntroduction:A better understanding of the local immunesystem is necessary to be able to generate aneffective immune response within the femalerabbit reproductive tract. This includesknowledge ofthe presence and distribution ofthe immunocytes.Material and methods:A panel of monoclonal antibodies directedagainst rabbit CD45, CD43, CD5, Ig ~-chainand MHC class II was used to labelimmunocytes in frozen sections of oviduct,uterus, cervix and vagina of the normal rabbitreproductive tract before and after inductionofovulation by HCG.Results:Positive cells were mainly found in themucosa throughout the reproductive tract.Significantly, more CD45+ leukocytes,CD43+ T cells and MHC class II positivecells were present in the vagina, cervix andoviduct than in the uterus (Table 1). Inductionofovulation remarkably increased the numberof CD45+, CD5+ and MHC II+ cells but notCD43+ cells in endometrium (Figure 1). Subepithelialand intra-epithelial CD45+, CD43+and CD5+ cells were the dominant cellpopulation in the mucosa (Figure 2).Table 1. Cell numbers in mucosa areas(mm 2 )~_------------CD45+ CD43+ MHCIIOviduct 89±3I.4 43±3.5 42±I1.6Uterus 19±6.7a 12±7.8a 20±I2.7bCervix I07±29.5 84±34.8 48±7.5Vagina 145±40.6 IIO±30.6 UCNotes: the values are mean ± S.D for fouranimals. A:P

UTERUSOBSERVATIONS ON OESTROUS SUPPRESSION USING HYDROXYPROGESTERONEHEXANOATE IN OESTROGEN CHAL~ENGEDOVARIECTOMISED CATTLEA.M. Padula, K.L. MacmillanUniversity ofMelbourne, Veterinary Clinical Centre, Princes Hwy, Werribee, Australia.SERIAL PLASMA PROGESTERONE CONCENTRATIONS IN OVARIECTOMISEDHOLSTEIN COWS TREATED WITH NEW AND REUSED CIDR DEVICESA.M. Padula~ K.L. MacmillanUniversity ofMelboume, Veterinary Clinical Centre, Princes.Hwy, Werribee, Australia.UTERUSINTRODUCTIONHydroxyprogesterone hexanoate 1 (HH) isavailable in a number of registered veterinarypreparations in Australia. Few publishedstudies are available regarding its efficacy as aprogestagen in cattle. HH was found to beineffective in maintaining pregnancy inovariectomised mares given 500 mg every 7days starting 15 days post insemination (1).HH was able to delay uterine involution inpostpartum dairy cows when given 100 mgdaily from calving to 22 days postpartum (2),suggesting progesterone like activity but couldnot maintain pregnancy when given weekly inpregnant ovariectomised cows (3).The objective of this study was to investigatethe progestagenic activity ofHR in cattle.MATERIALS AND METHODSI Alternatively known as hydroxprogesterone caproate(Merck Index 1998).RESULTS AND DISCUSSIONJugular blood concentrations of progesteronewere below the sensitivity of the assay in allsamples assayed. It would appear that in cattle,HH is not converted into free progesterone.HH clearly suppressed ODB induced oestrousbehaviour when challenged at 24 hours.Oestrous response to the 2.0 mg ODB challenge(see Table 1) appeared to have returned tonormal by 8 days post HH with the samenumber of cows detected in oestrus aspreviously. Common protocols for using HH insupporting pregnancy advocate treating atintervals of 14 to 28 days. Based on the dataobtained in this experiment this interval mayneed to be shortened to less than 7 days.Table 1. Oestrous response at 24hrs post ODB in cowstreated on day 0 with 1000 mg HH.Day ofODBNo. in standing heatAn initial study was performed to determine if ------------------HH would be converted into free circulatingprogesterone. Six long-term OVX cows werepaired and injected intramuscularly once with acommercial preparation of either 250, 500 or1000mg HH in oil. Jugular blood samples werecollected daily for 7 days following HH.Plasma progesterone was assayed for using acommercial solid phase RIA (Coat-a-Count®;DPC, Los Angeles) with a stated cross reactivityto HH of3.4%.Twelve non-lactating OVX Holstein-Friesiandairy cows were injected with 2.0 mg oestradiolbenzoate (ODB) in oil as an oestrous challengetest. Visual observation at 24 hours post ODBwas used to detect cows in standing heat. Asingle 1000 mg injection of HH was then giveninto the gluteal muscles 3 days after the initialODB challenge. The ODB oestrous challengetest was repeated at 2 and 9 days after the HHinj ection. Proportional data was analysed bychi-squared test.-3 10/12 a+1 0/12 b+8 10112 aa vs b, t ;p< 0.0001.HH appears to be a progestagen in cattle by itsability to suppress oestradiol induced oestrousbehaviour although its duration of actionappears to be relatively short.ACKNOWLEDGEMENTThank you to Jurox for supplying the HR.REFERENCES(1) (1) McKinnon, A. 0., S. Tarrida delMarmoI Figueroa, et al. (1993). EquineVeterinary Journal 25(2): 158-160.(2) Fosgate OT, Cameron NW and McLeodRJ. (1962). Journal of Animal Science21:791-793(3) Johnson KR and Erb RE. (1962) Journal ofAnimal Science 21 :633-639Email: a.padula@pgrad.unimelb.edu.auINTRODUCTIONVaginal delivery of progesterone using a CIDRdevice is frequently used in combination withoestradiol to control ovarian follicledevelopment in order to facilitate oestroussynchronisation. The device contains 1.93 g ofprogesterone in a silicone matrix with an innernylon-stiffening core (1). Oestroussynchronisation programs commonly involveresynchrony ofcows that are not pregnant to theinitial mating and a similaroestradiol/progesterone combination is used todetect these cows subsequently. The issue ofreusing the progesterone CIDR device becomesan economic one when large numbers of cattleare to be treated.The objective of this study was to determine ifpreviously used CrDR devices could be used tomaintain similar plasma progesteroneconcentrations to a single new device inovariectomised cattle.MATERIALS AND METHODSTwelve long term ovariectomised Holstein­Friesian dairy cows were randomly allocatedreceive either a single new device (CIDR-B~;Genetics Australia, Bacchus Marsh) (n=4); asingle device that had been previously used for7 days (n=4); or two previously used devices(n=4). Each cow was pre-treated for 3 dayswith a single previously used device to inducehepatic enzymes involved in progesteronebreakdown and to reduce initial variationbetween cows. All previously used devices wereprepared by soaking in cold water overnightfollowed by gentle scrubbing with chlorhexidineand then autoclaving for 15 mins at 115°C.Blood samples were collected from thecoccygeal vessels into 10ml lithium heparinvacuum tubes (Vaccutainer®; Becton Dickson,USA) on the day of allocation to treatment (day0) and again on days 1,2,3,7 and 14. Plasmawas immediately separated by centrifugationand frozen at -20 c C until assayed. Progesteronewas assayed using a commercial solid phaseantibody RIA (Coat-a-Count®; DPC, LosAngeles). Progesterone levels across groups .ateach time point were analysed by one-wayANOVA using Bonferroni's method formultiple comparisons at each time (SPSS V9).RESULTS AND DISCUSSIONTable 1. Mean plasma progesterone concentrations(ng/ml) ± s.e.m. by day after device insertion.Day 1 x new 1 x prevo used 2 x prev. used0 1.74 ± 0.09 1.99 ± 0.39 2.40 ± 0.291 2.50 ± 0.52 2.98 ± 0.59 2.84 ± 0.512 1.75 ± 0.43 1.69 ± 0.26 2.77 ± 0.573 1.92 ± 0.21 2.14 ± 0.48 2.63 ± 0.817 1.59 ± 0.16 a 1.41 ± 0.23 a 2.58±0.17 b14 0.95 ± O.12 a 0.89 ± O.13 a 1.55 ± 0.08 bMeans within rows with different subscripts differsignificantly (P

UTERUS·FSA PLENARY LECTURESSUPEROVULATION OF THE BRUSHTAILPOSSUM DURINGSEASONAL ANOESTRUS WITH PORCINE FSHIUIF.C. Molinia, A.M. Glazier and IV. MyersCooperative Research Centre for the Conservation and Management ofMarsupialsLandcare Research, PO Box 69, Lincoln 8152, New ZealandINTRODUCTIONManipulation ofthe reproduction ofcaptive marsupials is an on-going strategy ofthe Marsupial CRC to facilitate assisted breedingofendangered species and control ofoverabundant populations (1). Superovulation has been achieved in the brushtail possum usingPMSGILH during the breeding season (2) and porcine (P) FSHlLH during seasonal anoestrus (3). However, doses ofpFSH used inthe latter study resulted in overstimulation. This study examined the dose ofpFSH that yielded the maximum number ofproperlymatured oocytes, the timing ofovulation after pLH treatment and the competence ofthe ovulated eggs to fertilize following artificialinsemination (AI).MATERIALS AND METHODSEffect ofpFSH dose: Female possums (n = 12 per group) were treated by i.m. injection with 6,3 or 1.5 mg pFSH (Vetrepharm,Canada) twice daily (12 h apart) for four consecutive days followed by 4 mg pLH (Vetrepharm, Canada), 12 h after the last pFSHtreatment. A control group (n = 12) was superovulated with a single i.m. injection of 15 iu PMSG (Intervet, The Netherlands)followed 78 h later by 4 mg pLH. Ovaries were examined and eggs recovered by flushing oviducts and uteri with heparinised (12.5iulml) PBS, 2 days after pLH treatment. Eggs retrieved were stained with Hoechst 33342 (Sigma, USA) and assessed for nuclearmaturation status using fluorescence microscopy.Timing ofovulation: Possums (n = 24) were superovulated by 8 x 3 mg pFSH/4 mg pLH treatment as described above. Animalswere euthanased 27-39 h after pLH treatment and the ovaries examined to determine the timing ofovulation onset.Artificial insemination: Possums (n = 12) were superovulated as for the timing of ovulation trial. On the day of insemination,spermatozoa were flushed from the cauda epididymides of3 males with EMEM media (4). Samples were pooled and stored at 4°Cbefore insemination. Uterine (n = 5~ 5 x 10 6 motile sperm!uterus in a 0.2 ml dose) and vaginal (n = 5~ 100 x 10 6 motile sperm in a2 ml dose) insemination was performed with the aid ofa laparoscope, 27.5-28.5 h and 13-14 h after pLH treatment, respectively.As a control, an additional 2 females were inseminated into the uterus or vagina with EMEM media alone. Ovaries were examinedand eggs recovered as detailed above, 2-3 days after AI. Eggs retrieved were stained with Hoechst 33342 and assessed for fertilityby fluorescence microscopy.RESULTS AND DISCUSSIONAll females responded to pFSH/pLH treatment, although optimal stimulation occurred in those receiving 8 x 3 mg pFSH, with 13-14ovulations and recovery of 11-12 eggs per female (8 x 1.5 mg pFSH: 13 ovulations, 8-9 eggs~ 8 x 6 mg pFSH: 7-8 ovulations, 4-5eggs per female). In contrast, only 7/12 females responded to the standard PMSG/pLH protocol and ofthose responding, only 2-3ovulations occurred and only 1-2 eggs per female were recovered. However, around 80% of eggs recovered after PMSG/pLHtreatment were classed as nuclear mature (Metaphase II + 1polar body) compared with around 60% ofeggs from the pFSH/pLHtreated animals.Possums superovulated by 8 x 3 mg pFSH/4 mg pLH treatment, began ovulation around 30 h after pLH treatment. However, recentdata from another 48 similarly treated females (as part ofIVF and egg maturation studies) indicates that the onset ofovulation maybe as early as 27 h after pLH treatment. Following uterine and vaginal insemination, 11/26 and 21/53 eggs recovered were embryos,respectively. No embryos were obtained from control animals inseminated with EMEM media alone. These results confIrm that atleast a proportion ofeggs generated from this refmed pFSH/pLH protocol are properly mature and competent offertilization. Evenhigher embryo yields using pFSH/pLH could be expected ifthe yield offully matured eggs ovulating could be increased and/or ifuterine or vaginal insemination was performed 6 or 21 h before expected ovulation, respectively (1). Work is currently focussed onthese issues, and in the longer term on generating viable young using superovulation, AI and embryo transfer teclmology.REFERENCESI. Mate KE, Molinia FC, Rodger JC (1998) Anim Reprod Sci 53, 65-76.2. Glazier AM, Molinia FC (1998) J Reprod Fertill13, 191-195.3. Fletcher TP, Molinia FC, Glazier AM, Rodger JC (1998) ProG Aust SOG Reprod Bioi 29, 28.4. Molinia FC, Myers JV (1999) PrOG Aust SOG Reprod BioI 30, 134.INITIATION OF HUMAN PRIMORDIAL FOLLICLE GROWTH IN VITRO IN ULTRA THIN SLICES OFOVARIAN CORTEXIH. M. Picton, IA.:Mkandla, 20. Salha, Ip. Wynn & IRG. Gosden.IDepartment ofObstetrics & Gynaecology, University ofLeeds, Leeds, UK, 2Assisted Conception Unit, LeedsGeneral Infirmary, Leeds, UK.IntroductiOli: The human ovary is endowed with hundreds ofthousands ofprimordial follicles at birth, but verylittle is known about the biology ofthe events which lead to initiation oftheir growth. Follicles in vivo are closelysurrounded by a complex and dynamic milieu, including ovarian stroma and·branches ofthe systemic circulation,nervous system and scavenger cells. Furthermore the architecture of the ovary shows that quiescent primordialfollicles are located in a thin, relatively avascular, layer in the ovarian cortex, whereas growing follicles arefound in the cortico-medullary border which is richly vascularised. This suggests that primordial follicleactivation and growth may depend on the provision of nutrients, hormones, and/or growth factors from thesurrounding tissue. These observations could account for why conventional methods of isolated follicle culturehave proved incapable ofsupporting human primordial follicle growth in vitro. A simpler alternative strategy, isto grow early follicles (of

FSA PLENARY LECTURESOVARIAN FUNCTION AND FOLLICLE DEVELOPMENTEXPRESSION OF THE TELOMERASE CATALYTIC SUBUNIT (TERT) IN BOVINE OVARIAN FOLLICLES ATDIFFERENT STAGES OF FOLLICLE DEVELOPMENT.Tina C. Lavranos and Raymond J. Rodgers.Department of Medicine, Flinders University of South Australia, Bedford Park. South Australia. 5042Decline ofinhibin B in Seminal plasma parallels suppression ofspermatogenesis in amale contraceptive study.RA Anderson 1 ., C W Martin 2 , 8 C Riley., N P Groome 3 ., and D T Baird 21MRC Reproductive Biology Unit and .'2Dept ofObstetrics and Gynaecology., Centre for Re~roductive Biology., Umverslty ofEdinburgh, 37 Chalmers 8t EdInburgh UKSchool ofBiological & Molecular Sciences, Oxford Brookes University, Oxford UKInhibin B is a product ofthe seminiferous epithelium, and is present in both blood.andseminal plasma (SP). That secreted into blood is invol~ed in the feedb~ck.re.gulatlon ofFSH secretion. We have previously reported a correlatIon betwee~ ~p Inhlbln Bandsperm concentration, suggesting that inhibi~ B m~y ref1ec~ the a~tIVI~ ofthe .seminiferous epithelium. We have further InvestIgated thIS relatIO~S~IP b~ measunngchanges in inhibin B in both blood and seminal plasma during. admimstration ofa .combination ofthe pregestogen desogestrel with testosterone In a male contraceptIvestudy.30 normal men were randomly allocated to receive 300mg testosterone pellets sc withone of3 doses ofdesogestrel: 75J..Lg, 150J.lg or 300J.l daily po for 8 weeks. Inhibin Bandthe proaC containing monomeric a-submit forms were m.easured by specific ELISA.Gonadotrophins were rapidly and profoundly suppressed In all groups, followe~ bypartial recovery particularly ofLH in th 75~g group. Median sperm co~centratlons ~ft~r3 weeks treatment were 24, 10 and 0.1 x 10 Iml in the 3 groups respectIvely. SP InhibinB showed a wide range pre-treatment «15pg/ml to 6.2ng/ml)., and while concentrationsfell in all 3 groups, this reached statistical significance only in the ~OOl-lg group in whomit becaine undetectable in all men. There were significant correlatIons between SPinhibin B and both sperm concentration (p=0.025) and FSH (p

OVARIAN FUNCTION AND FOLLICLE DEVELOPMENTOVARIAN FUNCTION AND FOLLICLE DEVELOPMENTDIFFERENTIAL EXPRESSION OF GM-CSF RECEPTOR rriRNA SUBUNITS IN FOLLICULAR CELLSAND THE EFFECT OF GM-CSF DEFICIENCY ON OOCYTE MEIOTIC COMPETENCE.RB Gilchrist, LJ Ritter, DB Rowe, S Fujii, RJ Norman, SA Robertson and DT Armstrong.The Reproductive Medicine Unit, Department ofObstetrics & Gynaecology, The University of Adelaide, TheQueen Elizabeth Hospital, Adelaide.Introduction : Granulocyte-macrophage colonystimulatingfactor (GM-CSF) is a cytokine known toinfluence ovarian cyclicity and embryo development.GM-CSF acts through a cell surface receptorheterodimer expressed by many cell lineagesthroughout the ovary. Mice deficient in GM-CSF (GM-/-) generated using gene targeting techniques exhibitimpaired reproductive capacity, including smallerlitter sizes (1). The aims ofthis study were to examinethe expression of GM-CSF receptors in follicular cellsand the effect of GM-CSF deficiency on oocytemeiotic competence.Materials & Methods : Cumulus-oocyte complexes(COC) and mural granulosa cells (MGC) werecollected from GM -/- or wild-type mice (GM +/+).Mice were treated with SID equine chorionicgonadotrophin (eCG) and 48h later with SIU humanchorionic gonadotrophin (hCG) and cells werecollected either just prior to hCG treatment(immature), 9h after hCG (preovulatory) or 24h afterhCG (ovulated). In vitro matured (IVM) COC wereassessed for cumulus expansion and meioticprogression after culture in media supplemented with5% fetal calf serum, FSH and LH. Total cellular RNAwas extracted and reverse transcribed using randomhexamers and a Superscript II kit (Life Technologies).eDNA was amplified by PCR utilising a HotStarTaqDNA polymerase kit (Qiagen) and previouslydescribed primers (2). Negative RT and PCR controlsconsisted ofwater substituted for reverse transcriptaseand eDNA, respectively. Reaction products wereanalysed by gel electrophoresis and visualised overultraviolet light.Results & Discussion: MGC populations from bothgenotypes were found to express mRNA for the GM­CSF a and ~ receptor subunits (Figure I). Detectionof the ~ subunit was somewhat unexpected asexpression of this subunit is typically restricted tohaemopoietic cells. Contamination with leucocyteswas proven unlikely as murine leucocyte commonantigen mRNA was not detectable in MGCpreparations. Immature COC expressed the a but notthe ~ receptor subunit. However, expression of the ~subunit by COC was found to be developmentallyregulated, with expression occurring during the courseof oocyte maturation, both in vivo and in vitro (Figure2). This suggests that GM-CSF might playa-' ......... ......,GM-CSFRa.IilIiiIlIIIlif 'i' '~

OVARIAN FUNCTION AND FOLLICLE DEVELOPMENTOVARIAN FUNCTION AND FOLLICLE DEVELOPMENTANALYSIS OF RAT OVARIAN INHIBIN SUBUNIT mRNA EXPRESSION BY "REAL TIME" peRAnn E. Drummond, Mitzi Dyson and Jock K. FindlayPrince Henry's Institute ofMedical Research, PO Box 5152, Clayton, Victoria, 3168, AustraliaINTRODUCTIONInhibin is a dimeric molecule composed of an a subunitlinked to one oftwo ~ subunits (A or B), whereas activin isa dimer of the ~ subunits. These subunit mRNAs havepreviously been shown to be present in the ovary, localisedto granulosa cells (1). The relationships between the steadystate levels ofthe respective subunit mRNAs, the stages offolliculogenesis and the production of estrogen have notbeen well defined. Thus, the aim of these studies was toquantify the expression of steady state mRNAs for theinhibin a, ~A and ~B subunits by post-natal rat ovariesduring defined periods of follicular development (l) andby granulosa cells isolated from diethylstilboestrol (DES)­treated rats, using "real time" polymerase chain reaction(PCR) technology.MATERIALS & METHODSRNA was purified from pools of ovaries of 4(primordial/primary follicles), 8 (preantral follicles) and 12(antral follicles) day old rats and granulosa cells isolatedfrom the ovaries of 25 day old rats, which were eitheruntreated or treated with DES for 4 days prior to excisionof the ovaries. The RNA was reverse transcribed aspreviously described (2). A eDNA pool prepared from theovaries of 22 day old rats was used to generate a standardcurve. Standard and sample cDNAs were amplified usingprimer sets specific for the individual inhibin subunits, in areal time peR format (LightCycler: Roche, Mannheim,Germany) (3). The data was corrected for glyceraldehydephosphate dehydrogenase (GAPDH) expression.RESULTSOvaries containing follicles primordial to antral in nature,expressed all three inhibin subunit mRNAs. Expression ofthe inhibin subunits relative to GAPDH, was maximal inovaries of 8 day old rats in which primordial, primary andsecondary follicles predominate (Fig 1). The appearance ofantral follicles which are capable of producing estrogen, atpost-natal day 12, led to a decline in the mRNA levels ofeach inhibin subunit that was most evident for the ~subunits, particularly ~B. Direct comparison of theexpression levels of each subunit at post-natal days 4, 8and 12, revealed 16-, 32- and 64-fold more a subunitmRNA in the ovary relative to ~B mRNA, respectively.The level of expression of ~A subunit mRNA in 4 and 8day old ovaries was similar to that of ~B but by day 12,there was 2-fold more ~A mRNA relative to ~B expressedby ovaries containing antral follicles. Administration ofDES to immature rats prior to the isolation of granulosacells from the ovary, led to ~A and ~B mRNA expression(corrected for GAPDH) being down-regulated in theabsence of any significant change in a subunit expressionby granulosa cells. Direct comparison of the levels ofexpression of each subunit within groups, revealed 8-foldmore a subunit mRNA relative to either ofthe ~ subunits.The ratio of a:~A:~B subunit expression, determined bycalculation of fold changes from crossing point-cyclenumbers, was not noticeably effected by DESadministration. The exponential nature of PCR, in that adoubling of product occurs with a single PCR cycle, mayhave masked small changes in the absolute level ofexpression of the subunit mRNAs, which were apparentwhen the data was normalised for GAPDH expression.1.20.8oe0.6 pB subunit b:t:oa.~ 0.4-~r::.E 0.2:::l1Il.5~ 0.5 (4)0.6 pA subunit0.40.2o(3)b(3)(3) (3) (3)4 8 12Days Post BirthFig 1. Expression of inhibinsubunit mRNAs by post-natalrat ovaries. Different letterswithin subunits denotestatistical significance, P

·FSA SYMPOSIUM ON EMBRYOLOGYFSA SYMPOSIUM ON EMBRYOLOGYCURRENT KNOWLEDGE OF OOCYTE PREPARATION FOR OVULATION ANDFERTllJISATIONGayleMJonesCentre for Early Human Development Development, Monash Institute ofReproduction andDevelopment, Monash Medical Centre, Clayton AustraliaAt birth, the human ovary contains a pool of non-growing follicles that contain an oocyte arrested at thediplotene stage of the first meiotic prophase. Follicle growth occurs throughout life until the onset ofmenopause. At the onset of puberty, follicles and their occytes are recruited to resume growth. Thehuman oocyte grows from a diameter of 35 J.1.g to 120J.1.g over several months. During this period ofgrowth the oocyte synthesises and stores large amounts of mRNA and proteins that are essential to thecompletion of maturation and to the subsequent acquisition of embryo developmental competence andviability. It is also at this time that the oocyte secretes large amounts ofglycoprotein that condenses toform the zona pellucida. The zona separates the oocyte from the surrounding follicle cells but contactwith follicle cells is maintained via cytoplasmic processes that pass through the zona and end in gapjunctions at the oocyte surface. The zona pellucida also has an important function in subsequentfertilization events.The acquisition of developmental competence requires the completion of· the meiotiC programme(nuclear maturation) and the completion of separate set ofmolecular and structural events often termed'cytoplasmic maturation'. It is possible to have nuclear and cytoplasmic maturation proceedindependently but full developmental competence is only conferred when these events are integrated.Maturation in vivo is, in the main part, controlled and co-ordinated through the actions ofFSH and LH.Aberrations in the maturation process will result in failure downstream i. e. failure to mature toMetaphase IT, failure to cleave or failure to develop to blastocyst. Attempts to mature human oocytes invitro have resulted in increasing success rates and ongoing pregnancies. However a significantreduction in embryo developmental competence has been observed following maturation in vitro whencompared to in vivo, and this may be in part associated with the absence of the synthesis of specificproteins during the maturation process.EMBRYO METABOLISMPeter KayeDepartment ofPhysiology and Pharmacology, School ofBiomedical Sciences, The University ofQueensland, Brisbane, Queensland, 4072One of the earliest reports of culture of embryos was that of Hammond in 1949. Metabolic studiesawaited the design ofculture media capable ofsupporting embryos for the necessary incubation periods.Indeed, this may have been the primary cause of the development of culture media, in contrast to thepresent situation at the end of the century. Whitten's development of suitable culture systems in themiddle of the twentieth century permitted true metabolic studies with assays of oxygen consumption,enzyme activities ant he incorporation or oxidation of radiolabelled substrates, mainly in mice. Theresults established the fundamental parameters of current knowledge, which were· expanded andsupplemented as new technologies became available in succeeding decades. Its clear that availability ofsuitable technology hampered the growth of our knowledge of this crucial aspect of preimplantationphysiology. Techniques such as Lowry's metabolite assays suitable for single embryos,microspectrofluorimetric analysis, HPLC and confocal microscopy have each provided significantincrements in knowledge. The other notable contribution has come from the diversity of mammalianembryos under study. Whilst the first studies ofoxygen consumption used rabbit embryos, we now haveresults for humans, several rodents, ungulates and marsupials. This comparative data illustrates that, justas development ofthe species differs, so does the underlying metabolism.What ofthe new millennium? Knowledge ofmolecular genetics and the regulation ofgene expression israpidly expanding on the wave created by the genome studies. As we learn about the genes involved wewill gain further insight into metabolic physiology. However, it is most likely that understandingmetabolic fluxes and end points will continue to be limited by technologies, because ofthe necessity tostudy the metabolism of embryos with so few cells. New knowledge requires intellectual innovation;that will determine the future.Understanding the mechanisms involved in both nuclear and cytoplasmic maturation is intrinsic to thesuccessful application ofhormonal stimulation protocols for superovulation and subsequent fertilizationin vitro and to the development of culture medium and culture conditions for the successful maturationofoocytesin vitro.112113

FSA SYMPOSIUM ON EMBRYOLOGYFSA SYMPOSIUM ON EMBRYOLOGYMOLECULAR BASIS OF EMBRYO DEVELOPMENT LEADING TO IMPLANTATIONRobert DanielsMonash Institute ofReproduction and Development, 27-31 Wright Street, Claton, Victoria 3800The widespread use ofin-vitro fertilisation techniques to treat infertile couples has opened up new areasofresearch in human development. Molecular analyses ofhuman preimplantation embryos from the 1­cell to the blastocyst stage have been carried out in order to determine the genetic basis ofearly humanembryo development. A greater understanding ofthe mechanisms controlling human preimplantationdevelopment may lead to improvements in the culture conditions used in IVF procedures. To date,analyses ofgene expression at both the mRNA and protein level in human preimplantation embryoshave demonstrated the expression ofa number ofgrowth factors and cytokines, and/or theircorresponding receptors, which may influence the development ofhuman preimplantation embryos or~ffe~t th~ir implantation capabilities following transfer. The significance ofthese findings and theirImplIcatIons for embryo culture conditions will be discussed.SOME RESULTS OF IVF PROCEDURES IN ANIMALSBob SeamarkCSIRO Division ofWildlife and Ecology, Lyneham, ACT 2602(Abstract not submitted)CURRENT RESEARCH AREAS AND FUTURE PROSPECTS IN EMBRYOLOGYAlan TrounsonInstitute ofReproduction and Development and Monash IVF, Monash University,31 Wright St,Clayton, 3168Human embryology has made astonishing progress over the last two decades and the rapidly growingknowledge ofearly development offers new and interesting applications and demands increasing skillsoflaboratory embryologists working in IVF clinics.Major advances have been made in the culture ofhuman embryos and it is now possible to transferembryos to the uterus over the first six days after insemination without serious loss ofembryo viability.This provides the opportunity to study structure, morphology and function ofembryos and their viabilityand to examine embryo cell lineages formed by chromosomal aneuploidies during cleavage anddevelopment. Rapid advances have been made by scientists examining gene expression in the earlyembryo (Adjaye et aI., 1997) with new and novel genes discovered that will re-modify culture conditionsand provide gene expression screens for developmental competence. It is also possible to DNA fingerprint single sibling embryos and this technology will enable the identification ofthe particular embryoamong a number transferred, that developed to term. The ability to separate viable and non-viableembryos within a successful pregnancy is a very powerful analytical tool because it eliminates any effectofuterine receptivity for implantation and pregnancy.Human embryos that would have otherwise been discarded have been used to establish humanembryonic stem (ES) cells. The characteristics ofthese undifferentiated cells and their need to bemaintained in this pluripotential state are also informative for human embryology. The directeddifferentiation ofthese cells in vitro will be a major focus ofresearch over the next two decades.New cryopreservation systems have been developed that utilise vitrification (glass formation) and these.techniques are likely to replace conventional freezing techniques very quickly. Important new researchon evaporative drying with sugars is likely to revolutionise preservation ofcells in the near future. Thepreservation ofovarian tissue for patients with cancers that are likely to result in sterility as a side...effectofthe treatment is well advanced and work is presently focused on the methods for autotransplantation.Nuclear transfer and embryo micromanipulation techniques continue to be explored in embryology.Cytoplasmic transfer, nuclear transfer in eggs to avoid aneuploidy and the long-term prospect ofmakingartificial gametes are interesting options. It has also been argued that "therapeutic cloning" may beneeded to make ES cells compatible for use for tissue and organ transplantation in the future. Thisinteresting option is under threat from legislation at the present time in Australia and is already unlawfulin the State ofVictoria. Strong representation from science and medicine is needed to preserve thisoption for the sake ofterminally ill and severely handicapped patients.Adjaye J, Daniels R, Bolton V, Monk M (1997) DNA libraries from single human preimplantationembryos. Genomics 46: 337-344.114115

FSA SYMPOSIUM ON EMBRYOLOGYFSA FREE COMMUNICATIONS-SPERM AND EMBRYOSSTIMULATION PROTOCOLS AND RESULTS-EFFECTS ON EGG AND EMBRYO QUALITY,IM:PLANTATION RATES AND FUTURE IM:PLICATIONS.Nick S. MacldonDivision ofReproductive Medicine, Department ofObstetrics and Gynaecology, Erasmus UniversityMedical Centre Rotterdam, 3015GD Rotterdam, The Netherlands.Since the early days ofIVF an impressive array of ovarian stimulation protocols has been employed withthe aim ofobtaining as many oocytes as possible in order to allow multiple embryo transfer and improvepregnancy rates. The contemporary approach to ovarian stimulation in IVF treatment involves prolonged,complex and expensive treatment regimens, which are not without risk, while singleton pregnancy ratesremain disappointing. Present ovarian stimulation protocols result in supraphysiological levels ofprogesterone and estrogen in the luteal phase which act directly and indirectly to mature the endometrium,influencing receptivity to implantation. The development of endometrial receptivity is a complex process,and may be altered by inappropriate exposure to sex steroids. Alteration in the estrogen/progesterone ratio,growth factors and cell adhesion molecule profiles may occur following ovarian stimulation, potentiallyaffecting endometrial receptivity. Recent clinical IVF studies have shown implantation rates and corpusluteum function to be influenced by early luteal phase estrogen concentrations. While well carried outcomparative studies are scarce, there appears to be a reduced implantation rate and a higher pregnancyloss rate prior to clinical detectability following ovarian stimulation compared to natural cycleconceptions, and both the antiestrogenic endometrial effect of clomiphene or supraphysiological estradiollevels associated with gonadotropin treatment may be implicated. The maturation of the oocyte itself isinfluenced by sex steroid concentrations, while the true impact of supraphysiologic exposure to estrogenson embryo quality has not been fully elicited.Increasing our knowledge ofthe physiology of follicle development and dominant follicle selection mayenable the design of less complex, safer and cheaper ovarian stimulation regimens for IVF. Decrementalserum follicle stimulation hormone (FSH) levels during the follicular phase of the menstrual cycle arerequired for single dominant follicle selection. Multiple large preovulatory follicles can be induced by theadministration of small doses of exogenous FSH during. the mid to late follicular phase, preventing thephysiological decrease in FSH stimulation. Intervention with decremental serum FSH levels to extend theFSH 'window' in combination with gonadotrophin releasing hormone (GnRH) antagonists to prevent apremature rise in serum luteinizing hormone (LH) may induce ongoing growth of multiple folliclessufficient for IVF without the need for luteal support. Our recent data support such a shift in approach toovarian stimulation, and we are now engaged in studies designed to test and develop this approach, whichcould also provide an attractive clinical context for single embryo transfer.THE EFFECT OF MORPHOLOGY AND MOTILITY OF TESTICULAR SPERM ONFERTILIZATION AND PREGNANCY RATESWR Edirisinghe, JE Dowd, and JA AllanThe Wesley IVF Service, Wesley Hospital, 40 Chasely Street, Auchenflower, Queensland.It has been shown that similar fertilization and pregnancy rates can be achieved using either ejaculated or testicular spermshowing various degrees ofteratozoospermia (1) when intracytoplasmic sperm injection (leSI) is employed. On the otherhand, sperm motility seems to· affect fertilization rates and pregnancy outcome (2). In this study we have analysed thefertilization and pregnancy data for testicular sperm in relation to the motility and morphology of individual sperm whenused in the lesl programme.In a total of 51 lesl cycles, fresh (9) or cryopreserved (42) testicular sperm were used. A single embryologist performedall the leSI procedures and the morphology and motility of sperm used for injection were recorded. The spermmorphology according to whether normal, single or multiple abnormality (according to strict criteria) was later correlatedto the fertilization and pregnancy rates. Similarly, sperm motility graded according to WHO {Grade (Gd) 0, 1 and 2} wasalso correlated to the fertilization and pregnancy rates. As there was no significant difference in the pregnancy ratesbetween the fresh (12/47, 25.5%) and frozen (6/31, 19.4%) embryo transfer cycles, a total of 78 combined fresh andfrozen embryo transfer cycles were analysed for the study. The findings are given in the table below.Category Type oftesticular sperm used P valueMorphologyMotilityNormal Single Multiple GdO Gd 1 Gd2Fertilized oocytes (n=242) 44 117 81 94 109 39 ns(18.2) (48.3) (33.5) (38.8) (45.0) (16.1)Unfertilized oocytes (n=64) 10 32 22 30 26 8 ns(15.6) (50.0) (34.4) (46.9) (40.6) 02.5)Embryos transferred in pregnant 10 (21.7) 25 11 5 a 25 16 c a vs bcycles (n=46) (54.5) (22.7) (10.9) (54.3) (34.8) (P

FSA FREE COMMUNICATIONS-SPERM AND EMBRYOSFSA FREE COMMUNICATIONS-SPERM AND EMBRYOSTESTICULAR ASPIRATION: FREEZING SEMINIFEROUS TUBULES AND ICSI;A VIABLE ALTERNATIVE TO VASECTOMY REVERSAL.JA Allan , A Catakovic and WR EdirisingheThe Wesley IVF Service, Wesley Hospital, 40 Chasely Street, Auchenflower, Queensland.Testicular sperm have been used successfully in achieving fertilization and pregnancies in managing infertility due toazoospermia (1). In this study we have attempted to compare the success and cost involved in performing this mode oftreatment to the conventional vasectomy reversal procedures.147 testicular aspirations were performed between March 1995 to June 1999 for the investigation and the treatment oftheazoospermic male. 61 aspirations were performed on 41 patients under local anaesthetic on an outpatient basis on menwho had obstructive azoospermia resulting from vasectomy or failed vasectomy reversal. The IVF outcome data and thepatient costs in these cases were reviewed and compared to the current costs and pregnancy outcomes followingvasectomy reversal.Seminiferous tubules were found in all of the 61 aspirations and viable sperm was found in 60 of these .cases. Theaspirated seminiferous tubule material was prepared and frozen for subsequent use in an IVF cycle. Eight patients werenot included in the outcome analysis as they have not as yet proceeded to IVF treatment. The seminiferous tubularmaterial was graded at the time of aspiration and then related to· the fertilization and pregnancy rates and the time sincevasectomy (table 1).Table 1- Seminiferous tubule quality in relation toa) FertT I Izaf IOn and pregnancy outcome b)T Ime . mt ervaI between vasectomy and asplrafIonQuality Male Female ICSI fert. Preg. Quality Number of Average timeAge (yr) Age (yr) rate (%) rate (%) patients Interval (yr)Good 43.7±3.6 a 34.3±4.9 118/206 e 4/20 Good 10* 9.0±6.0(57.3) (20)Fair 47.9±4.4 b 30.9±3.7 c 46/115 t 4/10 Fair 8* 9.5±6.5(40.0) (40)Poor 44.8±6.8 35.0±4.4 d 2011327 g 9/47 Poor 19* 13±5.3(61.5) (19.1)P

FSA.FREE COMMUNICATIONS-SPERM AND EMBRYOSFSA FREE COMMUNICATIONS-SPERM AND EMBRYOSAN ASSESSMENT OF FACTORS ASSpCIATE:p WITH IMPLANTATION INEMBRYOS GENERATED FllOM RE-INSEMINATIONNadine Richings 1 , Harold Bourne 2 and HW Gordon Baker l ,2IMelbourne IVF, Victoria Pde, East Melbourne and 2 Reproductive Biology Unit, Royal Women's Hospital,Grattan St, CarltonIntroduction: When there is a failure offertilisation following insemination by conventional IVF,unfertilised oocytes can be re-inseminated via ICSI. Although fertilisation and implantation rates are oftenlow, pregnancy and live birth can be achieved (1, 2).The aim ofthis investigation was to assess various factors associated with implantation following thetransfer ofembryos resulting from re-inseminated oocytes.Materials and Methods: Mature oocytes that had failed to fertilise following conventional IVF were reinseminatedvia ICSI, 20-24 hours after the initial insemination, using I-day old motile sperm from the IVFinsemination well. Re-insemination was employed in 115 cycles (750 oocytes) where no pronuclei wereobserved in any oocytes 16-20 hours after the initial insemination (Complete Fertilisation Failure = "CFF")and in 31 cycles (250 oocytes) where pronuclei were observed in a small proportion ofoocytes within thecohort following the initial insemination (Low Fertilisation = "LF"). Re-inseminated oocytes wereobserved for signs offertilisation 16-22 hours after injection and embryos were evaluated 40-46 hours postinjection.Results: A total of 1000 unfertilised oocytes from 146 cycles were injected and a normal fertilisation rateof39% was achieved, with (3PN and IPN rates of14% and 9%, respectively. Following re-inseminationthere were no significant differences between the "Complete Fertilisation Failure" and "Low Fertilisation"groups in the normal fertilisation rate ("LF" 35%, "CFF" 40%; p>O.OI), cleavage arrest ("LF" 15%, "CFF"14%; p>O.OI) and the proportion ofpoor quality embryos (>30% fragmentation: "LF" 20.1%, "CFF"21.6%; p>0.01). There was however a significant difference in the amount ofslow-growing embryos (2­cell stage: "LF" 45.5%, "CFF" 24.1%; p

FSA FREE COMMUNICATIONS-SPERM AND EMBRYOSFSA FREE COMMUNICATIONS-SPERM AND EMBRY~'ANALYSIS OF ANEUPLOIDY IN EMBRYOS FROM 53 IVF PATIENTS WITHPOOR PROGNOSIS OF PREGNANCYL. R. Gras \ G. M. Jones 2, A. P. Kausche 2 and A. O. Trounson 21. Monash IVF, 252 Clayton Rd., Clayton, Victoria, 31682. Monash Institute ofReproduction and Development, Monash University, Clayton, Victoria, 3168IntroductionNumeri~al chr~mosome abno~alityrates of57% have previously been reported in morphologicallynormal embryos fromIVF patIents wIth poor progn~~lSofpregnancy (i.e. ~ 37 years ofage, repeated IVF failures and/or altered karyotype) (1).T~ese chromosome abnorm~htl~s have been postulated as a possible cause ofpoor embryo viability, resulting in IVFfallures ~he~ embry~ selectIOn.1S based purely on morphological criteria. Selection ofchromosomally normal embryos fortransfer slgmfica?tly l~cr~ased Implantati~n rates. Comparison ofembryo morphology and corresponding aneuploidydemonstrated a higher l~cldence ~fnumencal chromosome abnormalities in both slower (5-6 cell, 61% aneuploidy) andaccelerated.(>8 c~ll, :6Yo aneUplOI?y) c!eavage stage embryos, than in embryos at the 7-8 cell stage (42% aneuploidy) onday 3 po~t-InsemmatlOn (1)..IdentlficatIOn ofembryos that possess a greater potential to develop to term is beneficial inIVF, pa~tlcularly when selectmg fortransfer from a large cohort ofembryos. Parameters likely to reflect increased risk ofaneuplOIdy, and hence reduced embryo viability are therefore advantageous.Materials and MethodAneuploidy was investigated i~ a group of53 IVF patients with poor prognosis ofpregnancy. Embryo biopsy wasperf~rr.ne~on day 3 embryos WIth ~ 5 cells, by laser zona drilling, removal of 1-2 blastomeres and fluorescent in situhybndl~atlOn (FISH) analysis for chromosomes X, Y, 13, 18 and 21 (25 patients) and in addition, chromosomes 16 and 22(28 patIents).ResultsA total of.3 62 em~ryos were biopsied. FISH analysis was possible from 314 embryos (87%), resulting in identification of158 euplOId (50.3 Ya) ~d.15? ane~ploid (~9.7%) embryos. No FISH result was obtained from 48 embryos (blastomeres~ere anucleate or hybndlsatlOn faded). SIxty-five aneuploidies involved single monosomies or trisomies (41.7%), 81Involved ~o:e than one chromosome error (51.9018cell) embryo~ was 54.3 Ya, however thIS was not slgmficantly greater than in 7-8 cell embryos (P>0.05). Aneuploidy inslower cle~vmg embryos (5-6 cell) was 61% when associated with high (>113) fragmentation and 55.7% with little or nofragmentatIOn (P>0.05). More ~lastomer~~biopsied from 5-6 cell embryos were considered anucleate (14/129, 10.9%) thanfrom !-8 cell embryos (7/176, 4Ya). AddltlOnaIIy, of17 biopsied muitinuceated blastomeres, only 4 were found to beeuplOId. .Trans~er ofa total of 118 embryos identified as normal for the chromosomes tested resulted in 7 ongoingpregnancIes, WIth a pregnancy rate of 14% and implantation rate of6%.ConclusionWe confi:~ that embtyos from patients with poor prognosis ofpregnancy have a high incidence ofchromosome numericalab~ormahtles. EXclu~lOn from tr~sfer of cI:r0mos?~llyabnormal embryos has the potential to improve prognosis in thesepatIents. MorphologIc~1 obs~rv~tlOns co~bmed WIth Information derived from preimplantation diagnosis provides furthervaluable embryo sel~ctlOn cn!ena. SelectlOn for transfer ofgood quality 7-8 cell embryos on day 3 in preference to thosethat are slower cleavmg, may mcrease chance ofchromosome normality andtherefore improve implantation potential.EXPERIENCE WITH THE INTRODUCTION OF A COMPREHENSIVEPREIMPLANTATION DIAGNOSTIC SERVICE FOR SERIOUS GENETIC DISEASEMcArthur, S.J., de Boer, K.A., Leigh, D., Roberts, C.G., Catt, J.W., Bowman, M.C., Persson, J.A., Anderson,J.C., and Jansen, R.P.S.Sydney IVF, Sydney, NSW 2000.Introduction. In the 1990s Sydney IVF considered that a comprehensive preimplantation genetic (PGD) service wouldrequire optimum laboratory practices in the otherwise different fields ofin vitro fertilization (including embryo culture to atleast the morula stage to permit transfer ofmetabolically uncompromised embryos after biopsy), cytogenetics with rapiddetection ofchromosomes using fluorescent in-situ hybridization (FISH - for rapid identification ofcertain autosomes andthe sex chromosomes in removed blastomeres and/or polar bodies) and accurate polymerase chain reaction (PCR)techniques with reliable products derived from singles copies ofDNA. Therefore, before the introduction ofPGD, threedistinct service were developed to functional efficiency, after which they were integrated to comprise the PGD project.Methods. Development ofstage specific culture medium and optimum embryo culture methods have been describedpreviouslyl. The PGD laboratory team was integrated with the clinical genetics team to consider each couples PGDrequirements. Pregnancies followed the abandonment ofacid Tyrode's for opening ofthe zona pellucida and the useinstead ofa FERTILASE® near infrared laser. As well as for avoiding sex-linked genetic disease, IVF with selection ofembryos on the basis ofsex and apparent euploidy has, for some families, been for the (non-HIC-rebated) purpose ofaddinga child ofthe other sex to existing children.Results.PGD ResultsOverall AssistedConception results atSIVF,1998Overall PGD Sex Selection Genetic PGD

FSA FREE COMMUNICATIONS-IVF CLINICAL. FSA FREE COMMUNICATIONS-IVF CLINICALDIETARY-DERIVED ISOFLAVONES ARE PRESENT IN OVARIAN FOLLICULAR FLUIDIN WOMEN UNDERGOING TRANSVAGINAL OOCYTE COLLECTIONDC Knight, JPP Tyler and GL DriscollCity West IVF, 12 Caroline St, Wes1meacL New South Wales, Australia 2145!n Australia the trend toward dietary change by the general public in an attempt to improve health has resulted in anIn~rease~ consumption.ofsoy based products. Dietary supplements containing isoflavones have also been marketed andWIth.the mt~rest regarding herbal or natural reme~ies gaining lay media prominence, consumption ofthese products maycontInue to Increase. lsofl~vones have been descnbed as both oestrogen agonists and antagonists but recent data hassuggested that the mechamsm ofoestrogen action is not the same in all cells and that classification ofcompounds asoestrogen agonists or antagonists is tissue or cell dependent.The aim ofthis ~tudy was to assess whether isoflavones from normal dietary intake were detectable in the follicular fluid ofwome~ un~ergoIngcontrolle~ovarian hyperst.imulation in a cycle ofassisted reproductive technology (ART). Benisteinand daldzem w~re m~asured In serum an? folhcular fluid by HPLC and each woman completed an isoflavone foodfrequency questIOnnaIre (developed at CltyWest IVF) to assess background dietary intake ofisoflavones.Ninety-two samples o~ follicular fluid were collected from 38 women. Genistein was identified in follicular fluid from threewo~en. b~t not all folhcles from an ovary had detectable levels. Only one woman had detectable concentrations ofgemste~nm both ~er s.erum (8.03 ng/ml) and follicular fluid (15.58 and 14.65 ng/ml from 4.5 and 3.5 ml volume folliclesrespectIVely). Daldzem was not detected in the serum or follicular fluid ofany woman.Sinc~ oestr~gen ~nd selective oestrogen receptor modulators (SERM's) such as clomiphene citrate, are concentrated withinov~n~n folhcles lsoflavones may also concentrate there. In this study, only a single subject had both serum and folliculartlUld lsoflavon~ measurements. ~ile a co~ce~tration effect was seen, no conclusions can be drawn from this piece ofdatum. SERM s are nota?le for dlff~rences m tissue selectivity, partly explained by the presence ofoestrogen receptors~~6.' ERB and ERp. WhIlst oestradIOl has equivalent affinity for both receptors, genistein is relatively more selective forA PROSPECTIVE, RANDOMISED, CONTROLLED, DOUBLE-BLIND, DOUBLE-DUMMYCOMPARISON OF RECOMBINANT AND URINARY bCG FOR INDUCING OOCYTEMATURATION AND FOLLICULAR LUTEINIZATION IN CONTROLLED OVARIANHYPERSTIMULATIONGL Driscoll, IPP Tyler, JT Hangan, PR Fisher, *MA Birdsall*, and DC KnightCity West IVF, City West House, 12 Caroline Street, Wes1mead, NSW, 2145, and *Fertility Associates, 90Greenlane Road East, Remuera, Auckland, NZA randomised, controlled, double-blind, double-dummy, phase III clinical trial was conducted in 84 women to compare theefficacy ofsubcutaneous injection of250 Jlg r-hCG (Ovidrel) to an intramuscular injection of5,000 ill u-hCG (profasi) ininducing final follicular maturation, resumption ofmeiosis in oocytes and early luteinisation in women undergoingsuperovulation with r-FSH (Gonal-F) prior to ART. The primary endpoint ofthe study was comparison ofthe total numberofoocytes retrieved per patient who received either r-hCG or u-hCG. Secondary comparisons included the number ofpatients with at least 1 oocyte retrieved; the number ofoocytes retrieved per number offollicles aspirated; the number ofmature oocytes (ie metaphase I + metaphase II); the number or normally fertilised oocytes and the number ofcleavedembryos (embryo utilisation). Endocrine profiles and ultrasonic evaluation ofendometrial thickness during treatment wasalso compared as were the incidence and severity ofadverse events, local tolerance to r-hCG or u-hCG administration andthe incidence ofmild, moderate or severe OHSS in each group. There were no statistically significant differences betweengroups for the primary endpoint (mean ± SD number or oocytes retrieved 10.75 ± 4.45 for r-hCG vs 10.28 ±..5.10 for u­hCG) and each ofthe secondary endpoint's (P

FSA FREE COMMUNICATIONS-IVF CLINICALFSA FREE COMMUNICATIONS-IVFCLINICALTHE EFFECT OF LH PRIMING PRIOR TO OVARIAN STIMULATION WITHFSH IN GnRHa TREATED WOMENPeter Benny Anoma Gooneratne and Sue HurdNew Zealand Centre for Reproductive Medicine, St Georges Medical Centre, Christchurch, NewZealand.Evidence suggests that when desensitisation is achieved with ·long acting GnRH analogues more FSH isrequired to stimulate follicle growth than when short acting analogues are used. We have hypothesised thatthe effect ofthe profound desensitisation could be reversed by LH administration prior to the use ofFSH andallow improved cycle quality.61 cycles were studied in a randomised placebo controlled trial. All received 200J.1g buserelin twice daily for14 days from day 21 ofthe cycle. They received either placebo or 150iu LH (Lhadi) for the last 5 days ofdesensitisation. Buserelin was then reduced to 200llg daily and stimulation with recombinent FSH (GonalF)was started at the indicated dosage for that woman. Oocyte recovery was preformed when at least 3 follicleswere greater than 17mm.53 cycles that fulfilled protocol criteria proceeded to embryo transfer. Comparison between LH group andplacebo group showed no significant differences in; FSH dosages 29.0 75iu amps vs 32.5 in placebo, peakestradiol 5.9 vs 5.8 nmolll, oocytes recovered 11.6 vs 11.2, or cleaving embryos 6.2 vs 4.9 (p=0.17)The embryos in the LH group were significantly more advanced at day 2 p=O.021 X2 test and significantlymore were ofsuitable quality to be frozen in the LH group, 42 (28%) vs 19 (13.2%) p=0.006.Ofthe 53 women who had embryo transfers there was no difference in the number oflive births, LH group 5+ 1frozen embryo birth, placebo group 5 live births.We concluded that LH priming does not increase the number ofoocytes recovered for IVF but in this studyappeared to improve embryo quality.Study sponsored by Serono Aust, Pty Ltd.A RETROSPECTIVE REVIEW OF LUTEAL PHASE HORMONE CONCENTRATIONS INWOMEN USING DIFFERENT FORMS OF LUTEAL SUPPORT FORASSISTEDREPRODUCTIONMazen Rawashdeh, Lija Arthur; 'Howard Smith, Peter illingworth'Department ofReproductive Medicine, Westmead Hospital.Current evidence suggests that the choice of luteal support between progesterone and hCG has limited effect on ARTconception rates provided sufficient progesterone is administered. (Akande etal, 1996). However many patientsrepoItdifficulty and discomfort in using progesterone pessaries. In this study, hormone concentrations were investigated in ol1derto check the absorption and hormonal consequences ofluteal support in a group ofwomen undergoing ART.All women were treated by standard ART long-downregulation protocol. HCG (1500 ill at three day intervals) was usedfor luteal support unless there was evidence ofovarian hyperstimulation (>10, OOOpmol/L oestradio~ >15 oocytes collected,clinical suggestion or past history of OHSS) in which case progesterone pessaries (200mg x2 per day) were used. Allwomen located geographically close (n=I44) to the Westmead Fertility Centre, had a serum sample collected between Days7 and 10 ofthe luteal phase and stored for later assay. Each sample was assayed for progesterone, oestradiol and inhibin A.According to this protocol, 45 women used progesterone pessaries (10 conceived) and 99 women used hCG (21 conceived).The progesterone concentrations in the group using progesterone pessaries ranged from 47-742 nmollL (median 81nmol/L).The progesterone concentrations in the women using hCG ranged from 38-2525nmollL (median 376nmollL). 87 (87%) ofthe women receiving hCG had progesterone concentrations in excess of the reference range (mean+ 2SD) for thephysiological menstrual cycle while 21 (48%) of the women receiving progesterone pessaries had progesteroneconcentrations in excess of the reference. The inhibin A concentrations in the group using progesterone pessaries variedfrom 4-649pg/mL (median 47pg/mL) and ranged from 16-1 I18pg/mL (median 272) in the group using hCG support. In thegroup using progesterone pessaries, 10 women (22.7%) had inhibin A concentrations below the reference range for thenormal menstrual cycle.Conclusions:All women using progesterone pessaries were found to have progesterone concentrations that are at least in thephysiological range with only a few having extreme supra-physiological concentrations. Most women using hCG lutealsupport have significantly supra-physiological progesterone concentrations. Endogenous luteal hormone output issuppressed in approximately 23% of women using progesterone pessaries with some cases exhibiting no detectableendogenous luteal function. The clinical significance ofthese endocrine variations is not clear.126127

FSA FREE C9MMUNICATIONS-IVF CLINICALFSA FREE COMMUNICATIONS-IVF CLINICALFRIDAY STARTS: A MANAGEMENT ADVANTAGEC Princehom, P.O'Donnell, J. StangerLingard Fertility Centre, Newcastle, NSW. AustraliaLingard Fertility Centre has been operating since 1984. It is situated within a 125 bed private hospital. The clinic currentlyundertakes 420 OPU's per year. For 15 years, until June 1999, OPU's were attended 7 days per week. The decision for theadministration of HCG was made when the patient reached "criteria". This was when 2 follicles reached an averagedimension of20mm. The clinic was operating under restricted access to an operating theatre to attend OPU's. All OPU'shad to be completed by 8:30 am Monday-Friday. The advantage ofthis system was that:1. Patients could start their FSH injections when it was convenient to them,2. There were rarely more than 3 OPU's on anyone day.3. Decision for the administration ofHCG was not compromised.The disadvantages were:1. The need to roster weekend staffat penalty rates2. Staffing the clinic in all disciplines ofthe clinic was inefficient3. Patients whose OPU fell on a weekend were commonly exposed to the doctor "on" for the weekend rather than theirown clinician4. Ultrasound scans were unpredictable.5. Waiting time tosee the nurse was unpredictable.In June 1999, the clinic had the opportunity to move all the OPU's out of the theatre suite and into a registered minorprocedure room. (Only 3% of our OPU's require general anaesthesia). This gave us the opportunity to attend OPU's laterthan 8:30am. We had the added problem of finding our own staffto run the minor procedure room. With these issues inmind a method to maximise midweek OPU's was sought. After assessing all our OPU's since beginning down regulationcycles (n=2318), 88% ofour OPU's occurred 11-15 days after commencing FSH injections. This information led us to startevery patient with their FSH injection on a Friday.The advantages ofthis, now in practice for 8 months has been more than we had first thought:1. Day 1 Starts. Ability to offer appointment times. All patients book in for a 0.5hr appointment time with a nurse forinstructions for injection giving with their partner or support person. Nursing manhours are allocated for this.2. The first ultrasound scan also occurs on a Friday (Day 8). These are also by 0.5hr appointment. The number ofscans ona Friday are often 10-15. This gives the advantage of employing an ultrasonographer for the whole day. Nursingmanhours are also allocated to accommodate this.3. Patient Benefit. Patients now have the ability to predict the day oftheir egg collection from the onset oftheir previouscycle. This has been helpful to both partners, especially in terms of organising time off work and accommodation forcountry clients.4. Staffing of the Minor Procedure Room has been streamlined with the greater percentage of OPU's occurring onTuesday, Wednesday and Thursday.REGNANCY FOLLOWING ICSI OF THE WRONG SPERM? - DE NOVO MUTATION ORPPUBLIC RELATIONS DISASTER?Robert Nonn~ Christine Kirby and Jeremy ThompsonReproductive Medicine Unit, The University of Ad.el~de,The Queen Elizabeth Hospital and Wakefield Chmc,Adelaide S.A. 5011. ous trust in the rocedures occurring during IVF, especially in specimen identity of ~ocytes orPatients place :::o~etra al ofthis trusfhas the potential to have enormous national and international reJ?efeusslO~. o~ .1l1espermatofzoa.. status 0 asslster. dYeprodYuctl'on In this case history,we discuss how such a potential problem of mIxed genetics. washandled.C h· t ry A couple who had previously had a female child following leSl became pregnant with fro~en e~~ryos.an~ase IS; ~ amniocentesis because of high risk of Down's syndrome. The fe~ was male "."d was .IIDScam severunderwen F \I .n a further cycle offresh leS!, a frozen pregnancy was conceIVed and amruocentesls performed: ~:~~:~:~~~~:~~:~~~;~:~:t;:Z~=o~n~~fi~y~~=C:~~~~oi;'~:::ti~~~;l~~o~?nr~·P:~gn~ly certain that the wrong sperm had been used in the laboratory..o.n the same d~y the lCSl was pe. orm: ' an0.~~~~:a::'d ~;~:~:~dt:ltsit~a~o:~fe:plaining to the clients, re~latoryVI ua ..h r anc now 36 weeks advanced ralsmg the questIon of 2 pregnancIes 0 uncertambodies and the press about a potential seriouslaboratory error.Aft kenThe decisionwas made to be open and honest with the couple and paternity testing was performed. ~~e~£~1 f~:~~;~~i~~~~~:~~U~~~~~~~:J::I~;=J:~;l::::::~:J=~~=1::~~~5=£:~organised in the event that the result was unfavourable.Result. Paternity was shown to be correct and the change was shown to be a rare de novo mutation.Conclusion This case highlights the enormous risks involved in handling gametes and the t~st pla~ed b~ patients ~?- t.?;W~~:::is:~=r;;~:::~e~~~:r;~~:~:e~. ~e~7~e ~~~~~:~n~~I~;~~~:::~t;~:~:.o r:~~~t;~~U P~;iCar;. q d ed be developed by all groups to handle such situations. In a SItuatIOn such as this, the FertilIty~:~~~~;sa~~o~:e ~~ ~~rectors Group should consider providing advice to smaller units who may not have the resources todeal with potentially disastrous situations.~rFriday starts has established minimal waiting time for clients, organised work units on specific days of the week, lessmanhours on weekends, and overall a better provision ofservice.128129

FSA FREE COMMUNICATIONS-NF CLINICALFSA SYMPOSIUM ON REPRODUCTNE SURGERYWHY DO PEOPLE WHO COMMENCE IVF TREATMENT STOPGab Kovacs, Alison Meeking, Vivien MacLachlanMonash NF, 4 th Floor, Epworth Hospital, 89 Bridge Road, RICHMOND 3121It has long been recognised that the ultimate success ofIVF depends on the number ofattempts. Previous life table analyseshave suggested that halfthe couples conceive within 3-4 cycles and 70-80% within 6. Although Medicare funds up to 6stimulated cycles, many couples leave the program before achieving a pregnancy or before using their full quota ofcycles.The aim ofthe study was to determine why couples do not continue with IVF treatment.During 1993 1629 couples registered with Monash IVF with view to having ART treatment. Ofthis group 1294 coupleshad at least one cycle oftreatment and their outcomes are presented here. These couples were asked to complete aquestionnaire, a sample ofwhich is presented here.Ofthe 1294 questionnaires sent outto couples who had IVF treatment 579 (45%) were returned. Ofthese, 120 (21%)wereno longer at the address given at the time oftheir last treatment. A total of459 (35%) completed questionnaires werereturned. A random sample of 105 questionnaires has been analysed and is being presented as a preliminary report.Reasons for not continuing with treatment included relationship break-up, switched to a different treatment (eg.donor.insemination), pursuing adoption, low chance ofpregnancy, IVF too emotionally stressful, financial constraints, concernabout long-term physical effects, switching to a different IVF clinic and completion offamily.The most common reasons for not continuing with IVF treatment were "family considered complete" (46% ofcouples),"IVF is to stressful" (18% ofcouples) and "financial constraints" (17% ofcouples). 47 couples (46%) achieved at least 1child through Monash IVF. The findings ofthis survey may have implications for areas offocus in counselling ofIVFpatients.THE PLACE OF SURGERY, SELECTION OF CASES AND RESULTS. Ossie PetruccoUniversity ofAdelaide, Women's and Children's Hospital, 72 King William Street,North Adelaide, South Australia 5006.(Abstract not submitted)CURRENT TRENDS IN ADHESION PREVENTION, RESEARCH AREAS AND RESULTSOF ANTI-ADHESION AGENTS TO DATEOssie PetruccoUniversity ofAdelaide, Women's and Children's Hospital, 72 King William Street,North Adelaide, South Australia 5006(Abstract not submitted)ENDOSCOPIC SURGERY FOR REPAIR OF PELVIC DAMAGEDavid MolloyQueensland Fertility Group PIL, Watkins Medical Centre, 225 Wickham Terrace, Brisbane, Queensland 4000(Abstract not submitted)130131

FSA SYMPOSIUM ON REPRODUCTIVE SURGERYFSA SYMPOSIUM ON REPRODUCTIVE SURGERYLAPAROSCOPY VS MICROSURGERY FOR REPAIR OF PELVIC DAMAGEAndrew Speirs eREII see the pair oftalks which David and I are giving not as a battle to decide on a winning instrument, but rather as anopportunity to remind ourselves ofthe issues. As surgeons we have a great array ofinstruments and techniques available tous to accomplish a given task. One particular task in recent focus is female sterilization reversal.I will compare the laparoscopic or microsurgical approach to this procedure under the following headings. The effectivenessofthe surgery, the risks, the cost ofsurgery, time in hospital, time offwork for the patient, and the training new surgeons.The effectiveness ofsurgery must be ofprime concern. When microsurgery was introduced it was rapidly evident that itmarked a great step forward in sterilization reversal. It was not just the magnification, but also the parallel recognition ofthe clumsiness and tissue damage caused by the old techniques. The microsurgical principals which evolved included;good vision (and lighting), fine sutures, non-reactive sutures, accuracy ofsuture placement, needle diathermy to minimizenecrosis, and protection ofthe peritoneum. All today's laparoscopic surgeons would strive to follow these same principals.However, it is easy to show the laparoscopic approach handicaps significantly.For repair ofother pelvic damage the greater precision possible with microsurgery may provide little added benefit to thepatient. Ifcuffsalpingostomy is contemplated, the briefer hospitalization and recovery time with endoscopic surgery willgenerally make this preferable to laparotomy for microsurgery.For female sterilization reversal, however, I believe that conventional microsurgery through the smallest practical incisionserves most patients best.ENDOSCOPIC TUBAL REANASTOMOSIS: RESULTS AND COSTSBruce DowningMonashIVFThis is a technique that is coming ofage and provides benefits both in cost and recovery to patients with pregnancy rates inexcess of75 per cent.has im roved in relation to laparoscopic cameras, fine microsurgical instruments along. ~th th~ developmenti()fTechn~logyk·ll . Pth·e hand·s ofmicrosurgeons More recently the use ofrobotics has shown to facllttate this surgery.knottymg SIS m .·k and Yoon have developed the technique ofaccess to the surgical field, tube preparation and surturing techniques.Ko,am h J . ·lkill· ·dA blend ofadvanced laparoscopic skills and microsurglca s s IS reqUIre .The benefit ofthis technique is shown clearly for the patient in terms ofmorbility, financial cost and for the surgeon inhospital visits, follow up and reimbursement.MICROSURGERY FOR TUBAL REANASTOMOSIS: RESULTS AND COSTSOssie PetruccoUniversity ofAdelaide,.Women's and Children's Ho~pita1, 72 King William Street,North Adelaide, South Austraha 5006(Abstract not submitted)132133

FSA SYMPOSIUM ON REPRODUCTIVE SURGERYFSA SYMPOSIUM ON REPRODUCTIVE SURGERYSTOP: NON INCISIONAL FEMALE STERILISATION UNDER MINIMAL SEDATION(FALLOPOSCOPY: DIAGNOSTICALLY USEFUL BUT TECHNICALLY CHALLENGING)Kerin John*,Plummer Helen, University ofAdelaide and STOP ResearchCentre,Ashford Medical CentreAdelaide S.A. AustraliaIntroductionThe single biggest threat to Earth's animal and plant. survival is human overpopulation in the next 50 years. Worldwidethere are 200 million human pregnancies per year and 13 million female sterilisations carried out byincisional surgery under general anaesthesia (WHO, 1998). However expensive and complex, sterilisation procedures aremostly unavailable to women who most need it in developing countries. Previous hysteroscopic methods failed to meetsafety and efficiency expectations (Kerin, Int. 1. Gynaecol. & Obstet. 51,29-39,1995).Materials and MethodsThe STOP system, developed by Conceptus Inc., San Carlos, California, USA, delivers a proprietary micro-coil device tothe tubal lumen using a small hysteroscope and refined tubal access technology. Upon release, the outer coil ofthe deviceexpands to gently wedge itselfagainst the individual tube's lumen and configuration across the utero-tubal junction. Thedevice is made ofinert materials and is theorized to have a permanent contraceptive effect due to its space filling design thatincites a local benign tissue reaction, which altersthe function and architecture ofthe tube (Valle & Cooper etal, AAGL, Nov 1999). The patient can observe the 10 to 15minute procedure on a video monitor and return home after a brief observation period. This world first Phase II ClinicalStudy began in July 1997 following approval by TQEH and Ashford Research and Ethics Committees and complies withthe US Food and Drug Administration (FDA) guidelines.ResultsPresently 87 women are happily wearing STOP devices. A hysterosalpingogram (HSG) 3 months post insertion showsbilateral proximal tubal occlusion in all cases. There has been no apparent change in menstrual cycle pattern, abnormalbleeding,pain or coital discomfort. No pregnancies have occurred to date. In 6 women,unsuspected proximal tubal stenosis or obstruction prevented placement. In 2 women asymptomatic tubal perforationoccurred and was detected at the 3 month HSG where tubal occlusion was also demonstrated. These devices were easilyremoved at subsequent clip sterilization. There were 3 technical failures and 2cases ofunilateral migration ofthe devices further along the tube detected at HSG again with tubal occlusion demonstrated.ConclusionTo .date th~ Pha~e II study data deJ?onstrates promising clinical outcomes in terms ofpregnancy prevention and very highpatlent satisfactiOn levels. Recent lmprovements in device design have markedly reduced technical difficulties andimproved stability in the tube. To date there have been no serious adverse outcomes.SELECTIVE SALPINGOGRAPHY, TUBAL CATHERISATION AND TRANSCERVICALTUBAL RE-CANALISATIONRobert WoolcottLingard Fertility Centre, Newcastle, New South Wales, 2291Selective salpingography remains a specialised investigation confined to tertiary referral institutions. This is largely due tothe perceived technical difficulty oftrans-cervical approach to the Fallopian tube associated with the inability to guide thecannula to the internal tubal ostia and the lack offamiliarity with fluoroscopic image intensification equipment Thetechnique is simple, easy to learn and inexpensive. There are implements available however such as the Fallopotorque tubalcatheterisation set which make it simple for almost every clinician with an average knowledge ofuterine anatomy andtechnical skill to perform this procedure. Most clinicians with hospital appointments have access to the necessaryfluoroscopic equipment to perform the procedure. Equipment and performance costs are only a fraction ofthat associatedwith laparoscopic or microsurgical procedures. Selective salpingography is superior to laparoscopic dye studies as a test ofFallopian tube patency.A randomised prospective controlled study oflaparoscopic dye studies versus selective salpingography as diagnostic tests offallopian tube patency, involving 278 female patients undergoing investigation ofinfertility, demonstrated selectivesalpingography to be a superior test ofFallopian tube patency. In 16/135 (11.9%) laparoscopies diagnosed proximal tubalocclusion (PTa) versus 5/138 (3.6%) at selective salpingography (p=0.018). Furthermore, when apparent bilateral proximaltubal occlusion is present as determined by both laparoscopic dye studies and hysterosalpingogram (in combination),selective salpingography will enable the demonstration ofpatency in approximately 35% oftubes.There is a potential therapeutic effect ofselective salpingography alone and the use ofthe technique permits immediatetreatment ofproximal tubal occlusion. Selective salpingography involving tubal catheterisation with or without wire guidecannulation is deemed by the American Society ofReproductive Medicine to be the management ofchoice where PTO ispresent alone without distal tubal disease. (American Fertility Society. 1993). There appears to be a significant therapeuticeffect ofselective salpingography alone, which is consistent with apparent benefit ofHSG on pregnancy rates. There isadifferential impact ofselective salpingography, tubal catheterisation and wire guide re-canalisation on pregnancy rates whenthese procedures are performed sequentially on a single occasion for the diagnosis and treatment ofapparent PTa. Crudepregnancy rates of20% for selective salpingography, 39% for tubal catheterisation and 13% for wire guide re-canalisationhave been achieved.Falloposcopy is a logical investigative extension ofabnormalities discovered by selective salpingography. Shouldmicrosurgical resection and re-anastomosis be contemplated due to an inability to achieve patency via trans-cervicalmethods then trans-fimbrial salpingoscopy is a wiser method ofendosalpingeal assessment. Apparent proximal tubalocclusion as diagnosed by selective salpingography identifies a sub-group ofpatient at high rsik ofhaving endometriosis.The association between endometriosis and PTa needs further investigation. Endometriosis is present in approximately70% ofpatients with proximal occlusion and otherwise normal tubes and PTa is present in 5-10% ofwomen withendometriosis.Selective Salpingography should be considered the 'gold standard' testofFallopian Tube patency. It has bothdiagnostic and therapeutic advantages over other methods and as such should be adopted more widely into routine clinicalpractice by general gynaecologists and infertility specialists alike.134135

FSA SY~POSIUM ON REPRODUCTIVE SURGERYFSA FREE COMMUNICATIONS-IVF ADMINISTRATION AND OUTCOMESENDOSCOPIC SURGERY VS LAPAROTOMY FOR ENDOMETRIOSIS AND FmROIDSArnaud WattiezClennont-Ferrand, France(Abstract not submitted)THE USE OF A COMPUTERIZED DATABASE VERSUS MANUAL COLLECTION OFDATA FOR THE NATIONAL PERINATAL STATISTICS UNIT AND THE REPRODUCTIVETECHNOLOGY ACCREDITATION COMMITTEE.Linda Robinson, Jennifer A. Crittenden, Peter. 1. Illingworth".Department ofReproductive Medicine~ Westmead Hospital, Westmead. N.S.W. 2145Many ofthe clinical indicators required by the National Perinatal Statistics Unit and the Reproductive TechnologyAccreditation Committee are difficult to collect. It is generally the role ofthe infertility nurse to collect and collate thisinformation which can be frustrating and time consuming.WHAT TO ATTEMPT AND WHAT NOT TO ATTEMPT ENDOSCOPICALLYArnaud WattiezClennont-Ferrand~ France(Abstract not submitted)WHERE DOES IVF FIT IN?Three new databases are presented which have been developed by Westmead Fertility Centre for the above purpose. Thesedatabases connect with existing databases containing clinical information from background or treatment cycles for eachpatient which is then transferred to the new databases, which are :Pregnancy Outcomes for all successful Fresh or Frozen Embryo Transfers (required by NPSU)Cancelled Oocyte Pickup cycles, (required by RTAC) andCancelled Frozen Embryo Transfer cycles.The advantages ofusing a computerised system are many. The program is easy to use with prompt buttons and pop upmenus. The information is easily transferred from each relevant database to another using minimal patient identification tocreate new records. Summaries ofall treatment cycles are easily obtained for statistical reports. This system is also veryadaptable with the potential to accommodate changes in the information requirements ofNPSU, RTAC, orfor auditingpurposes within the Westmead Fertility Centre.With the easy transfer ofinformation from one database to another much ofthe frequent record searching for requiredinformation is alleviated. Less time is required by the nurse using this system in compiling the information required forNPSU and RTAC than previously, decreasing the anxieties associated with deadlines; and allowing time for the moreenjoyable aspects ofinformation gathering - the pregnancy outcomes!... . . David MolloyQueensland FertIlIty Group PIL, Watkins Medical Centre, 225 Wickham Terrace, Brisbane, Queensland 4000(Abstract not submitted).......136137

FSA FREE COMMUNICATIONS-IVF ADMINISTRATION AND OUTCOMESCONTRIBUTIONS OF A NURSES DATABASE TO INFERTILITY PRACTICE.Elizabeth Pearce, Jenny Crittenden, Peter IllingworthDepartment ofReproductive Medicine, Westmead Hospital.In an infertility unit, the nursing staff have responsibility for the planning, co-ordination and individual management insome key areas. These areas include: Ovulation Induction, Intrauterine Insemination and Donor Insemination.It is critical for any practice to be able to audit outcomes, but due to the diffiIse nature ofthese treatments, it can be moredifficult to collect results in a systematic way as compared to more advanced assisted reproduction treatments. In addition itis important for nurses to be able to define the type ofdata collected and to be able to access and use the results, to modifyand improve clinical practice.W~ have therefore developed a database to study these areas. A Macintosh PC and commercially available software~l1eM~er Pro) were used. Each treatment has its own database and is linked to a couple database with demographicInfOrmatIOn on the couple. Treatment cycles are entered onto the respective database at the time of an inseminationprocedure or RCG administration.!he database is easy to use with pop up menus on many of the data entry points. Information collected on all programsInclude: date oflast menstrual period, previous pregnancy, ovulation induction details, number ofprevious cycles since lastpregnancy, maximum oestrodial, ultrasound findings, date ofLR peak! RCG administration. For the Donor Inseminationprogram the donor number is entered, type ofinsemination and cervical score. For the Intrauterine Insemination programthe semen param~ters are e~tered. All programs enter the luteal progesterone (ifperformed), pregnant (yes/no), date ofbirthand sex ofthe chIld. There IS also a comment box for any further information as required.All database~ h~~e specifically designed summaries. Summaries by patient or sperm donormean that an mdividual treatment record or semen performance can be quickly summarized. Summaries bv treatment variables such asmonth, year, age, previous pregnancy etc enable us to rapidly and easily review clinical performance and the"factors involved.In conclusion the nurses .database has proved to be user friendly and has improved time management as the data are beingentered on a .regular bas~s. ~nd are not left to well after treatment for collection. The success of the database depends onnurses assummg responslbIhty for both the data entry and collation ofstatistics. Control over the content and format ofthedatabase enables us to closely audit data entry ensuring the high quality ofthe information output.In summary this database is an effective tool for clinical review thus contributing to good patient care.FSA FREE COMMUNICATIONS-IVF ADMINISTRATION AND OUTCOMESRISING CONTRIBUTION OF ASSISTED CONCEPTION TO MULTIPLE BIRTHSIN AUSTRALIAPaul A. L. Lancaster and Tara HurstAIHW National Perinatal Statistics Unit, The University of New South Wales, Sydney, NSW 2052AustraliaTransfer ofmore than one embryo or oocyte in IVF and other types of assisted conception has resulted in highmultiple pregnancy rates. In 1988, the Fertility Society of Australia recommended that no more than threeembryos or oocytes should be transferred in each treatment cycle, aiming to reduce multiple births and avoid thehigher risks offetal death and neonatal mortality and morbidity that occur in multiple births.We analysed trends in multiple pregnancies after assisted conception in Australia and New Zealand andcompared multiple pregnancy rates for the various types of assisted conception (other IVF: fresh, other IVF:thawed, ICSI: fresh, ICSI: thawed, and GIFT) and for maternal age groups. For births occurring in Australia in1991 to 1997, we compared births after assisted conception with population data from the perinatal datacollections.Among 23,321 assisted conception pregnancies of at least 20 weeks' gestation conceived between 1979 ~d1997, there were 4,343 (18.6%) twin pregnancies, 559 (2.4%) triplet pregnancies and 33 (0.1%) pregnanCIesresulting in quadruplets or quintuplets. Twin pregnancies for all types ofassisted conception declined from morethan 20% in the late 1980s to 17.1% in 1994 and then gradually increased to 18.8% in 1997. Tripletrates fellfrom more than 3% in the 1980s to the lowest rate of 1.6% in1997. While higher order multiple pregnancies alsodeclined, 5 quadruplet pregnancies (4 after ICSI) were conceived in 1996. Women aged less than 35 years :weremore likely to have multiple pregnancies than those in older age groups. In 1991-1997, GIFT had hIghermultiple pregnancy rates than transfer offresh embryos after ICSI or other IVF. Multiple pregnancy rates werelower after transfer ofthawed embryos than after fresh embryos.Among all multiple births occurring in Australia between 1991 and 1997,11.1% oftwins,.44.6% of~riplets and55.4% ofhigher order multiple births resulted from assisted conception. The number ~ftnplet and hi~er ordermultiple births after assisted conception varied each year, but twin births rose progressIVely from 594 In 1991 to1,020 in 1997.While the policy oftransferring no more than three embryos or oocytes has been successful in reducing ~ripletsand higher order multiple births, the decline in the incidence of twin pregnancy has been less substantIal. Asclinical services for treating infertile couples by assisted conception have continued to expand during the 1990s,the actual number of multiple births has increased. This accounts for the relatively larger contribution thatassisted conception has made to all multiple births in Australia in recent years. As more women have fewerembryos transferred, multiple pregnancy rates should diminish but it is important to consider the woman's age,especially for ICSI, when deciding how many embryos will be transferre~. Furthe~ ~tudies ~re needed todetermine the impact on multiple birth rates offertility drugs that are used outSIde IVF chmcal servIces.138139

FSA F~EE COMMUNICATIONS-IVF ADMINISTRATION AND OUTCOMESTRENDS AND VARIATIONS IN PERINATAL DEATH RATES AFTER ASSISTEDCONCEPTIONTara L. Hurst and Paul A. L. LancasterAIHW National Perinatal Statistics Unit, The University ofNew South Wales, Sydney NSW 2052AustraliaP~evious s~dies have shown that fetal, neonatal and perinatal death rates after assisted conception are muchhigher than In !he general population. Contributing factors include multiple births, differences in maternal age,~he typ~ ofassIsted conception procedure, maternal medical conditions, and other underlying causes oflllfertIhty. As new proced~res such as ICSI are introduc~d, and the relative use ofother procedures changes, it isnecessary to evaluate the Impact ofthese changes on pennatal outcomes, especially perinatal mortality.We anal~sed fetal, ne?nat.al and pe~natal deaths ofat least 20 weeks' gestation that occurred among all birthsfrom aSSIsted conceptIon In AustralIa and New Zealand. We studied trends in three conception cohorts ofbirths(1979-~8,.1989-94, an~ 1995-97),.variations by type ofpr~cedure, and then compared births occurring inAustralIa III 1993-97 WIth populatIOn data for the same penod, analysing data by maternal age (20-29 years 30-34 years, and 35 years and over). 'A;m0ng 28,884 infants conceived between 1979 and 1997, there were 613 fetal deaths (21.2 per 1,000 totalbIrt~S), 397 neonatal deaths (14.0 per 1,000 live births) and 1,010 perinatal deaths (35.0 per 1,000 total births).Pennatal death rates.we~e 100~er in 1989-94 (34.6 per 1,000) and 1995-97 (30.0 per 1,000) than in 1979-88 (48.9per 1,000). The declIne III pennatal death rates was more pronounced for singleton births than for multiple births.In comparing the outcomes for the various types ofassisted conception among infants born in 1993-97 perinataldeath rates aft~r transfer offresh embryos (other IVF, 34.8 per 1,000; ICSI, 37.0 per 1,000) and GIFT (z9.0 per1,000) were ~Ighe: than the rates after transfer ofthawed embryos (other IVF, 24.9 per 1,000; ICSI, 10.3 per1,000). Mu~tIpl.e bIrths a~counted for at least halfofthe perinatal deaths in each ofthese groups, but perinataldeath rates In smgleton bIrths were also higher after transferring fresh embryos than after transferring thawedembryos. There w~s no consis~ent relationship bet~een mat~mal age and outcome for the various types ofprocedure. In 199,,-9:, the p:nnatal death ~ate ofSIngleton mfants born after assisted conception (17.9 per1:000) was almost twI~e as hIgh as ~or all smgleton births in Australia (9.0 per 1,000). However, there was littledI:fferen~e ~etween aSSIsted concep~IOn and the general population in the perinatal death rates for twins (assistedconcept~on. 44.2 per 1,000; AustralIa: 44.1 per 1,000) and other higher order multiple births (assistedconceptIOn: 104.1 per 1,000; Australia: 101.0 per 1,000).Total perinatal death rates after assisted conception have declined but multiple births still account for more thanhalfofthese deaths. Infants born after tr?Jlsfer ofthawed embryos have lower perinatal death rates than thoseborn aft~r transfer o~fresh embry.os, a~n~ut~ble to better outcomes in singleton births and fewer multiple births.Th: pennatal mortalIty ofICSI bIrths IS SImIlar to that for other IVF births after transfer offresh embryos' ICSIpennatal death rates are lower after transfer ofthawed embryos.'FSA FREE COMMUNICATIONS-IVF ADMINISTRATION AND OUTCOMESHEALTH AND DEVELOPMENTAL OUTCOMES OF IVF CHaDREN DURINGTHE FIRST FIVE YEARSG.I. Leslie, C.A. McMahon, 1. Cohen, F. Gibson, C. Tennant, andD.M. Saunders.Northern Clinical School, University ofSydney at Royal North Shore Hospital, Pacific Highway, St.Leonards, NSW, AustraliaIntroduction: This paper reports preliminary findings for health and child develop~enta~ outcome~ up to ?:eyears from a larger prospective study of health, developmental and psychosocIal adjustment III famIlIesconceiving through IVF.Aim: To compare health and developmental outcomes at one and five years for infants conceived using in-vitrofertilisation (IVF).Methods: Primigravid couples were enrolled at 30wk gestation for this prospective ~dy o~ parental adjustmentand child health and developmental outcomes of singleton and twin children conceIved usmg IVF. At one andfive years ofage children had a compreh~nsive.physical exa~nation and developm~ntal a~ses~ment and par~ntscompleted a range of psychosocial que~tlOnnatres. At ~~e t~me of assessments detatled hISto~~S were obtamedfrom the parents concerning childhood Illnesses and utIlIsatIOn of health care resources. CogmtIve developmentwas assessed using the Bayley Scales of Infant Development (second editi~n) at one year and the WechslerPreschool and Primary Scale of Intelligence (Revised) at five years. Compansons of outcomes at one and fiveyears were performed using the paired t-test.Results: Seventy-one children have been assessed at both one and five years to date. Their mean (ran~~) birthweight was 3106 (1300-4535)g and gestational age was 38.6 (33-42)~k. Mean (range) number of VISItS to ~medical practitioner was significantly less during the fifth year than dunng the ~~st year [4.~ (0-25) '! ~.3 (~-25),P = 0 000] The difference was due mainly to a decrease in the number of VISIts to medIcal specIalIs~S. III thelatter year~ [0.7 (0-10) v 1.9 (0-6); P = 0.000], although there was also a decrease in the number of VISItS to ageneral practitioner [3.5 (0-15) v 5.9 (0-24); P = 0.013].The mean (range) Bayley Mental Development Index (:MDI) at one year was 103 (82-122). At five years themean (range) verbal IQ was lOy (?7-148), th.e performance IQ was 1~2 (81-~41~ and the full scale lQ was lIl r(77-149). The MDI correl.ted Sign'fic.olly WIth both the verbal IQ (r - 0.358, P - 0.002) and the full scale IQ (= 0.381; P = 0.001), but less so with the performance IQ (r =0.309; P = 0.01).Conclusions: These preliminary data suggest a decreasing utilisation of health care resources ?y IVF ~hildrenafter the first year up to the age of school entry. Our findings also suggest that most I': chI~dren ~Ill havenormal cognitive development and should therefore not be at increased risk for learmng dIfficultIes uponcommencing their schooling.140141

FSA FR,EE COMMUNICATIONS~INFERTILITY(CLINICAL)FSA FREE COMMUNICATIONS~INFERTILITY(CLINICAL)THE INFLUENCE OF BODY MASS ON FECUNDITY OF WOMEN DURINGASSISTED REPRODUCTION TREATMENTMJDavies, JXWang, RJNormanReproductive Medicine Unit, Department ofObstetrics and Gynaecology, University ofAdelaide, The Queen Elizabeth Hospital, Woodville, SA 5011, AustraliaWomen who are excessively under or over-weight have a higher incidence ofabnormalreproductive function, such as menstrual dysfunction and anovulation, which is associated withinfertility. However, there is no conclusive published evidence demonstrating that variation inbody mas~ is associated with the capacity for conception (fecundity) in women receiving assistedreproductlOn treatment. ·This study investigated the effect ofBMI (kg/m 2 ) on fecundity inwomen undergoing assisted reproduction treatment at the IVF clinic at the Queen Elizabeth and'Yakefield Hos~itals, Ad~l~id~, South Australia. Treatment included in-vitro fertilisation (IW)!IntracytoplasmIc sperm IllJectlOn (ICSI) treatment and gamete intrafallopian transfer (GIFT)treatment (together called ART group). Data were analysed for 3586 women who received freshART from 1987 to 1998.The B~ / fecundity relation was examined using multivariable logistic regression. Thefecundlty assum.ed an inverted "U' relationship with BMI. Compared to the reference group(BMI 20-24), thInness (BMI

FSA ~REE COMMUNICATIONS-INFERTILITY (CLINICAL) .FSA FREE COMMUNICATIONS-INFERTILITY (CLINICAL~AN EFFECTIVE GROUP APPROACH TO LIFESTYLE MODIFICATION ININFERTILE WOMEN WITH POLYCYSTIC OVARIAN SYNDROME (PCOS)INTRODUCTIONJenny Marks, Kate Stem, Sandra Quinn, Ruth Simmance, John McBainMany ?ve~eig~t women who hav~ polycys!ic ovarian syndro~e are anovulatory or oligoovulatory. WhileovulatIOn m~?ctIOn agents and SurgICal ovanan cautery may stImulate ovulation, there are risks associated withthe~e modalItIes and t~ey do not address the broader health issues for women with peos.WeIght loss ~d exercI~have.been shown to res~lt in resumption ofspontaneous ovulation, as well as reducinglonge: term nsks aSSOCIated WIth PCOS and obeSIty, however many women find it extremely difficult to adhereto weIght loss programs.The Big Girls' .Group (B~) aims to provide a holistic group program ofnutritional education, regular exerciseand psy~hOSOCIal support m order.to help ~o~en to make effective lifestyle modifications which triggerresumptIOn ofspontaneous ovulatIOn or faCIlItate safe use ofovulation induction regimens.METHODSOverweight anovulatOly/oligoOvulato~ wom~n with peas are eligible to participate in a six month program,:whIch compnses three hours of edu~atlOnsemmars and aerobics each week. Clinical and biochemical evaluationbut IS conducted at t~e start ~d conclu~IOn ofthe program. Seminars include predominantly nutritional seminarsdunng .alsohcounsellmg,t e program. medIcal, phYSIOtherapy and relaxation sessions. No additional fertility treatment is providedRESULTSA total of100 ~omen commenced the pr~gram ofwhich 71 (71%) completed it, with 66(66%) women whoenrolled attendmg at least 60:0 ofthe seSSIons. 911100 (91%) women were anovulatory at the commencemen7~~ethe program, the rest ,,:ere oltgoovulatOly. The average weight, BMI and waist -hip ratio at the commencementOf~h;&:~ respectIVely were: 116 kgs, 43 and 0.84 and at the conclusion ofthe program were 109 4 40 6~ . . e average weIght loss was 5.9 kgs with a range of-3.4-44.8kgs. The weight loss BMI and'WI! . f~~provement were more substantial in women who attended more frequently, with average ~eight loss bein ra 10gs. At the end ofthe program ~5 (55%) women were OVUlating regularly. Follow up ofpatients was avaifable;~~ up ~o 12 months after co~pletIon ofthe ~rogram, during which time additional fertility treatment was utilised(32:J) u ~ed. ~fwomen stIll Wishing to conceive at the completion ofthe program (48/71 67% ofgroup) 25o ave one so to date, and 13 (52%) ofthese have been spontaneous. ' ,CONCLUSIONA grou l p approach to ~eight loss, exercise and lifestyle modification is effective therapy for overweightanovu atory women WIth pcas ,EVALUATION OF A LIFESTYLE DEHAVIOUR MODIFICATION PROGRAM FORCOUPLES EXPERIENCING INFERTILITYJulie Dalrymple, Fran Leahy, Alan TrounsonMonash IVF,·4 th Floor Epworth Hospital, 89 Bridge Rd., Richmond, Victoria, 3121Introduction: Many researchers have reported that physical, environmental ~d !ifestyle influences cannegatively affect the male and female reproductive system [1,2}. So~eART clImcs.have responded by .. 'establishing successful weight loss programs to manage obese Infertile women. A.pdot program,was e~bs~edfor couples experiencing infertility at Monash IVF to investigate the effects ofa Lifestyle BehaVIor ModificatIonProgram.Objectives: This pilot program was implemented to assess whether an estab!ished ~~er~ial '~t ~uster'program, with 'in-house' customised initiatives, would assist couples to modIfy their behaVIor which m turnwould result in pregnancies.Participants: 9 couples and 2 'unaccompanied' women (due to family commitm~nts~ enr~lled into the p~o~am.Either partner had a Body Mass Index greater than or equal to 28 and were expenencmg drfficulty conceIvlllg.Intervention: A 19 week Lifestyle Behaviour Modification Program was implemen~ed. ~e i~tial 6 weekcomponent was outsourced to an independent group whic~ endor~~ .'~~ busters Waist loss philosophy [3].The remaining program component consisted of'healthy lIfestyle Imtlabves.Measurements: Pre and post program surveys, a 3 month assessment ofphysiological data together with amenstrual and infertility history were collected.Results: 8 couples and 2 'unaccompanied' womencompleted the program. Ofthese;- 9 out of 10 (90%) offemale partners decreased body measurements8 out of8 (100%) ofmale partners decreased body measure~ents . .3 pregnancies were reported (2 ended in first trimester abortions and 1 IS on-gomg).Conclusion: Our first program demonstrated that in this small group, a commer~i~ 'Gut Buster' ~rogram couldbe successfully adapted for patients accessing ART p~ograms: w.e found that shIftmg the emp~sls .away fromdiets and exercise, towards modifying lifestyle behaVIors, mamtamed pregnancy outcomes. DeSIgnIng aprogram for couples assisted motivation through participation.References: '. 1023[1] Gray A Feldman, H.A., McKinlay, J.B., and Longcope, C., (1991), J Chn Endo Metabol. 73.1016-, .[2] McCiur~, N., McQuinn, B., McDonald, 1., Kovacs, G.T., Healy D.L., and Burger, KG., (1992), Fertd Stenl.~3~ :~g~er, G., Bolton, A., O'Neill, M., and Freeman, D., (1996), International Journal ofObesity. 20: 227-231.144145.

FSA FREE COMMUNICATIONS-INFERTILITY (CLINICAL)FSA FREE COMMUNICATIONS-INFERTILITY (CLINICAL)INSEMINATION ELICITS AN INFLAMMATORY RESPONSE IN THE HUMANCERVIX1Sarah A. Robertson, IDavid J. Sharkey, IKelton P. Tremene~ 2Kristina Gemzell DanielssonIDept. Obstetrics & Gynaecology and Reproductive Medicine Unit, University ofAdelaide, SA 5005,and 20bstetrics & Gynaecology, Karolinska Hospital, S-171 76 Stockholm, Sweden.In mice, deposition ofsemen into the female reproductive tract elicits a local inflammation-like response,characterised by pro-inflammatory cytokine synthesis and recruitment ofleukocytes into the stroma and lumen oftheuterine endometrium (1). In women, an accumulation ofneutrophils in the superficial cervical mucous afterintercourse has been described, suggesting that a comparable response may occur in this site. The aim ofthis studywas to examine the cellular events occurring in human cervical tissue following intercourse during the peri-ovulatoryperiod.Small punch biopsies were collected from the ectocervix ofsubjects randomly allocated to one ofthree groups;intercourse (n=6), abstinence (n=6) or condom-protected intercourse (n=2). Initial ('before') biopsies Were takenduring the peri-ovulatory stage ofthe menstrual cycle (LHO - LH+1) and repeat ('after') biopsies were taken 48 hlater, at 12 h following intercourse or abstinence. Tissue was frozen in OCT and processed by standardimmunohistochemical methods (1) using a panel ofthirteen monoclonal antibodies (mAbs) specific for leukocytecommon antigen (CD45) or various leukocyte lineage-specific antigens. The number ofmAb+ cells in sections werequantified by video image analysis (1). Data were analysed by students (paired) t-test.abstain intercourse abstain intercoursebeforeafter+46+11%5040+5.:t.5%beforeafteribeforeafterFig. 1 Effect ofinseminationon the number(% positivity)ofCD45+ cellsin cervicalepithelium (A)and stroma (B).THE EFFECT OF INTERCOURSE ON PREGNANCY RATES DURING ASSISTEDHUMAN REPRODUCTIONKelton P. Tremellen l , Diana Valbuena 2 , Jose Landeras 2 , Agustin Ballesteros 2 , Javier Martinei, Sergio. Mendoza 2 , Sarah A. Robertson l , Carlos Simon 2 , Robert 1. Nonnan l1Reproductive Medicine Unit, University ofAdelaide, South Australia2Instituto Valenciano de Infertilidad, Madrid, Spain.IntroductionIntercourse during an IVF cycle has the potential toimprove pregnancy rates since exposure to semen isreported to promote embryo development andimplantation in animals. Conversely, coitus induceduterine contractions or introduction ofinfection mayhave a detrimental effect. The aim ofthis study wasto determine ifintercourse during the peri-transferperiod ofan IVF cycle has any influence on earlypregnancy success.MethodsA prospective randomised control trial wasconducted in which participants were randomised toeither abstain or engage in vaginal intercourse aroundthe time ofembryo transfer. Participants includedcouples undergoing thawed embryo.transfer at theReproductive Medicine Unit, University ofAdelaide,Australia and couples undergoing fresh embryotransfer at the Instituto Valenciano de Infertilidad,Spain. The principal outcomes measured were therates ofembryo implantation (positive serum or urineohCG) and viable clinical pregnancy assessed by a6-8 week pelvic ultrasound examination.Table 1. Pregnancy outcome at 6 - 8 weeksgestation.variable / study Intercourse Abstaingroupno. cycles 242 236no. embryos 654 689biochemical 5 6singleton 25 24twins 16 12triplets 3 1quadruplets 1 0non-viable 5 5singleton1 viable /1 2 2non-viableIntercourse was found to result in an increase in the number ofCD45+ leukocytes present in both the epithelialand stromal compartments (mean ±,SD increase in 'after' compared with 'before' biopsies = 46 + 11% and 87 ±6% respectively, P

FSA'FREE COMMUNICATIONS-INFERTILITY (CLINICAL)FSA FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOCIALPREIMPLANTATION GENETIC DIAGNOSIS OF J3-THALASSEMIANicole D. Hussey, Tenielle Webb, Jenny Hall* and Zbiguniew. Rudzki*Dept ofObstetrics and Gynaecology, University ofAdelaide, Queen Elizabeth Hospital, SA5011; *Dept ofMolecular Pathology, IMVS, Frome Rd, Adelaide, 5000Introduction: Preimplantation genetic diagnosis (pGD) is a technique available to couples who are at riskof passing on an inherited disorder to their children. PGD as an alternative to conventional prenataldiagnosis, requires the couple to undergo conventional IVF to enable their 8 cell embryos to be availablefor biopsy. During the biopsy a single cell (blastomere) is removed from the embryo and analysed. Afterbiopsy the embryo remains in culture until a diagnosis is reached at which time healthy embryos are eithertransferred or frozen. Even though the risk of producing an affected child is significantly reduced,conventional prenatal diagnosis after PGD is still advised to ensure the correct diagnosis was made. PGD isavailable for many disorders including cystic fibrosis, Duchenne muscular dystrophy, Huntington'sDisease, X-linked disorders, and ~-thalassemia. Two of the main limitations of PGD is that Allele DropOut (ADO), where only one of a pair of alleles amplifies successfully, and contamination can lead to themisdiagnosis ofembryos. ~-thalassemia is a blood disorder caused by a large number ofdifferent mutationsin the ~-globin gene. Within the ~-globin gene there are also a number of polymorphisms common tovarious ethnic groups that could be used to increase the accuracy ofdiagnosis by indicating when ADO hadoccurred. Rationale: The aim ofthis study was to develop a method for use on a single cell to detect thepresence of multiple ~-thalassemia mutations as well as determine the status of that cell wjth respect tomultiple polymorphic loci.Materials and Methods: A nested polymerase chain reaction (PCR) protocol based on that of Kuliev etal., (1998) was employed to amplify two regions of the ~-globin gene. The PCR products were thensequenced using ABI Prism Bigdye Terminator Cycle Sequencing (perkin Elmer) and electrophoresed onan ABI Prism 377 Automated Sequencer. Sequencher® was used to determine the genotype of the PCRproducts.Results: Two couples un~erwent 3. cycles of PGD, in two cycles an embryo transfer was achieved (2embryos transferred each tIme) and m one cycle a pregnancy was achieved. At scan a fetal heart beat wasnot present and a miscarriage ensued. Both couples are planning to undergo further PGD cycles.Conclusions: The amplification ofgenes from a single cell has typically been optimised for the productionofshort PC~ fragments which are more difficult to sequence. We report here the successful amplificationand sequ~ncI~g of PCR products from a single cell. This method has enabled us to offer PGD for ~­thalassemIa WIth greater accuracy as ADO and some types ofcontamination can be detected.Kuliev, A., et. aI., (1998). Preimplantation diagnosis ofthaIassemias. J Assist Reprod Genet, 15,219-25.INFERTILITY AND ASSISTED REPRODUCTIVE TECHNOLOGY - COMMUNITYUNDERSTANDINGAllison M. Macdonald and Roger H CookDepartment ofPsychology, Swinburne University ofTechnology, Hawthorn, Vic, 3122This study investigated community attitudes toward the use ofa range ofassisted reproductivetechnologies (ARTs) in a number ofdifferent family configurations. It also investigated thelevel and nature ofstigmatising attitudes held by the community toward infertility. FinaIly,thestudy sought to determine the relationship between these two factors.Two hundred and one people between the ages of25 and 45 completed a survey which presentedten stories where people in different personal circumstances found themselves confronted byinvoluntary childlessness. Respondents were asked to indicate their degree ofapproval ordisapproval ofthe people undertaking the ART options available to them as well as the option ofintercountry adoption.Exploratory and confirmatory factor analysis was applied to the items in the survey and factorsrelating to approval ofARTs and stigma ofinfertility were identified. The study.found very highlevels ofapproval ofthe use of ART options in situations involving age appropriate marriedcouples that allowed for gestational and biological parenthood. The study found relatively lowlevels ofstigma ofinfertility. Structural equation modelling was used to investigate therelationship between the factors. It was found that increasing levels ofapproval ofARTs wasfound to bear a moderate relationship to increasing levels ofstigma ofinfertility.In terms offrequency data, the general order ofapproval ofARTs in the different scenarios wasfound to be IVF GIFT and ISCI followed by overseas adoption and freezing eggs. Whilstsurrogacy and d~nor gametes were least approved of, acceptance levels still indicated a lot ofsupport in the community. In terms ofthe differing personal.circu~sta~ces pr~sented to .participants in the stories, the order ofapproval was couples In thelr 30 s, marned couples Intheir 40s, single women in their 30's, the lesbian couples and gay couples. The least preferredscenario involved a single, 18 year-old woman.This study concluded that how infertility is viewed, is influenced ~y the current societal con~extin which the use ofARTs is extremely highly approved. The findlngs promote the hypotheslsthat people who highly approve the use ofreproductive technology are more likely to makenegative attributions about a person's infertility when that person is untreatable or does not seektreatment. Explanations are offered for this finding.148149

FSAFREE COMMUNICATIONS-eOUNSELLING & PSYCHOSOCIALFSA FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOCIALPREGNANT AGAIN FOLLOWING THE LOSS OF AN IVF PREGNANCY"I FEEL LIKE I HAVE BEEN PREGNANT FOREVER"Kate BoumeMelboume IVF320 Victoria Pde, East Melboume.3002. AustraliaThe experience·ofpregnancy following years ofinfertility is supposed to be a joyous time full ofhopes andexpectations ofa healthy baby and tremendous reliefto be freed from the grind ofinfertility treatment. What is it likehowever for the women who have suffered a previous IVF pregnancy loss?This paper examines the experience ofpregnancy for seven women whose previous IVF pregnancies ended due tomiscarriage, ectopic pregnancy, stillbirth or neonatal death.Each describe the fear that they will again lose their baby in a similar way. All experience heightened anxiety duringtheir pregnancy. For.one who had a previous early pregnancy loss and for another who has a healthy'child previously,this tended to ease as the pregnancy progresses. Not surprisingly for those who lost their baby later, or who have hadseveral pregnancy losses; the whole period ofpregnancy is fraught with tension anxiety and feelings ofdread.Subjects also described the feeling ofbeing pregnant yet still infertile and a sense ofisolation and difference from thoseothers who had conceived naturally. As they were pregnant, however, they felt ambivalent about seeking support fromtheir IVF clinic counsellor, as they thought they weren't entitled to this.This paper explores coping mechanisms used by the women to get through the pregnancy. It describes the particularneeds they have during their pregnancy and the implications this has for counsellors in particular and healthpractitioners in general.As six ofthe seven subjects describe emotionally detaching themselves from their baby during the pregnancy and allsubjects anticipate being more protective than other parents; it would be interesting to investigate whether this translatesinto a higher rate ofparenting difficulties following the baby's birth.THE WELFARE, OF THE CHILD - PHILOSOPHY BECOMES PRACTICELooi, K.Flinders Reproductive Medicine, Department ofObstetrics, Gynaecology and Reproductive Medicine,Flinders Medical Centre, Bedford Park. South Australia.Introduction: The South Australian Reproductive Technology Act 1988 asserts that the interests ofchildren created bythe assisted reproductive technology are of paramount importance. Recommendations developed by the Council onReproductive Technology suggested pract~c~ o~tions for translating this phi~osophical int~ntion it.tto p~actice.:, "~response to these recommendations our umt mstltuted a new protocol, scheduhng a counsellmg session with the tmltcounsellor after the routine 8 week pregnancy scan.Aim: This study sought to determine the couples' focus at this stage ofpregnancy. In p.articular, w.e wishe

F~A FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOCIALFSA FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOCIALIntroduction - RationaleTHE EFFECT OF FIRST CYCLE FOLLOW UP BY COUNSELLORSLouise O'BymeMelbourne IVF 320 Victoria Parade East Melbourne 3002Over m~ny years our counselling unit has sought ways of improving access to counselling services for peopleundergomg IVF treatment. Although counselling is available free of charge, it is believed that the well documentedstr~sses oftreatment (depression, i~~lati~n, ~~f, s?ame etc) mean that. patients are less likely to seek the counsellingass1~tance they may need. In add1tion, mfertIhty 1S often the first major life crisis faced by a couple and the act ofseekmg couns~lling assistance may be quite foreign. Therefore improving the accessibility ofthe counselling servicedeserves attentlOn.MethodThe study te~ts the hypothesis that counsellor follow up after a patient's first cycle will increase subsequentco~sellor/pat1ent contaC!. Over a three month period, a total of 133 new patients were offered follow up contact bythe1r counsellor after theIr first treatment cycle. Subsequent contact with the counselling service was tracked over 8months and compared with that ofa ~ontrol group (~= 133). ~he data was also examined to measure the patient uptakeof the f~llow up offer; the capaCIty of the servIce to delIver follow up; gender differences; patterns of highcounsellmg need and the outcome ofdifferent types ofcounsellor contact.ResultsPatients in the follow up group were more t~an twi~e .as likely to have further counselling contact (34% v 14%), in thefirst 8 ~onths oftreatment. An overwhelmmg maJonty (96%) of patients wanted follow-up after their first cycle andthe:efVIce was abl~ t.o. meet 86% ofthese requests. The vast majority ofcounsellor contact was with women or couples(93 %) and no men InItIated contact on their own.Most contact (7()oJO) ~th counsellors occurred within the first 8 months of treatment. Counsellor initiated telephonecalls or.messages ~oth mcreased subsequent contact with counsellors (31% and 47% patients made further contact), butcareful mterpretatlOn ofthese results is necessary.. ConclusionTh~ study supports !he hypothesis that offering 1st cycle follow up will increase subsequent counsellor / patient contact.PatIents are. clearly m favour offollow up and the counselling service was generally able to deliver it. While 1st cyclefollow up I~ not a total ~olution in itself it ~ould be on of a number of measures used to improve counsellor access.The paper wIll also examme other study findmgs relevant to counselling service provision.SELF-CONCEPT DISCREPANCY THEORY: A MODEL FOR UNDERSTANDINGINDIVIDUAL VARIABILITY IN REACTIONS TO INFERTILITY.Meredith 1. Krust-McKayReproductive Medicine Unit, The Queen Elizabeth Hospital and Wakefield Clinic, University ofAdelaide,Adelaide, SA, 5000.Purpose: Empirical investigation of the psychological sequelae of infertility has so far produced an inconsi,stentcharacterisation of the emotional demands of this experience. It has been suggested that such inconsistencies mayreflect individual variability in the response to infertility, which few studies have considered to date. This resea.iChinvestigated Higgins' (1987) Self-Discrepancy Theory (SDT) as a conceptual alternative for understanding individ~variability in psychological reactions to infertility. Higgins' theory proposes that people possess more thanone~nPe,~tof themselves and that discrepancies between these self-concepts results in either dejection or agitation relatedemotional discomfort. SDT is therefore proposed as a model for understanding the type and magnitude of emotionalreactions a person may experience, based upon the type and magnitude ofself-concept discrepancies.Aims: The research aimed to (a) validate the application ofSDT to the problem ofinfertility and (b) draw conclusionsabout the clinical utility ofSDT as a model for predicting the type and magnitude ofindividual emotional responses toinfertility.Method: Participants were 20 male and 26 female fertility patients, diagnosed with primary and secondary infertility.Participants completed a postal questionnaire containing self-report measures ofdepression, anxiety, psychologicalsymptomatology, and self-concept discrepancy.Results: The results ofthis research indicate that people with fertility problems do experience discrepancies betweenthe different self-concepts proposed by SDT. Correlational analyses revealed that as the magnitude ofself-conceptdiscrepancies increased levels ofemotional discomfort also increased (r's ranged from.32 to .41, p

· FSA FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOCIALFSA FREE COMMUNICATIONS-eOUNSELLING & PSYCHOSOClA:L,PGD ~THE ROLE OF THE GENETIC COUNSELLORPamela Hutchins, Kay Oke, Jan Craig and Leeanda WiltonMelbourne IVF Pty Ltd, 320 Victoria Parade, East Melbourne, 3002Pre-implantation genetic diagnosis (pGD) has been performed at Melbourne IVF for nearly twelve months. This paperreviews the counselling records for the first 50 couples interviewed for PGD. It identifies some ofthe significant issuesfor patients at the time ofPGD, and looks at the role ofthe genetic/infertility counsellor.All couples are seen by an infertility counsellor who has genetic counselling qualifications prior to PGD. Thecounsellor explains the procedure, giving the couple some understanding of the possible genetic findings andconsequences ofthe test. At the same time, couples seem to have particular issues, often related to the stage of theirfertility treatment, that have emerged from our review as themes that may be important to raise in future interviews.The study identifies two main groups of people considering PGD - those with recurrent miscarriage or implantationproblems, and those with known familial genetic problems.Analysis ofthe interview records shows a variety of stressors which may impact on the couple's ability to cope withPGD. These include previous reproductive losses; age factors; varying coping styles; length of treatment andoutcome; other children in the family; the marital relationship; and other life stresses. There is an observable level ofanxiety and distress in a significant number ofthese patients.PGD introduces further stressors for couples who wish to undertake it. It raises hopes, but more statistical evidence isneeded to provide more detailed information as to its usefulness. Genetic information about the couple may be learntthat is difficult to deal with. Decisions may have to be made about embryos with abnormalities or with inconclusiveresults.PGD is an exciting new technique, that, however, has implications for the emotional lives for the couples consideringusing it. This paper looks at the needs ofcouples and the role ofthe counsellor at this stage.A PILOT STUDY: THE EFFECT OF WRITTEN EMOTIONAL DISCLOSURE ONCONCEPTION RATES IN PATIENTS UNDERGOING IN VITRO FERTILIZATION?Tiare B. Tolks, Joi Ellis, Robyn Irwin & Keith PetrieThe Health Psychology Research Group, University ofAuckland, Faculty ofMedicine and Behavioura!lScience,Private Bag 92019, AucklandABSTRACT . eal'th P ••Written emotional disclosure is shown to have a positive influence on physical. a.nd psychological h ', .. ", 0S}~~routcomes include improvements in immune function, fewer health centre VISlt~, l~ss .self-r~?orted Illness" ,.,..~improvements in mood! psychological wellbeing. Research also suggests that expenencmg mfertllIty and undergomginfertility treatment can have adverse psychological effects on the individuals involved.A pilot study was conducted to investigate w~~ther written emot~onal disclosure could influence ~~ncep~i~n rates.~dpsychological health in IVF patients. Participants were.recrUlted fr~m a New Zealand fertllI~y chOlc, Fertl.htyAssociates. Eighty-one IVF patients were randomly aSSigned to wnte about pen)~~al traumatic events or ti~emanagement topics for half an hour on four consecutive days. Immediat~l~ after ~~tmg, mood states and so~atlcsymptoms were assessed. Before and after completing the four days ofwntmg, participants com?lete~ .the Percelv~Stress Scale and Mental Health Index. Participants were also required to evaluate aspects of their wntmg after eachwriting session. This enabled the level ofdisclosure in writing to be assessed. Pregnancy tests were recorded at the endofthe IVF cycle.Post interventions, no significant differences between groups were observed for. eit?er ofthe psychological measur.es.Immediately post writing, the emotional disclosure participants experienced slgmficantly ?lg?er levels.of ne?atlvemood and reported more symptoms. As would be expected, emotiona~ disclosur~ was SI.g?lficant1~. higher !n theexperimental condition. A trend for a higher rate of pr~gnancywas not~~ m the emotlOnal wntmg condltlOn. EVidenceof a relationship between emotional disclosure, expenence of post wnt~ng sympto~s and subsequent pregnancy" wasobserved; emotional disclosure was associated with more symptom expenence and higher pregnancy rates.These findings suggest that further investigation needs to be conducted to d~termine the signific~ce. ofthe re!ationshipbetween written emotional disclosure and conception rates. It may be that this form ofpsychological mterventlOn servesas a useful adjunct to physical infertility treatment.154155

FSA FREE COMMUNICATIONS-eOUNSELLING & PSYCHOSOCIALFSA FREE COMMUNICATIONS-eOUNSELLING & PSYCHOSOC:w.;,THE USE OF A JAR METAPHOR IN WOlUUNG WITH DEPRESSIONM.E. Evans MontroneCity West IVF, 12 Caroline Street, WESTMEAD NSW 2145It is well documented that symptoms ofdepression are exhibited by a significant number ofinfertile patients. 12 One ofthe .greate.st chanen~es. ofa ~o?nsellor working in infertility is to help with the intense sadness at the possibility ofnothavmg children whIch IS exhIbIted by these (mostly female) patients.In a cohort study at this clinic of343 patient couples who commenced Assisted Reproductive Technology treatment in1997, 11% ~~ve presented forthe~apyto the end of 1999. The majority, 50 cases, required coping and support therapy.In the remaI.mng 20 case~, approxI.mately 5% of~he total patient cohort, periods oftherapy were required ranging from75 to 545 mmutes for aSSIstance WIth overwhelmmg and uncontrollable depressive symptoms.The challenge ~or the counsellor is to a.ssist these patients without pathologizing what can be seen as a normal responset~ u~~anted ch~ldlessness. F:o~ a.famIly therapy perspective symptoms ofdepression can be framed as sadness, not todImInIsh the pam b~t to ~ut It WIthm a manageable perspective. In this context this therapist has developed the use ofaJar Metaphor to aSSIst WIth the management ofthis overwhelming and incapacitating sadness.Using 5 illustrations. ofJars dra~ by patients during the course oftherapy this presentation will illustrate the use oftheJar Metaphor to clanfy boundanes, to make concrete and externalise symptoms, and permit patient management oftheirsymptoms.From an init~al patient presentation oftmcontrollable and unmanageable sadness, it is this therapist's experience that, inthe large maJo~ty ofcases, s~ptoms can. beco~e ma?ageable.within.a period ofbrieftherapy. Whilst these patientsnumber only 5% o~the total pattent. b~dy, m th.e mten.sIty oftheIr emotIOnal pain they can be challenging to work with.The Jar Meta!!hor IS one therapeutIc mterventIOn WhICh has been found to assist in the management of the depressivesymptoms which accompany infertility.BEiNG A NON-MOTHER: THE POLITICS OF SOCIAL CONVERSATIONde Lacey.. S LSchool ofNursing, Flinders University, Adelaide, South Australia, 5042IntroductionHave you ever wondered how things turned out for women who were engaged in infertility treatment, who didn't getpregnant and then didn't return? Commonly it is assumed that they have chosen either to adopt or to live 'childtree'since these are the options construed to be available to them. This study sought to examine how IVF fail~e. andchildlessness are constructed and how women who had withdrawn from infertility treatment situated themselves wi.regard to not being a mother. This paper reports the results ofone part ofthe larger study.Materials and MethodA post-structural discourse analysis was undertaken using textual data collected from published and unpublishedsources. Data consisted oftwenty-six books that were defined as key texts in infertility self-help literature, print mediamaterials from the 1993, 1994 and 1996 Infertility Awareness Weeks held in Australia, plus the transcripts ofinterviewswith ten women recruited from the community in response to a feature article submitted to the Community Healthsection ofa local newspaper. Deconstruction techniques were employed in interviewing and in textual analysis.ResultsIn social conversation women who are not mothers must locate themselves through their response to the question 'Doyou have children?'. Whilst non-motherhood is constructed in self-help and media materials as 'childfree living', tbewomen who were interviewed in this study located themselves as childless but not childfree and indeed were careful toavoid this positioning in conversation. However the discourse ofnon-motherhood limited the locations they could takein conversation and despite their attempts to position themselves as having resolved their childlessness, they weretypically positioned as having failed.ConclusionThe discourse ofnon-motherhood locates childless women as either infertile (and actively trying to become pregnant)or 'childfree' - a situation that is founded by choice and reliant on the ability to reframe life circumstances. Womenwho have withdrawn from treatment and have not chosen a childfree lifestyle are mis-located in social conversation.11fai~~:~ne~i~'ye1aSI ~:~I~tChVOlOlgsl'c 4al Nasse Sss Nment and follow-up after in vitro fertilization: assessing the impact of2' n 1yo, 0 , ovember 1990H Slade PRet al: d A ~rospelctive, longitudinal study ofemotions and relationships in in-vitro fertilization treatmentuman epro uctIOn vo 12 no 1 pp 183-190, 1997 .156157

· FSA FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOCIALASSESSMENT OF STAFF ATTITUDES TO ISSUES IN ASSISTED REPRODUCTIVETECHNOLOGIES1 Baxter E.~, Daniels K.. 1 , G~oneratne A. 2 , Phillipson 0. 2 , Scrimshaw K. 2 and Sin 1. 2Department ofSOCIal Work, UmversIty ofCanterbury, Christchurch, NZ and 2 New Zealand Centre forReproductive Medicine, 249 Papanui Road, Christchurch, NZIntrod.uction: Adva~ces.in reproduc~ive technologies have raised many ethical issues which society is struggling todeal WIt~.. Sta:rworkIng In reproductIve technology are faced with these on a day to day basis. The XX is a relativelysmall clImc WIth .20 staff members and a tradition of.close personal contact and support for patients. We are attemptingto assess .staff attItudes ~o ART ~~ the degree to WhICh stafffeel they have input into policy making. We feel theseresults WIll be of~alue m.determmmg areas where ongoing education ofexisting staffwill be ofvalue in guiding newstaff and perhaps m definmg areas ofclinic practice which should be modified.'Material and Methods: We planned a t~ee st~ge process. Initially all staffwere canvassed to identify issues of~once:n. Two approaches were used for thIS: an mformal one-to-one discussion to explore what caused them concernsm theIr.work, and a request that they write down, in private and anonymously, things which were issues for themThes.e bsts w:r~ then collated and the issues identified split into six categories: interaction with patients and their'relatI~es, ~ohcIes, ~h~ stafft.eam, ethical issues, administrative matters and individual/personal matters. AquestlOnnaI~e contammg 54 Items was constructed and staffwere asked to select a response from: always, most ofthetIme, somet~mes, rarely ?r nev~rto each ~uestion. Staffwere guaranteed that their responses to the questionnaireswould remaIn confidentIal. ThIS was achIeved by having the responses collated by the researcher from outside the~entr~ (K.D.): For ~he seco~d stage w.e are devising specific scenarios for staffto respond to, and the third stage will beIntervIews to InVestIgate the Issues whIch have been identified for each individual. The preliminary results arepresented here.Res~lts: ~l q~estions elicited a range ofresponses. The majority ofour staffexpress concerns over the limitedc?nsIderatlOn gIv.en to the needs .an?.rights o~future children and the related issues oftreating lesbian couples and~I~gle women. HIgh :vorkloads lImItIng the tIme available to spend with patients, the difficulty ofgiving bad newsem~ affected by pat~en~ stress and the problems ofdealing with couples with a limited chance ofsuccess were all'~entlO~~d b~ the maJontl ofstaff. Most staffwo~ld also like the opportunity for more input into Centre policies. On~ ~ pOSItIve SIde, over 80X> ofstafffeel that there IS someone to give support when it is needed and all staffreportemg proud to be members ofthe Centre team and that they feel they are seen as caring profes~ionals.Conclusio~s:. The resul~s sho:v that sta~ of~ fe~iIityclinic hold a range ofopinions and in many wa s this reflects therang~dof 0dPIlllons found In SOCIety. The ImplIcatIons ofthis for the operating ofa fertility clinic need ~ be carefullyconSI ere.FSA FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOGIiM...CHARACTERISATION OF SEMEN DONORS ACCEPTING IDENTITY RELEASE ANDUSE BY LESBIANS AND SINGLE WOMENKeith Hamso!1 Michael Condon and Andrea HaywardQueensland Fertility Group, 225 Wickham Terrace, Brisbane, Q, 4000Even in States without semen donor registries there is a growing demand for donors prepared to release details oftheiridentity to progeny resulting from the use oftheir semen. Donors are also recognised to have a right to restrict the useof their semen to recipients in defined social circumstances. While traditionally donor semen has been used fOI'th~treatment ofinfertile heterosexual couples, there is a growing demand for tested and quarantined semen from lesbiansand single women. The purpose ofthis study was to characterise donors accepting their identity release and the usei!(i)ftheir semen by lesbians and single women to enable improvements in donor recruitment rates.Demographic data of27 semen donors recruited in Brisbane over the past three years were analysed according to th~jfdirections for the release oftheir identity and their willingness for their semen to be used by lesbians or single women.Overall, 15 (56%) accepted future release oftheir identity, 17 (63%) approved use by lesbians, and a slightly different17 (63%) approved use by single women. Only 6 (22%) approved of all three aspects. All had a clear personaldirection in life and all saw some satisfaction in helping people although the financial aspect was also important. Thewives ofall 8 married donors were aware that their partners were semen donors, as were the current partners of7 ofthe19 single donors. Only 2 had not told anyone else oftheir involvement with semen donation and this seemed unrelateato their desire for future release ofidentity details to recipients or progeny.Donors consenting to the future release ofdetails oftheir identity were more likely to:Be over the age of30 years with very low approval under 26 years ofageHave no qualifications or a diploma rather than a degreeBe involved in the sciences rather than the humanities or businessQuiet thinking people rather than extrovertsBe born in Australia with very low approval by migrantsBe marriedDonors consenting to the use oftheir semen by lesbians were more likely to:Be under the age of30 yearsBe involved in the humanities or business rather than the sciencesBe extroverted peopleNot be marriedDonors consenting to the use oftheir semen by single women were more likely to:Be under the age of26 yearsBe born overseasNot be marriedBe in current relationships ofrelatively short durationIt is clear from these results that semen donors approving the three aspects addressed by the study come from differentgroups within the community. This knowledge may help us to more efficiently recruit semen donors to meet theserecipient requirements.158159

FSA FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOCIALFSA FREE COMMUNICATIONS-eOUNSELLING & PSYCHOSOC~···MOTHERS OF FIVE YEAR OLD IVF CHILDREN: A COMPARATIVE STUDY OFPSYCHOLOGICAL ADJUSTMENT AND PARENTING STRESSMcMahon CAl, Gibson, F 2 , Leslie, GI 2 , Cohen l , J, Saunders, DM 3 , Tennant, CC I,.4Departments ofPsychological Medicine l , Neonatology, Obstetrics & Gynaecol ogy3, Northern ClinicalSchool, University ofSydney at Royal North Shore Hospital, St Leonards, 2065,Background~This paper is a five year follow-up report from a prospective study examining psychosocial adjustment toparenting for families who conceived their first child through IVF.Aim: The aim ofthis paper is compare IVF ~others with,comparison mo~hers on measures ofpsychological well-being~d also to compare mothers who have expenenced ongomg treatment faIlure subsequent to the birth oftheir first childWIth those who have not.Methods: 69IVF mothers and 35 matched controls were seen for assessment and interview when their first child wasfive years old. All the mothers,completed self-report questionnaires to assess current anxiety, marital satisfaction, self­~steem ~s a woma~ and parent~ng str~ss. Defensive responding (a bias towards presenting the most favourableImpressIOn ofone s psychologIcal adjustment) was also assessed, using a subscale ofthe Parenting Stress Index (Abidinet aI., 1986)Results: The only difference to emerge on comparison between the IVF and control groups was that IVF mothersrepo~ed a more ~xternallocus ofcontrol, F(1,97) = 7.34, P = .007, However, comparing the IVF group who hadexpenenced,perSIstent treatment fai!~re with those who had not, results showed that the IVF mothers with persistenttreatment faIlure reported more P,ositIvely on all psychological measures with significantly lower parenting stress(F(2,101 = 7.9~, P = .000), and. higher self-estee~ as a woman, (F (2,101) = 3.20, P = .05) than both comparison and IVFmothers who dId not have persisten.t treatment !aIlure. Interestmgly, those with persistent treatment failure reportedextrem~ly low scores ?n ~e defenSIve respondmg scale ofthe PSI relative to the other groups, F(2, 101) = 5.73, P =.004, WIth 35% reportmg m the "defensive" range.Conclusions: Consistent,with.our findings,during early parenthood, the current findings demonstrate that IVF mothersoffive year oIds ?O not dIffer 1ll psychologIcal adjustment to parenthood compared to a matched control group,~lthough t?e findmgs may to ~om~ ~xtent ~e accounted for by defensive responding. There are a number ofpossiblem~erpr~tatIOn~ofthe. ~ounter-IntUltI~e findmg ofinverse relations between psychological adjustment and treatmentfaIlure Includmg POSItIV~ self-reportIng, the stresses ofhaving more than one child and also the possibility that thoseIVF mothers who expenence repeated treatment failure are, in fact, highly motivated and very competent parentsBEHAVIOUR AT 5 YEARS OF AGE FOR'CHILDREN CONCEIVED THROUGH INVITRO FERTILISATION (IVF): A PROSPECTIVE STUDYFrances Gibson l Catherine McMaho~, Jenny Cohen 2 , Garth Leslie 1 and Douglas Saunders 3 ,Psychological Medicine 2 and Obstetrics and GynaecoloW, Northern Clini:c3.ISchool at Royal North Shore Hospital, S1. Leonards, Sydney 2065Departments ofNeonat~logyl,Introduction: Previous research has largely shown reassuring outcomes concerning IVF child development, be~Vi~~and parental adjustment. However, results have ~aried in both positi~e and less optimal directions, for e~l~lnassociation with multiple birth, mode ofconception and the use ofdIfferent m~~res (self-report, obse~atlO~) orinformants (mother, fathers, teachers). Reported here are ~ndings from a ~ongI~dmal follow-up offamlly adJ~~~1for couples who conceived through IVF. Results from earher phases ofthis project rev~aled that I~ m0t.hersj'~~:••.more anxious about their babies' well-being during pregnancy and they reported more mfant behaVlour dIfficulty ~~!first postpartum year. This study examined the subsequent behavioural adjustment ofthese children 5 years postpattut:B.Methods: Seventy primiparous couples and their singleton infants conceived through IVF, ~d a matched controlgr~~~of63 couples were recruited during pregnancy. To date, 50 IVF and 35 control group famIlIes have been seen ~orth~.~year follow-up. Both mothers' and fathers' adjustment were evaluated through a range of self-re~ort measur~ mcludl~gthe Parenting Stress Index. To evaluate child adjustment par~nts c~~pleted th~ Preschool BehavIOur ChecklIst .(screening everyday beha~iour), th~ Child Beh~viour ChecklIst (clImcal behaVlour problems), and the Sh0:t ChildhoodTemperament QuestionnaIre (emotlOnal, behaVIoural style). Tea~hers also co~pleted the Preschool Beh~vlOur.Checklist. Analysis ofvariance was used to analyse data controlhng for covanates (parental age, educatIOn, chIldgender) found.to have discrete influences on adjustment outcomes.Results: There were no significant IVF control group differences for mothers and fathers in ove:alilevel ofparentingstress due to either child (difficult) or parent (distress) factors and mean total stress ~cor~s were In the average.range.Neither was there a group difference for both mothers and fathers as a result ofmultlV~ate analyses ofbehaVlour andtemperament. However, on the checklist ofeveryday behaviour, completed by all three mformants, IVF mothers ratedtheir children with more behavioural difficulty compared to control group mothers (p = .028), whereas there were nosignificant IVF control group differences based on father or teacher ratings. In addition, 21 IVF mothers, ~m~ared to 5control mothers, responded that some oftheir child's behaviours cau~ed ~hem a p.r~blem (p = .009). DespIte thIs,level ofIVF maternal concern, notably fewer children were identified as sconng In!he chmcal problem range on the ChildBehaviour Checklist and there were no significant IVF and control group drfferences.Conclusion: Children conceived through IVF show comparable behavioural adjus~ment ~~ed on fathe: ~d teach~ratin s ofeveryday behaviour, maternal and paternal reports ofte~p~rament, ~d m the mClde~ce ~fchmcal behaVl~urprob~ms. However, IVF mothers' reports ofeveryday behaviour mdlcated a highe: level of c~lld dlfficul~ythan ~heIr 5year old peers as reported by mothers from a simila: back~ound. ,These apparent dI~er~nces In ,the behaVIOur ratmgs ofIVF children compared to controls are consistent With earlIer findmgs from the long~tudmal pr?Je~t. However, theyneed replication with the total sample, further substantiation across informants, and !ntetpre~atlon m th~ context ofotherparenting attitudes and concerns. Ifconfirmed, this pattern ofadjustment may have ImplIcations for chIld managementand guidance for IVF families.160161

FSA FREE COMMUNICATIONS-COUNSELLING & PSYCHOSOCIALFSA FREE COMMUNICATIONS-eOUNSELLING & PSYCHOSOCIA.DFIVE-YEAR FOLLOW-UP OF IVF FATHERS: PRELIMINARY FINDINGS FORPARENTAL ADJUSTMENT.Cohen, J., McMaho~ C., Gibso~ F., Tennant, C.C., SaWlders, D.M. and Leslie, G.!.Departments ofPsychological Medicine, Obstetrics and Gynaecology and Neonatology, Royal North ShoreHospital, St Leonards 2065. Australia.Introduction : The aim of this study was to investigate at five-years postpartum the psychosocial adjustment andparenting stress of fathers of children conceived by IVF with appropriately matched control fathers. Previousinvest!g~tion of IVF ~athers r~veal~d their tend~ncy to ~eport more psychosocial ~istress compared to naturallycon~lVmg fathers, dunng the third tnmester oftheIr partner s pregnancy. By four months postpartum, these differenceshad dIsappeared. At twelve months postpartum, IVFfathers reported less marital satisfaction and lower self-esteem thanthe natura1l~ ~onceiving cOI~parison fathers. Data presented here are preliminary findings from a larger longitudinal~tudy,. eXB:mlI~mg parental adjustment, parent/child relationships and child outcomes amongst IVF families. The currentmveStIgatIOn mcludes both cross-sectional comparisons and longitudinal trend analyses.Materials and Methods: To dat~, 46 .(ofthe originally recruited 65 IVF fathers) and 26 (of the original 61 controls)have .com~leted self-repo~ questIOnnaIres at five years postpartum. Psychosocial indices were based on self-reportquestI?nnaI~es. General adJ~stment ~as ~easured using the Spielberger State Anxiety Inventory, the General HealthQueStIonnaIre and the. SpanIer Dya~Ic Adjustment Scale (measuring the quality of the marital relationship). Parentingstress w~s assessed usmg the Parentmg Stress Index (S.hort Form). Groups were compared using multivariate analysesof covanance and repeated measures analyses of varIance (where the four measurement occasions were 30-weekspregnancy, four-months, 12-months and five-years postpartum).Results : At five y~ars postpartum, the~e were no. signi~cant .differences between the IVF and naturally conceivingfat?ers for state anxIety, general well-bemg or mantal satIsfactIOn. Both groups displayed profiles reflecting adequateadJu~t~ent, well within the clinical normal ranges. When the IVF and control fathers were compared across thet~ansition t~ou?h early parenthoo~ (from 30-w~ks pregnancy through to five-years postpartum), both groupsdIsplayed a sIgmficant trend to expenence more anxIety (p=.OI) and a less satisfactory marital relationship (p=.OOI) atthe five-year stage compared to the earlier assessment phases.Conclusions: IVF fathers .reported satisfa~ory levels ofpsychosocial wellbeing and marital adjustment at the five-yearpostpartum stage, appeanng to be managmg as well as other naturally conceiving fathers from a similar background.When the. pathwa~ throu~ ~arly paren~ho~d was examined more closely, IVF fathers appeared to mirror the controlfathers, In reportmg a slgmficant dech~e m marital satisfaction and heightened (though non-clinical) anxiety. Thechallenges ofearly parenthood s~em to Im~ac~ upon IVF ~d naturally conceiving fathers similarly by the five-yearstage, pOSSIbly outweIghmg any speCIfic effects related to fertility status.SEX SELECTION FOR FAMILY BALANCING- THE CASE AGAINSTHenry WellsmoreLingard Fertility Centre, Merewether NSWThe determination ofthe sex ofa fetus has been available to patients through CVS and amniocentesis since the middleseventies. This technology has enabled prospective parents to abort a fetus when there is a risk ofa serious sex link~genetic disease. Such use has been generally viewed as ethically acceptable however to use the technology to discov~the sex ofthe fetus and to abort because the fetus is the 'wrong' gender has always been controversial. Thiscontroversy was associated with the ethical concerns surrounding both abortion and sex selection.In the last 5 years the abortion concerns have been removed from the discussion because preimplantation genetic :t~gcan now discover the gender information. Such testing is carried out on a biopsied cell of the embryo and isd~.Iebefore the embryo is replaced into the woman's body. This enables doctors to replace only the embryos ofthe 'des~'gender.The development and proliferation of this technology requires that the ethics of sex selection for so called 'faIl'JJ1ly'balancing' be explored.Recently there has been media discussion about the ethics of selecting embryos on gender for 'family balancing' andmuch ofthis discussion has concluded that social sex selection is ethically acceptable. The arguments are based onapresumption in favour of reproductive freedom. They also argue that respect for autonomy requires that moralcondemnations be directed at actions that can be shown to be harmful or clearly violate the rights ofother persons.The arguments surrounding the issue of sex selection are examples concerning the rights of individuals versusconsiderations about what is in the best interests ofsociety.This paper seeks to examine some ofthe issues that may be considered as "in the best interests ofsociety". Issues suchas the apparent philosophical shift in medicine, the medical profession's obligations to society and the discriminationinherent in social gender selection.This paper then argues that there are compelling reasons why society interests should take pr~edent and concludes that.'£eX selection for social or non medical reasons should be condemned.162163

FSA MINIPOSTER SESSIONFSA MINIPOSTER SESSIONTHE GRADING OF EMBRYOS AT ONE DAY AND THREE DAYS POST­INSEMINATION ARE COMPARABLESue E Watso~ Stephen M Junk, Jeanne MYovich, John L YovichPIVET Medical Centre, Cambridge Street, Leederville, WA 6007IntroductionThe embryologists at most IVF centres use cell stageand morphology for selection of embryos at 2 or 3?a~s post-insemination. However, recent reportsmdlcate that there may be an association betweenpronuclear embryo morphology and implantationrates.(I) Th~ aim of this study was to determinewhether there IS any correlation between the grades ofthe pronucleate and grades and cell stage ofthe 3-daold embryo.yMaterials and MethodsSixty cycles ofIVF and ICSI were analysed A~otal ~f 5~1 embryos were assessed at 20 ho~rs postm.semmatlOnand graded for nucleoli and pronucleiahgnme~t and presence ofan halo around thepronucleI. There is a maximum grade of 15 all t dto th " ...c-. " oca e.e peuect pronucleate embryo, with a range of 1to 15. The pr?nucleate embryos were divided into twosub-groupsbWIth scores.of] to 9 or ]0 to ]5.The sameem ryos were exammed at 72 hours post-inseminationand assessed for cell stage and graded according to cellstage, fragmentation, clarity and regularity ofblastomeres, with 4 being the maximum score Thetotal pronucleate score was compared with th~ cellsh tage ~d embryo score ofthe 72 hour embryo usingt e chI-squared test.'15CDCD10l!enCj 5()0Figure 10I5 10 15 20IIPNScoreLr-i Figure 2'0 CD 4fI " 3o 2~1.fi 010 15 20PNScoreResultsOfthe 541 pronucleate embryos examined, 250 (46%)developed to cleaved embryos of>6 cellsComparison of pronucleate embryos t~ cleaved 72­hour ~mbryos indicated a significantly higherproportlOn of>6 cell embryos in the 10 - 15 total PNscore ~

FSA MINIPOSTER SESSIONAN ANALYSIS OF PREGNANCY LOSS FOLLOWING THE TRANSFER OFCRYOPRESERVED EMBRYOSDavid H. Edgar l ,2, Harold Boume l and John C. McBain l ,2 .I Reproductive Biology Unit, Royal Women's Hospital and 2 Melbourne IVF, Melbourne, Victoria, AustraliaIntroduction: Clinical application ofembryocryopreservation can greatly enhance the averagenumber ofimplantations achieved from cohorts ofembryos generated following ovarian stimulation forin vitro fertilisation/embryo transfer (IVF/ET) whilerestricting the incidence ofmultiple pregnancy.However, cryopreservation ofearly cleavage stageembryos can result in both complete and partial lossofthe blastomeres within individual embryos. Wehave previously reported that blastomere losssignificantly reduces the potential for developmentofcryopreserved embryos to the stage offetal heart(FH) detection following thawing and ET (1). Thepresent study was designed to investigate whethercryopreservation and/or loss ofbIastomeres isassociated with failure ofprogression from detectionofserum f3-hCG to confmnation ofa fetal heart beatand/or impaired subsequent development to livebirth.Methods: Analysis was carried out on pregnanciesresulting from the transfer ofembryos from both ourlaboratories between 1993 and 1999 and wasrestricted to day 2 (post-insemination) embryos.Only ET procedures in which all transferredembryos were equivalent in status were included inthe analysis. Embryos were cryopreserved using theslow freeze/rapid thaw, propanediol + sucrosemethod. Pregnancy was first detected by thepresence off3-hCG in serum and the implantationrate (IR) was defined as the proportion oftransferredembryos which progressed to the stage where a FHwas detected by ultrasound. Statistical significancewas assessed by X 2 •Results: The implantation rate ofall frozen/thawedembryos (All Thaw) is shown in Table 1 togetherwith the rates for intact thawed embryos only(Intact) and partially intact thawed embryos only(Partial). A control (Fresh) group ofnon-frozenembryos is also included for comparison. Thenumber ofcycles with a detectable serum f)-hCGand the number ofcycles with at least onedetectable PH are also shown. The mean numberofembryos transferred per ET was less than 2 in allgroups.Table 1IR(%) hCG+ve FH +ve (%)All Thaw 908/9406 (9.7) 1021 804 (78.7)Intact 465/387602.0) 510 411 (80.6)Partial 97/1591 (6.1) 123 91 (74.0)Fresh 958/8935 (I O. 7) 1068 814 (76.2)There were no significant differences in theproportions ofcycles with a positive f)-hCG resultwhich progressed to FH detection between any ofthe groups.The outcome following detection ofa FH is shownfor all groups in Table 2. Only pregnancies whichhad resulted in a recorded outcome were included.Table 2Total FH Liveborn LostFH (%)All Thaw 723 650 73 (10.1)Intact 367 330 37 (10.1)Partial 78 70 8 (10.3)Fresh 752 679 73 (9.7)The results demonstrate no significant differences inthe proportions ofFHs which subsequently abortedbetween any ofthe groups.In order to eliminate factors associated with multipleimplantation, we examined the rate ofloss ofsingleton FH pregnancies and also calculated themean birthweight of the babies born from each typeofembryo. Again only cycles with recordedoutcomes were included.The results are shown inTable 3.Table 3FHx1 Abort (%) Mean B.Wt. (g)All Thaw 562 60 (l0.7) 3398Intact 287 32(11.1) 3392Partial 67 6 (9.0) 3314Fresh 527 67 (12.7) 3264Again there were no significant differences in theproportions of FHs which did not result in a livebirth and the mean live birth weight was similar forbabies born from all classes ofembryos.Conclusions: The results demonstrate that, althoughpartially intact frozen/thawed early cleavage stageembryos have a significantly reduced potential fordevelopment to the FH stage, this reduction cannotbe attributed to an increased probability ofearlypregnancy loss. The reduced implantation rateassociated with blastomere loss is, therefore, likelyto be predominantly a reflection ofimpairedpreimplantation development. In addition, neithercryopreservation per se nor cryopreservationassociatedblastomere loss appears to influence theoutcome following establishment ofa clinicalpregnancy.1. Edgar et al.(2000), Hum. Reprod.,]2, 175-179SINGLE EMBRYO TRANSFER- A PILOT STUDYMark Bowman, James Catt, Mark LivingstoneSydney IVF, 4 O'Connell St Sydney NSW 2000FSA MINIPOSTER SESSleNBackground: Over the last two years at Sydney IVF, the twin pregnancy rate following routine twoembryo transfer, after IVF or IeSI, has been consistently about 300,10. Whilst many couples, especi~lywith primary infertility, welcome twins, there is a known increase in perinatal morbidity and mortahtyassociated with twin pregnancy in comparison with singleton pregnancy. Additionally, many couplesreturning to IVF for further treatment following a successful pregnancy, are anxious to avoid a twinpregnancy. These data examine the outcomes from single embryo transfer at Sydney IVF over a two yeartime period.Methods: All patients having single embryo transfer following stimulated cycles at Sydney IVF in thecalendar years 1998 and 1999 were identified using our embryology database. Two groups were assessed:one group having elective transfer of a single embryo, and a second group where only one embryo wasavailable for transfer. Single embryo transfers occurred at the cleaved stage (day 2 or 3) as well as at theblastocyst stage. These results were compared with our overall pregnancy rates obtained during 1998, forstimulated cycles.Results: The results obtained from elective single embryo transfer over 24 months were not significantlydifferent from our overall pregnancy rate for 1998:Elective single ET 1998/9 24Non elective single ET 1998/9 218Overall clin. pregnancies 1998 581# transfers # pregnancies (%)lOa (41.6)Significance (r)24 b (11.0) a&b P< 0.001200 c (34.4)a&c nsConclusions: Elective single embryo transfer is usually recommended to younger women, or to thosewho have successfully conceived previously. In this subset, the results compare favourably to tho~ewomen having two embryo transfer. Our preliminary data is also suggesting that blastocyst culture willallow a more intuitive choice in single embryo transfer. These pilot data have led to the commencementofa randomised trial comparing single (blastocyst) embryo transfer and two (cleaved) embryo transfer.166167

FSA MINIPOSTER SESSIONTHE FACTORS INFLUENCING COUPL~S TO DONATE THEIR EXCESS FROZENEMBRYOSBridget A Woodro~e, Sue E Watson, Stephen ~ Junk, Jeanne M Yovich, John L YovichPIVEY Medical Centre, 166-168 Cambndge Street, Leederville, WA, 6007.IntroductionIn an IVF. p~ogramme, the generation of embryos forcryo~reservatIon and the subsequent transfer of them isan mte~al part of successful treatment. TheRepr~ductIve Technology Council of Western Australiap~rnllts emb!y0s to be stored for a period ofthree yearsWIth. a pos~Ible extension of a further two years uponspeCIal applIcation. The dilemma that many couples faceon~e they have completed treatment is what to do withtheIr excess embryos. The two options available tocouples are to discard or donate. It is not possible toperform research onembryos in WA.Th~ aim of this paper is to review the couples whodecId~ to donate - their circumstances and reasons fordonatIon.Materials and MethodsThere were twenty couples in the study whose embryoswere frozen between 1992 - 1997. A total of 163embryos were donated (range 2 to 23 embryos for each~ouple). The a:v~rage maternal and paternal ages at thetIme of the ?ngmal treatment cycle were 36 and 38years, respectively.Prior to d?nat~on, couples had counselling during whichthe do.natIOn ~ssues were discussed and consent formsfilled. m. It IS from these forms that the reasons fordonatIOn were obtained.The factors examined were .(a) reasons for donation(b) num?er ofliving children in the donor family at timeofdonatIOn(c) use ofdonor gametes(d) pregnancy rate in the IVF attempt (includ·subsequent FET's) that generated the frozen embryos mgResultsReasons for donation are shown' m the:6 II0 owmg table:REASON(S ) FOR DONATINGNUMBEROFCOUPLESTo help another infertile couple9To give the embryos a chance oflife 5Discontinuing infertility treatment 2Completed family size4Coul'le separated4Nothing stated2* Range ofchildren in the donor family - 0 _3Average number ofliving children - 1.8* Number offamilies using donor sperm - 3/ 20Number offamilies using donor oocytes - 2/ 20* Pregnancies from. the attempt which generated thedonor embryos - 14/70NB .~wo pregnancies were biochemical and theremammg were ongoing clinical pregnanciesDiscussion!he co~ples who decide· to donate their embryos are anmter~stm~ subgroup. It is not just the stated reasons butalso lIfe CIrcumstances which explain why they donate.The ~xplanations couples give as their reasons fordonatIng can be divided into six categories (six coupleshave stated two. reasons for donation). The mostcommon reason gIven (9 / 20) is that they would like tohelp another infertile couple. Other couples (5 / 20) donot want to destroy the embryos and want to oive th"ch f I'e. " O' em aance 0 he. The reasons for donating also reflectwhy the couple. do ~ot wish to continue treatment iecompleted famIly SIze (3), ceasing all infertilitytreatment.(4) and the couple have separated (4). Twocouples dId not state their reasons for donating. It wasnoted that one of these women, had adopted out a babypre-IVF.The maj~ri~y of~ouples (13/20) had a family size oftwoor three hVIng chIldren and this was taken to demonstratea comp~eted family. Many of these were spontaneousconceptIOns follOWing a successful IVF attempt Fthe five couples with no living children, three don~~e~~fter ~~e couple separated and two decided to cease allmfertIlIty treatment.There. is a trend with the donor couples to continue thedonatIon proce~u~e. Many ofthese couples (25%) werethemselves reCIpIents of donor gametes. This grouphave a stronger understanding of donor issues and mayfeel that b~cause. ~omeone gave to them to enable themto have theIr famIlIes, they in turn would like to donate.There is a high pregnancy rate (700,/0) for the IVF attemptthat generated the e~bryos being donated. The couplesm~y the:.efor~ perceI~e these embryos more as "potentialchildren which contnbutes to their reasons for donation.!his stu~y has demonstrated that there are many issuesmfl.uencmg a couple, that lead to their decision to donatetheir excess frozen embryos.FSA MINIPOSTER SESSIONFROZEN EMBRYO PREGNANCY RATES:BENEFITS OF PRONUCLEAR STAGE FREEZINGA Catakovic, WR Edirisinghe, R Jemmott, JE Dowel, C Kirby and JA AllanThe Wesley IVF Service, Wesley Hospital, 40 Cbasely Street, Auchenflower, QueenslandINTRODUCTIONCryopreservation of embryos is a routine practice inmost in vitro fertilization (IVF) programmes and itimproves a couple's chance ofachieving a pregnancyfrom a single oocyte recovery. In this study we havecompared the pregnancy rates for different groups ofpatients in our IVF programme taking into accountwhether the patients had only fresh embryo transfercycles, only frozen embryo transfer cycles or bothfresh and frozen embryo transfer cycles.MATERIALS AND METHODSFor the study, 1342 IVF and frozen embryo transfer(FET) cycles performed between 1997 to 1999 wereanalysed. Majority of the patients consented to haveembryos cryopreserved. The embryos werecryopreserved at the pronuclear stage usingpropanediol as the cryoprotectant. The .embryotransfer with fresh embryos was performed on day 2(post egg pickup) and with frozen embryos on day 3.The frozen embryos were transferred mainly intohormone replacement cycles. The data was analysedfor the following patient groups: Group A) Freshembryo transfer (ET) cycle plus FET cycles. GroupB) FET cycles only (all embryos cryopreserved toavoid hyperstimulation) and Group C) ET cyclesonly (mainly due to 1-3 embryos generated).RESULTSThe embryo survival rate following freezingpronuclear embryos ·was 82% and the averagenumber of embryos transferred in all groups variedTable 1: The patient details, pregnancy rates and pregnancy wastageare given in the following table.Various Group A GroupB Group Cparameters ET FET ToW FETonly ETonlyNo. patients 323 268 323 73 193No. Cycles 413 502 - 154 276Age 34.0+4.4 a 34.5±4.2 c - 33.0±4.0 d 35.9±5.2 bNo. ongoing 73 51 124 20 48pregnanciesOngoing preg. 17.7% 10.2% - 13.0% 17.4%rate/cycleOngoing preg. 20.2%e 19.2% 38.3%1 27.4% 24.9%grate/patientNo. Preg. 27 34 - 14 18wastage (6.5%) (6.8%) (9.1%) (9.3%)(rate/cvcle)a vs b and e vs f, P < 0.0001; c vs d, P < 0.0002; fvs g, P < 0.002from 2 to 2.3. The details on patients, pregnancy ratesand pregnancy wastage are given in table 1. Of thetwo ET groups, the patients in Group C were olcierthan Group A. In the FET groups, the·· patieI1~;i;jjlGroup B were significantly younger than the'patfeRtsin Group A.When·the ongoing pregnancy rate was .sttId1'~~ercycle, the combined FET cycles gave signifireduced pregnancy rates (71/656, 10.8%) thaD ecombined ET cycles (121/689, 17.6%; P < 0.0004)(Figure I). However, the ongoing pregnancy ra~p~rpatient was the same for both FET (20.8%)ClJ;ld~'t(23.4%) groups (Figure 1).With the availability of additional FET cycl~s)1;ll.epregnancy rate per patient in Group A improvedsignificantly in comparison to ET only cycles (TableI). The pregnancy rate per patient achieved wit:hFHTonly cycles (Group B) was not significantly d.i:tiereJ.itto that obtained with a combination of ET andFE"fcycles (Group A). The pregnancy wastage wassimilar in all groups (Table 1).CONCLUSIONSAvailability of cryopreserved embryos can impl"()vepregnancy rates for couples by nearly 1.5 to 2 tiJ:nesmore than those achieved with only IVF-ET cycles.The patients who are at a risk of hyperstimulationappear to be younger and they can be successfullymanaged with cryopreservation of all embryos atpronuclear stage during the treatment cycle andtransferring them in subsequent PET cycles with()~taffecting their chances of achieving a pregnancy.-~ 15~~ 10~Figure 1: Ongoing ·preg. Ratesin combined FET & ET cycl~oPregrate/Cycle25i P

FSA MINIPOSTER SESSIONVAGINAL ULTRASOUND SCANNING PRATICE OFFERTILITY NURSES IN AUSTRALIAElizabeth Morris, Stephen SteigradRoyal Hospital for Women, Sydney, AustraliaFSA MINIPOSTER SESSIONWHAT IS THE PROBABILITY OF CONCEPTION FOR COUPLES ENTERING AN IVFPROGRAM?Gabor Kovacs, Susan Breheny, Vivien MacLachlan.Monash IVF, 89 Bridge Road, Richmond., Victoria 3121.IntroductionThe role of the fertility nurse is continually expanding.Vaginal ultrasound scanning represents one aspect of thisdeveloping role. At present there are no formal trainingcourses in vaginal ultrasound scanning in Australia or NewZealand.AimTo assess whether there was a significant interest inattending a formal course in vaginal ultrasound for thosenurses practicing in Australia and New ZealandResultsThe return rate was 77.4 % (41). Total number of nursesemployed was 176.Ninty-two (52%) nurses were interestedin a formal ultrasound scanning courseIn seventeen of the clinics nurses already performedvaginal ultrasounds (Chart 2 below).One ofthe most common questions asked by couples prior to commencing ART treatment is 'What arethe chances ofsuccess?'. ART results are typically expressed is in terms ofpregnancy rate per treatmeill'tcycle. However, this does not take into consideration that some couples may have only one atteml't,whereas others may try many times. In order to correct for this, the use of'life-table' analysis hasbee)i;introduced by several IVF programs. Expressing results in this manner, we believe, gives the bestestimation ofthe chance ofART outcome.The aim ofthis project was to analyze the outcome ofall couples who commenced treatment withstimulated cycles at our program between January 1992 and December 1997 and lifetable their outcomesfor treatment during the following five years. All pregnancies resulting from the thaw and transfer ofembryos frozen in theses cycles were included.MethodThe names and addresses of all known clinics were takenfrom the Fertility Nurses Register for Australia and NewZealand. Intota153 questionnaires were sent out (Chart 1).Chart 1: Questionnaire on Vaginal UltrasoundScanning Practices ofFertility NursesI.How many nurses are currently employed within theunit?2.Are follicular scans performed by nurses?3.How many ofthe nurses are currently trained inscanning?4.How many nurses are currently in training to scan?5.Who is teaching the nurses to scan?a) Other fertility nurses ~b) Ultrasonographerc) Doctord) Other, please specify6) How many ofthe nurses, within the unit would beinterested in doing a formal course in vaginal ultrasoundscanning?7) Would you travel interstate to do a course?YesNo8) Ifa course were available would you considercommencing in Vaginal Scarining?YesNoBooAny comments.no. ofnurses trained in vaginal ultrasoundII no. ofnurses training in vaginal ultrasoundiii no. ofnurse not trained in vaginal ultrasoundTeaching of vaginal ultrasound was carried out by mainlyby doctor (Chart 3 below).iiinurses taught by other fertility nurses1.1 nurses taught by ultrasonographersonurses taught by doctor• nurses where another resource was usedTwenty-seven clinics (88 nurses) said they would travelfor a course.Twenty-eight clinics (104 nurses) stated that if a coursewere available then they would consider vaginalultrasound scanning as a skill which could be peIformedby nurses.ConclusionCreation offormal course in vaginal ultrasound appears tobe ofinterest to many clinics.A detailed curriculum to the course requirements isnecessaIy before such a course can be success:ful1y created.During the six year period, a total of4,225 couples commenced their first stimulated treatment cycle. Ifapregnancy occurred in a cycle from the transfer ofeither fresh or frozen embryos, then this wasconsidered as a conception cycle and further cycles were not evaluated..outcomes 0ftreatmentbJy ruthl 1 e a e anaI YSIS. are shown In . the tablebelowCycle No. Couples Pregnant- Pregnant- Total Cumulativefresh frozen Pregnancies pregnancytransfer transfer rate1 4225 777 99 876 20.7%2 2156 393 42 435 36.7%3 1016 159 19 178 47.8%4 470 71 5 76 56.3%5 225 31 3 34 62.9%6 81 12 0 12 68.4%It should be noted that the outcomes expressed by this analysis are still somewhat an underestimation forseveral reasons. The first reason is that there are still many frozen embryos remaining from the reportedtreatment cycles which, when thawed and transferred, may add to the total numbers of pregn~cies. ~hesecond reason is that sometimes more than one pregnancy results from the one oocyte collectIon. Thisoccurs when a woman conceives from the transfer ofa fresh embryo and after birth, conceives from thetransfer offrozen embryos created in the same oocyte collection cycle.We believe that expressing results in this manner gives the best estimation ofthe chance ofART outcomefor patients prior to commencing ART.170171

FSA MINIPOS'TER SESSIONWHY DO COUPLES WHO REGISTER FOR IVF NOT PROCEED WITH TREATMENT?Ms Alison Meeking, Professor Gabor Kovacs, Ms Vivien MacLacWanAim: This study aims to identify reasons for couples not proceeding lvith IVF treatment once they have registered.Metho~: 435 couples who ~~ registered for IVF treatment but did not proceed with treatment were sent a questionnaire inthe.mail. Eac~ couple was mVIt~d to fill.out the questionnaire and return it in an enclosed, reply paid addressed envelo e~a~e~ts were informed that ~y information would be tr~ated co~dentially and that no data to be reported would idenEr;mdividuals. C~uple~ w:re notified that ill numbers were mcluded m the questionnaire to match up individuals with medicald~ta. The questt0m:rnrre mcluded a number of options why a couple may not have proceeded with treatment and patients weregIVen the opportumty to add any further reasons as necessary.Results: .105 (25%) completed questionnaires were returned and used for the analysis. The most common reasons for nocommenCIng IVF treatrn~t were "conceived naturally" by 72couples (69%), the thought ofIVF treatment as too stressful" bt14 couples (.13%) and "SWitched to a different IVF clinic (10%~. 88 couples (84%) subsequently went on to form families of ~lea~t one child ~y m~ans othe: ~ IVF. The mean length oftime that couples, who eventually conceived naturally had beentrymg to conceIve pnorto regIstenng for treatment was 3.2years. The results are summarized in the table below. 'Table.Reasons for not proceeding with IVF treatment after registeringREASON NO. PERCENTAGEConceived naturally (No need)72 69Thou.e;ht of undertakin.e; IVF was too stressful14 13Switched to a different IVF clinic10 10Chance of pre.e:nancv too low8 8Concerned about long-tenn physical side effects5 5Relationship break-up3 3Financial constraints3 3Age3 3Unhappy with IVF process2 2OtherDecided to remain child-freeSwitched to a different treatment (e.e; herbal remedies)Hysterectomy . 7 7Family problemsDefacto relationship (not eligible)No need (pregnant with honnones)Religious reasonsNB (some couples gave more than one reasonConclusion: These results indicate that man 1 .for a few years. For patients who did not acl~e~ouP es go o~ to ~on~;ve naturally even after ~ng to conceive unsuccessfullywas the neA1 most common reason for not r e ~regn~cIes natur y, the thought of ~dergolllgIVF as being too stressfulundergoing IVF treatment has decreased o~e~~~l~;t ~Ith treatment. The n~ber ofVISItS to hospital required by patientsfor excessive travel. These factors may h d d ew years and the estabhs~ment ofsatellite clinics has reduced the needave re uce some ofthe stressors preVIOusly experienced in the IVF process.FSA MINIPOSTER SESSIONTHE EFFECT OF HIGH INSEMINATION CONCENTRATIONS ON IVF OUTCOMESPam M. Matthews and David H. EdgarMelbourne IVF, 320 Victoria Pde., East Melbourne, 3002INTRODUCTIONAperennial problem in IVF is unexplained failure of oocytes to fertilize. Although the advent of ICSI has overcome tlrisproblem.in known male factor patients, there is still a group ofpatients with apparently normal semen parameters where P00for total failure offertilization occurs.METHODSOnly patients·scheduled for IVF, with greater than 6 oocytes recovered, were considered for this trial. Oocytesof each~1lteatwere randomly divided into 2 groups, to be either inseminated in the standard manner with 100,000 motilespermet'>wi1h500,000 motile sperm. Embryos were chosen for transfer on the morphological appearance withoutreferencetoJiii$.einsemination conditions. The outcome ofeach group was assessed in terms offertilization rate, 2PN and >2PN, cleavage tate,ie embryos at 2 cell stage and >2 cell stage at 48 hours, embryo utilization and implantation rate. X 2 analysis was1!isett'todetermine significance.RESULTSA total of 58 patients were included in the trial, with 323 oocytes allocated to the standard insemination group and 345 oocytesto tlle high insemination group. As indicated in table 1 there was no significant difference in any of the parameters measured.TABLE 1No FERT FERT EMB EMB EMB EMB EMB EMB *.Th1P2PN >2PN >2C 2C ARR ET FROZ USED RAWSTANDAR 323 174 21 104 54 16 47 101 148 14.0D 53.8% 6.5% 59.8% 31.0% 9.2% 27.0% 58.0% 85.9%INSEMIllGH 345 184 35 98 76 10 61 99 160 14.5INSEM 53.3% 10.1% 53.2% 41.3% 5.4% 33.1% 53.8% 86.9%X 2 NS NS NS NS NS NS NS NS NS*Only embryos where outcome could be determined were included in thisfigureOf the 58 patients, 7 had 25% or less fertilization, in 4 of these all oocytes failed to fertilize. Within the group ofoocytes thatwere inseminated with 100,000 motile spenn, 13 patients had 25% or less fertilization, whereas 9 patients in the group ofoocytes inseminated with 500,000 spenn had 25% or less fertilization. This difference is also not significant.DISCUSSIONFrom the results it would appear there is no deleterious affect on the IVF outcome when high numbers ofsperm are used forinsemination. At least in normospermic men there also appears to be no real benefit from inseminating with high numbers,with equivalent fertilization rates in the 2 groups. This is not a surprising outcome in patients with a good fertilization rate. Itis worth looking more closely at the patients with a poor fertilization rate. Ifwe consider patients that had 25% or lessfertilization in either the high or low insemination group, there is an 18% (14/76) normal fertilisation rate in the standardinsemination group and a 31% (28/90) fertilization rate in the high insemination group. This is not significant, but numbers arelow and further investigation may be worthwhile.CONCLUSIONSInseminating with high numbers of motile sperm has no deleterious affect on outcome in IVF, but in a small group ofnormospennic patients it may improve fertilization rate.172173

FSA MINIPOSTER SESSIONFERTILISATION RATE AND THE DURATION OF~SPERM-EGG lNTERACTIONA. Lakmaker, J.D. Stanger and K. Stevenson.Lingard Fertility Centre. Merewether NSW.Recent studies have suggested that decreasing the duration ofspenn-egg interaction promotes embryo quality and pregnancyafter cryostorage. The implication ofthis is that prolonged co-incubation ofgametes may have detrimental effects on embryoquality. Whether this is because ofthe accumulation ofwaste metabolites from spermatozoa or because ofnegative aspects ofzyg~te culture in glucose c?n~g media has not been explored. While glucose is required for gpenn motility, mouse embryostudies have found a negative nnpact on early embryo metabolism.Before reducing the duration ofinteraction, it seemed reasonable to confinn that fertilisation rates are not effected. Thereforein series ofpatients undertaking IVF [but not ICSI] where 6 or more ova were recovered, the duration ofgamete interactionwas equally split between 2 hours and 16 hours [paired trial]. The oocyte-coronal cell mass was removed with a largemicropipette and transferred to another well containing the same media as the 16 hour extended culture. Both groups wereexamined at 16 hours for evidence offertilisation and embryos with 2 pronuclei were pooled for further culture. Thefertilisation environment comprised RTF [Irvine] with 10mg/ml HSA [IVF Sciences] as 100ul microdrops containingspennatozoa ~der mineral oil ~VF Sci.ences]. Spermatozoa were initially collected 3 hours before insemination [group A:2hours preparation and 1 hour premcubation] and later at 4-5 hours before insemination [Group B:2 hours preparation and 2-3hours preincubation] and diluted to approximately 400,OOO/ml. All ova were collected between 7:00am and 8:30 am andadded t? the preincubated spermatozoa at 14:00 hours. Fertilisation rates [%] were compared using Students t-test -pairedcompanson.Preincubation [Hrs]* P

FSA MINIPOSTER SESSIONSPERM AUTOIMMUNITY IN A PATIENT WITH SEVERE ASTHENOSPERMIA AND SHORT SPERM TAILSGN Clarke1DY Liu z ,M Martic z ,M O'Bryan 3 and HWG Bake~1Andrology Unit,Royal Women's HospitaI,Carlton,3053;2Department ofObstetrics and Gynaecology,University ofMelbourne,Parkville,3052;3Monash Institute ofReproduction and Development,Monash MedicalCentre,Clayton,3168,Australia.INTRODUCTIONPrevious studies have defined a group ofpatients with primary infertility who have short sperm tails , dysplasia ofthe fibroussheath(FS) and axonemal anomalies(Chemes et al,1987).The syndrome shows familial incidence and often includesrespiratory pathology.We have recently identified two patients with this condition,one ofwhom was inadvertently found tohave a strong autoimmune reactivity to the sperm tail.This paper presents the results ofinitial investigations on this patient.METHODSThe immunobead test(IBT) was perfonued as described previously(Clarke et al, 1985).For indirect immunofluorescence spermwere washed twice in Tyrode solution,smeared,air-dried and fixed in 90% ethanol for 30 minutes and kept moist duringsubsequent steps.Patient serum(l/20) and conjugate(1I100)were diluted in Tyrode solution containing 5% BSA and 5%normal goat serum.The conjugate was Zymed goat anti-human IgG-FITC.SDS-PAGE was performed using Biorad redi-castminigels,transfer using a semi-dry blotter(Amrad) and western blotting detection with patient's serum diluted 1/100 and Dakorabbit anti-human IgG-HRP conjugate at 1I1500.Blocking and antibody dilution was in TBS plus 5% skimmed milk.Fig.l Electron micrograph ofthe patient's sperm(Mag-lO,OOO).FSA MINIPOSTER SESSIONOUTCOME OF OPEN BIOPSY SPERM RETRIEVAL IN SPERMATOGENICFAILURE:'PREDICTIVE FAauRE OF TESTICULAR HISTOLOGYS.Holden.*,P. Jackson*,M. Frydenberg# D.de Kretser**, R.McLachlan*&Monash IVF*,Monash Institute Reprod.& Develop**and Prince Henry's Institute Med. Res& Clayton, 3168and Cabrini Medical Centre, Malvern, Australia, 3144Open testicular biopsy fails to yield sperm for rcsr in -50% of men with non-obstructive azoospermia 1 Testicularhistology has been reported to be of limited predictive value for sperm isolation? We have advo~ated a pre-ARTdiagnostic needle biopsy as we have found it useful in predicting the likelihood of sperm recovery. This stlJdY SQught ~odetermine the relationship between the success of open testicular biopsy and the histological pattern of spermatogemcfailure with the aim ofproviding better counselling of couples considering this ART approach in the future.A retrospective review ofall men scheduled for open biopsy at Monash IVF :fro~ November 19,96 to D~epbe.r \~99was performed. Surgery was scheduled in men with non-obstructive azoospemna or extreme obgo~mna m 'rh. 01Ilviable sperm may not have been available in the ejaculate. In some m~n, a diagnostic.fine needle b~opsy (FNA) wasperformed prior to ART while fragments oftestis were also sent for histology at the time of op~n bIOpSY. The ..histological pattern was rigidly defined as: (i) Sertoli cell only (SCOS) - all tub~es must contam no genu cells:. (n)germ cell arrest (GCA) ifspermatogenesis in ~e entire biopsy ceased. at a specific.stage of genu cell developme!1t egospermatogonia or primary spermatocytes: or (111) hypospermatogenesis (hypoSG) ifany tubule showed progreSSIOn ofgenu cells to the elongated spermatid stage.75 men were scheduled for open biopsy however viable sperm was aVai~ble from fresh or ft:0z~n ejaculates in 2! men(28%) and thus 54 underwent surgery. In 23:54 men (43%) sperm were Isolated for ICSI while m 31:54 men (57~)none were found. A pre-ART FNA was performed in 22 men and histology was available ~om the ~y ofopen bIOPSYin 33 men. The relationship between these histological patterns and the recovery ofsperm IS shown m the Table.RESULTSThe patient had sperm counts of2 to 56 million/ml,motilities of0 to 1% and short sperm tails(15-30J.lm).EM showeddysplasia ofthe FS and disorganisation ofmitochondria(Fig.l).Axonemes were Usually normal,but many sperm had disruptedouter dense fibres(ODF).Most(-70%) were viable by eosin-nigrosin dye-exclusion.The patient's serum was negative byindirect IBT,but strongly positive by indirect immunofluorescence(titre--200) against sperm tails(Fig.2).Westem blottingunder reducing conditions gave a very strong band at-45Kda with whole human sperm,and cross-reactivity with purified ratsperm tails with main bands at -80Kda and 45Kda(Fig.3).Control serum from a sperm antibody negative male gave nostrong bands by western blotting.-80kDa45kDa----...HISTOLOGYIDIAGNOSTIC BIOPSYITYPE OF BIOPSYOPEN BIOPSYI Sperm found INo sperm Sperm found INo sperm found1at open biopsy Iat open biopsy IlIHypospermatogenesis 10 1 11 0Sertoli cell only 1 6 1 15~rm Cell Arrest 0 4 0 6INo histology0 0 1110LTOTAL11 11 23 31The diagnostic biopsy finding of hypoSG was associated with a 91% prospect of sperm re~ove~ at sub~equen~ openbiopsy while results for SCOS (14%) and GCA (0%) were very poor. Si~larly ~hen cons~denng the histolopcalpattern on tlle day of open biopsy, all 21 failed cycles where a histo!ogy dIagnOSIS was aVaIlable were exclUSIvely scasor GCA while hypoSG was found in 92% of the successful recovenes.IA different group of7 patient's had diagnostic needle biopsies and subs.equently unde~ent a testicular micro dissectionprocedure 3 as described by Schlegel for the recovery of spermatozoa pnor to commencmg an ART cycle.Fig.2 Immunofluorescence ofnormal humansperm tails with patient semm(Mag-500).DISCUSSION2 3 4 5Fig.3 Western blot with patient serum.Lanes 1&2,rat sperm tails;lanes 4&5,human sperm.The results ofthis study indicate that the patient had developed a strong autoimmune response to an internal component ofthesperm tail of-45Kda molecular weight.Work is proceeding towards identifying this component,but a possible canditate couldbe Spag4(Tarnasky et al,1998).This observation may lead to further understanding ofthe aetiology ofthis condition.REFERENCESChemes,HE et al1987 Fertil Steril.48,664-9.Clarke,GN et al1985 Am.J.Reprod.Immunol.Microbiol.7,1l8-23.Tamasky,H et al1998 Cytogenet.Cell Genet.81,65-7.FINE NEEDLE DIAGNOSTIC BIOPSY SPERM: RETRIEVED NO SPERM RETRIEVEDHYPOSPERMATOGENESIS 1 0SCOS 1 4GCA 0 1A further two patIents WIth Klinefelters syndrome also underwent the testicular nucro dissecti0n procedure and nosperm were recovered. . . . ..We conclude that the application of strictly defined lustoiogical c.ntena m the assessment of pre-treatment FNAprovides a clinically useful prediction ofthe likelihood of open bIOpSY spe~ recovery. The !avorabl~ outco~e ofhypoSG was confirmed bOtll prospectively with the FNA and also m th~ histolo~ at open bIOPS!. It IS. essential to fixtesticular tissue for histological assessment in Bouins or Clelands solution to achieve accurate diagnOSIS of germ celltypes. Finally the need for rigidly defined histological criteria cannot be over stressed.References: l.Tournaye et al.,(1996)Hum Reprod 11 127.2..Tournaye et al., (1997)Hum Reprod 12 803.Schlegel et al,(l998)Hum. Reprod 4 439.176 177

FSA MINIPOSTER SESSIONFSA MINIPOSTER SESSIONCompoundTrichloroethaneMethyl MethacrylateTolueneBenzenesXylenesCONTAMINANTS IN GAS LINES - BIG OR LITTLE DEAL?Catt IW and Henman M.Sydney IVF, Sydney, NSW 2000Aim. To determine the efficacy of 'in line' gas filters.Introduction. Volatile organic compounds are present in the environment at measurableconcentrations (1) which potentially can affect embryos (2). Expensive or inexpensive fixes to thiscan be purchased and installed into air conditioning units. This may improve the workenvironment but not necessarily the embryonic culture environment where compressed medicalgases are used to generate the correct mix (either totally or by mixing with ambient air). Althoughgases are supplied as "medical grade" with a guaranteed analysis there is a possibility that tracecontaminants could arise from the gases, cylinders or the tubing used to connect the gas to theincubator. We have investigated this using a simple methodology.Materials and Methods. An activated charcoal filter was placed in a gas line (a mix ofsilicontubing and oxygen 'bubble' tubing (Baxter» which fed two MINC (Cook) incubators. The filterwas a refillable "in-line" filter usually used for scrubbing ofgases used for gas chromatography(Alltech Associates, USA) and was filled with 60 g ofgranulated, activated charcoal. Aftereighteen months ofcontinual use, the charcoal was removed, mixed and sent for analysis. Theanalysis was conducted by a commercial company and the activated charcoal was desorbed intocarbon disulphide. This was analysed by gas chromatography using flame ionisation detection forhydrocarbons, alcohols/ketones and aliphatic chlorinated hydrocarbons with a detection limit of 1J-lg organic compound / g charcoal.Results.Concentration (J-lg/g charcoal)ExposedUnexposed12530610.43.537.3These figures equate to approximately 4 J-lg oforganic compounds passing through (hopefully!)the incubator per day.The efficiency oforganic carbon removal by charcoal is exceptional (>99.9% ofthe organicsmeasured) and the adsorption capacity ofthe charcoal is quoted as being 50% ofits dry weight,when tested with chloroform. Therefore under our test conditions the filter should last over 10years!ndndndndndSuccess in providing a comprehensive IVF Laboratory service to Regional NewSouth Wales.No OPU 70NoET# Preg per ET (%)# Clinical Preg (%)No FrozenET# Preg perET# Clinical PregMcArthur S.1., Cart l.W. and Henman MJ.Sydney I.V.F. Sydney. New South Wales 2000Introduction. Since it's inception, IVF has been a service readily accessible to people especially thoseliving in larger urban areas. Couples living in regional areas have had to rely on a combination oftraveland/or the provision ofa less than complete service, to access IVF .technolo~ies. At .Sydn~y IVF w~ haveprovided an IVF service, which we have now expanded further to mclude rntcromampu~atlon t:C~EJ.ues .for patients in regional centres. Medical and nursing requirements are provided by on site spectallsts. Thispaper demonstrates that Sydney IVF can offer a comprehensive IVF service to regional New South Waleswhilst maintaining a success rate equal to that achieved in our central Sydney laboratory.Results:Table 1 SIVF Regional Clinic Results v SIVF City Clinic Results.Overall AssistedConception results atSIVF Regional, 1998

FSA SYMPOSIUM ON THE MALEFSA SYMPOSIUM ON THE MALETHE ENDOCRINOLOGY OF MALE AGEINGRobert I McLachl@, Principal Research Fellow, Prince Hemy's Institute ofMedical Research, Monash MedicalCentre, Clayton, Victoria, Australia 3186Endocrine changes accomp~y. the ~ro~e~s of normal agein~ in men. The clinical challenge arises in decidingwh~~er observ.e~ changes Within an mdi~I~ual repre~ent bemgn associates ofnonna! ageing rather than significantV~tions req~~ treatment. The defimtion, detection and treatment of endocrine disorders in older men is madedifficult by our llIDlted understanding the process ofnormal ageing(ie: ~hen ~affected by concomitant illness), atypical or non-specific presentations of endocrine disorders, coeXIsting.medical.problems, the. over or ~der-reporting of symptoms, cognitive & psychosocial factors and finallythe paucIty ofeVIdence supporting a posItive cost-benefit outcome for interventions such as androgen therapy.Endo~rine refere~ce ran~es are u~u~.lly ~escribed for ~e young population while cross sectional data for the elderlyofte.n m~lu~e subjects With co-eXIsting Illness: The wIde between-individual variability of endocrine changes withagemg hmit ~e usefulness o! such cross sectional normal ranges. In addition, age-related endocrine changes varybetwe~n ~e different endocnne systems. In some cases, homeostasis is maintained ego The reduced production ofthyroXIne IS balanc~d by its diminished clearance and an unchanged TSH. In other systems acute stress reveals thereduced homeostatI~ reserves ego Glucos~ intolerance. Clear changes occur in the growth hormone axis and inadrenal adrogen section, the latter underlymg the extensive and unsubstantiated use ofDHEA therapy.I~ ~e reproductive axis, a range of subjective and objective changes occur with age. Well older men haveSlgn~C~tly 1000~er total an~ bioav~ilable se~ testoterone (T) levels than young men. There is a progressivedecl~ne m Leydig cell function (pnm~ t~stIcular failure) with advancing age. Cross sectional and longitudinalstu~es that co~trol for concurrent medicall1lness show a fall in serum T levels of 1-2% per year after the age of 30while. abo~t 5Yo of.men over age 60 years are clearly hypogonadal (defined as serum T level 90%) that occur tlrrough transformation ofthe secretory epithelial cells and result in theprogressive disruption of the glandular structure and invasion of surrounding stromal tissue. Histroicallyefforts have focused on identifying the molecular changes that occur within the epithelial cells in theinitiation and progression of malignancy. In contrast, little attention has been given to the surroundingstroma and its role in the malignant process. In nonnal development of the prostate, we have investigatedthe reciprocal interactions between the mesenchyme and epithelium leading to the maturation of theepithelium and the differentiation of stromal tissue. The regulatory processes involved in normal prostatedevelopment are re-activated in cancer and this model system has provided insight into the essential role ofthe stroma in disease progression. Recent advances have identified unique properties of stroma that areassociated with regions oftumour. The challenge for the future is to identify key mediators of the stromalchanges and how they direct epithelial cell transformation with a view to identifying future therapeutictargets.2. Cancer-bone cell interactions the development ofmetastases.In 1889 Paget provided the first data that breast cancer metastases display target organspecificity. Sincetllen, may types oftumours have been observed to preferentially metastasise to specific tissues. Forexample bone and lymph node metastases commonly occur in men with primary prostate cancer. In asmany as 75% ofmen with advanced prostate cancer metastatic lesions develop in bones ofthe spine, pelvisand femur. Paget proposed the 'Seed and Soil' theory ofmetastasis; ie. the microenvironment ofthe targetorgan provides conditions that support the development of the metastatic lesion. Our more recentunderstanding extends tlllS hypothesis and emphasises the importance of reciprocal interactions betweenprostate cancer cells and bone cells (particularly osteoblasts and osteoclasts) at the site of metastasis. Tostudy these cell-cell interactions we developed a model of invasion ofbone by prostate cancer cells thatresults in reproducible invasion and lesions with histological characteristics related to those observed inhuman bone metastases ofprostate cancer. The model provides a tool to study the mechanisms underlyinginvasion ofbone by prostate cancer cells. In addition, it provides a means to identify and analyse theeffects ofcompounds that may have tllerapuetic potential in the prevention or management ofskeletalmetastases in men Witll advanced stage prostate cancer.(Abstract not submitted)180181

FSA SYMPOSIUM ON THE MALEFSA SYMPOSIUM ON THE MALETESTICULAR CANCER: INCREASING FREQUENCY AND RECENTDEVELOPMENTSDavid M de KretserMonash Institute ofReproduction & Development,Monash University, Clayton, Victoria, AustraliaApproximately 400 new cases oftesticular cancer are diagnosed annually in Australia and the rates ofthis disorder are increasing throughout the world. The reasons for this increase remain unclear but is inkeeping with the increasing frequency ofa number ofother reproductive disorders such as hypospadiasand cryptorchidism. There are also significant geographical differences in the incidence oftesticularcancer; for instances rates are high in Denmark in comparison to nearby Finland and parallel thefrequency ofcryptorchidism and low spenn counts. As yet the reasons for these geographicaldifferences is unknown but may point to genetic or environmental factors.Testicular cancer is uncommon beyond the age of 40 years but available historical data suggest thatthere has been a shift from an older to a younger age of presentation over the past century. There is .now general acceptance that in the majority oftesticular cancers, the frankly neoplastic condition ispreceded by the presence of a precancerous condition termed carcinoma in situ (ca in situ). Ca in situis characterised by the appearance oflarge abnonnal germ cells resembling gonocytes in theseminiferous epithelium at multiple sites. Appearances suggest that these cells have the capacity foramoeboid movement along the basement membrane as they multiply and they progressively displacethe nonnal genn cells leaving tubules composed of Sertoli cells and Ca in situ cells. Follow up of menwith this histological condition suggests that 50% will develop an invasive tumour within 5 years.THE PROSTATE CANCER 'EPIDEMIC'(Current Trends In Incidence, Mortality and Treatmentfor Localised Prostate Cancer)Mark FrydenbergChairman, Department ofUrology, Monash Medical.Centre,. Centre Road, East Bentleigh, Victoria, 3165Analysis ofthe Australian and USA (SEER) databases will be presented. There has been a huge increase (> 100%)in the incidence ofprostate cancer, secondary to PSA testing. This peaked in 1992 in the USA and in 1995/6 inAustralia. This increase was most pronounced for men < 65 years ofage, who were diagnosed with modemte1ydifferentiated, localised cancers. There was a decreased incidence (56%) of distant disease and an increased use oflocal therapies (1983 - 10% RRP, 27% RT vs 1992 - 37% RRP, 32% RT). Morbidity ofbiopsy (including anxietycaused) is acceptable, cancers found on PSA screening are pathologically significant and there is significant risk ofexcess cancer mortality at 10-15 years for moderately and poorly differentiated cancers treated conservatively.Mortality has reduced 16% in USA (91-97), 17% in Australia (93-97),23% in Quebec, Canada and recently by 43%in Tyrol, Austria. Draft recommendations ofthe Australian Cancer Network working party in the treatment optionsoflocalised prostate cancer will be presented. PSA has left its mark on the vital statistics for carcinoma of theprostate. That early detection by PSA testing and treatment has reduced prostate cancer mortality is an extremelyplausible hypothesis based on current evidence (although other explanations may be possible), but cannot be provenuntil results ofRCTs are available (approximately 2008) Patients and doctors must have the autonomy to requestand order testing with information provided about the limitations.Diagnosis ofthis condition depends on adequate fixation ofthe testicular tissue since the abnormalchromatin patterns are easily visible in Bouins or Clelands fixatives but are obscured in formalin fixedtissues. Since this condition and testicular cancer is more common in association with cryptorchidismand infertility, it is essential that testicular tissues used for spenn retrieval for ICSI are examined, afterappropriate fIXation, for Ca in situ.Current views suggest that the age of onset and the presence of abnormal germ cells resemblinggonocytes raise the possibility that testicular cancers arise from gonocytes that have failed to Imdergotheir normal progression to spermatogonia. The reasons for this aberrant behaviour are now beingactively sought as are methods to simplify diagnosis. The concept that testicular cancer arises fromaberrant development of gonocytes raises the possibility that intrauterine exposure ofthe foetus tochemicals may be involved in the increasing frequency ofthis disorder. The current interest in theeffects of exposure ofthe male foetus to estrogen-like compounds may provide experimental evidenceto support such an hypothesis.182183

· FSA SYMPOSIUM ON THE MALEFSA SYMPOSIUM ON SURROGAC¥THE TREATMENT OF ERECTILE DYSFUNCTION - FROM SEX mERAPY TOGENE THERAPYBronwyn StuckeyKeogh Institute for Medical Research, QEII Med Centre, Nedlands, WAThirty years ago it was thought that most erectile dysfunction (ED) was psychogenic in nature. The elucidation ofthe mechanisms of the erectile response and identification of the disease states which affect it have led to greaterunderstanding of the organic nature ofED. In our ageing population, epidemiological studies have shown that theprevalence ofED is around 50% in men over 40, and higher in those with hypertension, diabetes and cardiovasculardisease. Today ED should be a stimulus to investigate for these underlying conditions rather than to reach for the"quick fix" .Endocrine abnormalities are an uncommon cause of ED and vascular and neurological disease constitute the mostcommon aetiologies. The maintenance of the flaccid state and the achievement of the erect state are botha delicatebalance of sympathetic and parasympathetic drive coupled with a vascular system capable ofresponse.The serendipitous discovery that intracavemosal injection (ICI) of papaverine produced smooth muscle relaxationand an erectile response led to the development ofICI therapy. ICI, using papaverine, phentolamine or prostaglandinEI, revolutionised the treatment of ED and brought effective therapy to the majority, irrespective of aetiology.More recently, serendipity has brought sildenafil, with an entirely different mode of action and requiring intactperipheral neurological mechanisms. This has brought treatment of ED into an era when the "blunderbuss"approach ofICI for all will be abandoned and treatment will be chosen according to aetiology. To this end new drugtherapies are being classified according to their site of action. Central nervous system initiators are those which actto initiate an erection e.g. the dopamine receptor agonist apomorphine. CNS modulators, such as testosterone, act tofacilitate the response to sexual stimuli. Peripheral initiators act within the penis to directly initiate an erection. TheICI vasoactive agents papaverine and PGEI fall into this category since they act independently of sexual stimulus.Phosphodiesterases, including sildenafil, are classified as peripheral modulators since they act within the penis tofacilitate a sexual stimulus but will not initiate an erection oftheir own accord. Is it possible that drug therapy willbe joined by gene therapy? The central role of K channels in potentiating the erectile stimulus throughout thecavernosa has been identified. Possible therapies have been eXlllored by the injection of genes encoding for themaxi-K channels into the corpora cavemosa.Prevention ofED should not be forgotten. Modifications of lifestyle risk factors such as smoking and treatment ofhyperlipidaemia should have benefits for erectile capacity as well as for other areas of healtll. A betterunderstanding of tlle mechanisms by which diseases such as diabetes impair erectile response may lead to effectiveprevention ofED in those disease states.CLINICAL INDICATIONS, EXCLUSIONS, TECHNIQUES AND RESULTSMartin Stafford-BellCanberra Fertility CentreFollowing the passage ofthe ACT Substitute Parents Agreement Act in November 1995 theCanberra Fertility Centre commenced a gestational carrier pregnancy program associated with itsIVF clinic in 1996. The program is a true gestational carrier pregnancy program and does notoffer traditional surrogacy. At this moment the program does not offer surrogacy arrangementswith donated gametes althoughthis is continually under review.In the ensuing years there have been 281 enquiries from around the country for the program 41ofwhich have been presented to the Ethics Committee for consideration and ofwhich 36·havebeen approved. The reason for not proceeding is usually the inability ofthe commissioningcouple to have a surrogate available and the law specifically prohibits advertising for surrogates.There are a small group ofpatients who have failed to proceed because they do not meet theselection criteria and a further small group have withdrawn because ofthe complexity oftheprocess.To date 25 commissioning mothers have undergone 31 cycles ofoocyte retrievals yielding apregnancy rate of24.5 and a live birth rate of 14.2%.A group ofpatients with previous radical hysterectomy and transposition ofthe ovaries havebeen treated. The majority ofthese patients have been shown to suffer from incipient ovarianfailure and have proceeded to egg collection at their own request despite the acknowledged highrisk offailure which has indeed eventuated. We have developed a policy ofnot treating patientswith incipient ovarian failure who have little or no chance ofsuccess. Revising our data withthat in mind would yield a pregnancy rate of22.5% and a live birth rate of 19.4% per embryotransfer procedure.Finally, although we now understand that most ED is organic in aetiology we should not lose sight ofthe significantcontribution from psychogenic factors or ignore them in therapy.184 185

FSA SYMPOSIUM ON SURROGACYFSA SYMPOSIUM ON SURROGACYCOUNSELLING OF PARTICIPANTS IN THE A.C.T SURROGACY PROGRAM.Kim RidingB.Th (Hons), B.S.W. MAASWCANBERRA FERTILITY CENTREIntroduction: Once a ~o~missioning (g.enetic) couple and surrogate (and partner ifapplicable)have me~ the set ofguIde.hnes f~r selectlo.n, they, plus any dependent children over the age of4,are reqUlr~d, by the hospItal ethIcs commIttee, to have counselling assessments. The children are?nly r~quIre~ to atten? assessment interviews with one counsellor, but all adults must attendmterviews wIth the clmic counsellor and an independent counsellor. Relevant information~erta~ning to these sessions will be presented, including an examination ofthe 13 issuesIdentlfie? by the John James Hospital Ethics Committee which must be addressed, and submittedto them m report form.P~ofessional Issues to be addressed This section will examine professional issues such as thed1fTere~c: between "asses~me.nt" and "co~nselling", identifying the different stages ofsurrogacyand theIr ~mpact, an exammatIOn ofwhat IS meant by "altruism", the role ofcoercion andvolunteenng vs duty.Issues arising fro.m "successful" surrogates: Discussion ofthe types ofissues encountered bysuccessful ~ases In the surrogacy programme. To date, 7 surrogates have given birth and handedover the chIld/ren to the genetic parents.THE FRUITS OF MY FERTILE MINDThe Patient's View: Deciding on a surrogacy arrangement is a difficult decision and thesurprise when a friend offers her assistance together with the joy attending a successful outcomeare well described. However, the lack ofappropriate legislation relating to the baby born as aresult ofsurrogacy means that with the birth ofthe baby we begin a long battle to have ourgenetic child officially recognised as ours.The Surrogate's Views: The decision to offer help as a surrogate comes after much careful andrational discussion and is followed by a considerable deal ofcounselling before embarking onthe procedure. Despite the joy deserved from helping a friend or a relative to have their ownchild by this process, surrgacy produced for me a totally unexpected series ofemotions bothduring and after pregnancy. Frank discussions ofsuch problems is required in order to helpthose seeking to provide a full and comprehensive counselling service for surrogacyprogrammes.Legal Considerations State and Federally (Colin Thomson)The laws which appear to obstenately obstruct commissioning couples in their desire to havetheir child recognised as theirs were passed a good many years ago as society's attempts torecognise other methods ofassisted reproduction and to protect the parties to these agreements.Surrogacy seeks to tum that response on its head. While that legislation is still required ego Incases ofdonor insemunation, donor eggs or embryos. There is also a political fear ofunregulated assisted reproductive technology and a certain amount ofcommunity concern. Acarefully constructed and advanced argument is required to secure legal change throughreassurance ofboth politicians and cominunity. Suggestions as to how that argument may bepresented are advanced in this presentation.186187

FSA SYMPOSIUM ON SURROGACYFSA SYMPOSIUM ON POLYCYSTIC OVARY SYNDROMEAVOIDING PSYCHOLOGICAL AND ETIDCAL PITFALLS IN GESTATIONALSURROGACYSusan CooperGestation and surrogacy while presenting obvious benefits also has many potential psychologicaland ethical problems. This paper will address some ofthese and attempt to suggest a pathwaythrough the difficulties.THE DIFFICULTY IN DEFINING PCO'SRick Legro(Abstract not submitted)LEGAL CONSIDERATIONS, STATE AND FEDERALLYColin Thomson(Abstract not submitted)ENVIRONMENTAL CAUSESJohn Eden(Abstract not submitted)GENETIC CAUSES - NEW GENESRick Legro(Abstract not submitted)LIFESTYIJE CHANGESAnne Clark(Abstract not submitted)188189

FSA SYMPOSIUM ON POLYCYSTIC OVARY SYNDROMEFSASYMPOSIUM ON POLYCYSTIC OVARY SYNDROMEINSULIN MODIFYING APPROACHESRob NormanReproductive Medicine Unit, The University ofAdelaideThe Queen Elizabeth Hospital and Wakefield ClinicADELAIDE, S.A. 5011Polycystic ovary syndrome (pcaS) is a condition in which insulin resistance is common,particularly for those patients who are anovulatory. The elevated circulating insulin aggravatesthe effect of LH and other growth factors of thecal androgen production from the ovary.Improvement of insulin resistance through diet and exercise, has been shown to be highlyeffective in restoring ovulatory function and decreasing circulating androgen levels.Pharmacological modification by drugs such as diazoxide have also been shown to reducetestosterone levels in pcas.Much attention has been focussed recently on the role of drugs such as metformin andtroglitazone as so-called "insulin sensitisers". There has been widespread use of metformin inthe United States of America to reduce the cost ofgonadotrophin ovulation induction. This talkexamines the evidence behind the efficacy of metformin and troglitazone as insulin sensitisingagents and indicates that there is still insufficient evidence to be sure that metformin and similardrugs are effective in inducing ovulation in pcas. There is only one randomised controlled trialfor which sufficient information is available to encourage the use of metformin. The vastmajority of publications on metformin are observational and are not conducted in the form of atrial. The use ofnewer agents such as rosiglitazone and chiro-inositol will be examined.OVARIAN DRILLINGGab Kovacs(Abstract not submitted)DISCUSSION AND CLOSING REMARKSRick Legro(Abstract not submitted)190191

AUTHOR INDEXAUTHOR INDEXAbdelnourG 69 Cheung T 32 Downing B 133AbdoMA 109 ChoongP 181 Driscoll G L 124, 125Adams S 34 ClarkA 189 DrummondAE 110Affandi.B 96 Clarke G N 53, 176 Duggal P S 75Aitken J 51,52,54, Cleary M 79,80 Dunlop M E 53,57Allan JA 117,118,169 Clow OL 86 Dyson M 110Anderson J C 123 Clulow J 35,37,41 Earl RA 95Anderson RA 106 Cohen GI 160 Ecroyd H 42Archer J 83 Cohen J 141,161, 162 Eden J 189Armstrong D T 75, 99, 108 Condon M 159 EdgarD 65,119,166,173Arthur L 127 CookRH 149 Edirisinghe W R 117, 118,169Attard M 119 Cooper N J 64 Ellis J 155Baird D T 106 Cooper S 188 Erwich J J H M 95BakerHWG 53, 57, 58, 12O, Cotticchio G 69 Evans Montrone M E 156176 Coveney DA 31 Farmer P J 31Ballesteros A 147 Cox S 101 Findlay J K 110Barry M 72 Cox S L 79, 80 Fisher J 181BaxterE 158 Craig J 154 Fisher M W 85Beagley KW 41,42 Cram D S 61, 89 Fisher P R 125Benny P 126 Crittenden J 137, 138 Fleming J 86BergD 36, 101 Dai Y 50 Fleming S 69Birdsall MA 125 Dalrymple J 145 Fletcher T P 38Blache D 28,29 Daniels K 158 Friese K 94Bourne H 120,166 DanielsR 114 Frydenberg M 177, 183Bourne K 150 Danielsson K G 146 Fujii S 108BowmanMC 123, 167 Davies MJ 142, 143 Gargett C E 44,92Breed W G 63,64 De Boar K 59 Garret C 53Breen T 68 De Boer KA 123 Gerdprassert 0 56Breheny S 171 De Kretser D 56, 177, 182 Gibson F 14 I, 160, 161Briese V 94 De Lacey S L 157 Gilchrist R B 108Browne R K 35, 37 DeedJ 24,25,26,27 Glazier AM 87, 104Catakovic A 118, 169 Dharmarajan AM 109 GookD 82,83Catt J 71,84, 123, 165, Diamente M 91 Gooneratne A 126. 158167, 178, 179 Dimitriades E 98 Gosden R G 105Celi P 29 DowdJE 117, 169 Gras L 122Graves J AM 43,46 Janssens PA 40 Lederman FL 44Greaves I K 46 Jemmott R 169 Legro Rick 189, 191Grkovic I 53 Jenkin G 79,80 Leigh D 123Groome N P 106 Jericho H 65 LeihyMW 30GuW 40, 100 Jeschke U 94 Leslie G 161Guerin M 24,25,26,27 Jones AR 39 Leslie G I 141, 160, }'62Hall J 148 Jones GM 112, 122 Lewis B 51 !Hall V 91 Jones R 40 Lewis I 91Hamilton H M 70, 73 Jones R C 41,42,52 LinM 54Handel sman D 180 Junk S M 38, 164, 168 LiuDY 53,57,176Hangan J T 125 KamaiR 99 Livingstone M 167Harris M S 55 KaneP 77 Looi K 151HarrisonK 68, 159, 175 Katz MG 61 Macdonald AM 149Harry J L 43 KauscheAP 122 Macklon N S 116Hartwich KM 67 KayeP 113 MacLachlan V 130, 171, 172Hayward A 159 Kerin J 134 Macmillan KL 102, 103Hearn J P 32 Kerr PJ 40, 100 Maddocks S 60, 73Hedger M P 56 Khong TY 95 Magarey G M 88Henman M J 178,179 Kilpatrick LM 45 Magoffin DA 75Holden S 177 Kirby C 129, 169 Mahony M 35,37Holland M K 40; 100 KneeR 41 Makovitzky J 94Hunter M 68 Knight D C 124, 125 Manuelpillai U 93Huntress S 38 Kola I 45 Marks J 144HurdS 126 Korfiatis N 90 Martie M 53,57,176Hurst P R 85,86 Kovacs G 130,171,172, Martin CW 106Hurst T L 139,140 191 Martin G B 28,29Hussey N D 148 Krust-McKay MJ 153 Martinez J 147Hutchins P 154 Lacham-Kaplan 0 90, 91 Mate KE 87,88Hutson J M 31 Lakmaker A 174 Matthews C 24,25,26,27Illingworth P 69,78, 127, 137, Lalic I 165 Matthews PM 173138 Lancaster PAL 139, 140 Mattiske D 81Irving-Rodgers H F II I Landeras J 147 McArthur S J 123, 179Irwin R 155 Lavranos T C 107 McBain J C 82, 144, 166Jackson P 177 Layfield S L 43 McClean RV 63Jansen R P S 123 Leahy F 145 McCully B 82192193

AUTHOR INDEXAUTHOR INDEXMcDonaldRM 34, 36 Parry L J 47 Renfree M B 30,31,43McLachlan R I 89, 177, 180 PaskAJ 43 Roberts C 59, 123McLeodB J 85 Pearce E 138 Roberts R D 34McMahon CA 141, 160, 161, Perrson J A 123 Rodger J C 55, 87162 Persson J 59 Rodgers R J 107, 111MeekingA 130, 172 Petrie K 155 Rogers P 92Meier S 74,101 Petrucco 0 131 Rogers PA W 44,93,96Mendoza S 147 Peura TT 70, 73 Roman S D 48Merry NE 119 Phillipson G 158 Rowe D B 108MkandlaA 105 Piccolo F 39 Rudzki Z 148Molinia F C 36, 87, 104 PlummerH 134 Salamonsen LA 45,98Molloy D 136 Ramsay A 97 Salha 0 105MonkM 49 Rawashdeh M 127 Saunders D M 141, 160, 161,MoorR 50 Rice G E 47 162Morris E 170 Richings N 120 Scarlett C 54Murdoch R N 52 Richter D U 94 Schmitt J 181Murray C T 71 Riding K 186 Scrimshaw K 158Myers JV 104 Riley C 82 Seamark R F 40Mylonas I 94 Riley S C 106 Sebastian LT 47Nelson A 82 Risbridger G P 181 Sebire K L 56Newcombe N 42 Ritter L J 108 Setchell B P 60Newton H 78 RobbL 98 Setiadi I 62Nieto F 65 Roberts C T 95 Sharkey D J 146Nikolic-Paterson DP 56 Robertson D M 58 Shaw G 30,31,47,81Norman R 129, 190 Robertson SA 66, 97, 99, 108, Shaw J 79, 80, 81Norman R J 72, 75, 108, 142, 146, 147 Sherrin D 68143, 147 Robinson J S 67, 95 Short R 33O'Bryan M 56,176 Robinson L 137 Sidhu K S 36, 87O'Byrne L 152 Robson S 72 Simmance R 144O'Connell A 97 Picton H M 105 Simon C 147O'Donnell P 128 Plunkett D 96 Sin I 158O'Leary S 99 Pommering M 37 Sistina Y 62OkeK 154 Princehorn C 128 Sjoblom C 66O'Neill R JW 43 Pugh PA 34 Slater H 121PadulaAM 102, 103 Quinn S 144 Smith H 127Smith J F 34,36 Tremellen K 146, 147 WebbT 148SnowM 79,80 Trijatmoko R 62 Wellsmore H 163Song B 89 TrounsonA 0 61, 89, 90, 91, Widiastuti R 62Speirs A 132 115, 122, 145 WiklandM 66Stafford Bell M 185 Tyler J P P 124, 125 Williamson R 121Stanger J D 174,128 Valbuena D 147 Wilson J D 30Steigrad S 170 Vercoe PE 29 Wilton L 121, 154Stern C J 58 Verkerk G 101 Winkler L 94Stem K 144 Vollenhoven B 93 Wolvekamp M 79, 80Stevenson K 174 Voullaire L 121 Woodroffe BA 27, 168Stuckey B 184 WadeMA 52 Woolcott R 135Sutton KA 48 Wakefield MJ 46 Wright D 59Svartman M 46 WalkerS K 67, 70, 73 WynnP 105Taggart D 46 Wallace C 48 XuD 45Tellam R L 29 Wang J X 142, 143 YaeramJ 60Tennant C 141, 160, 162 Washington D 27 Yanagimachi R 76Thompson J 101, 129 Watson S E 38, 164, 168 Yovich J L 38, 168Thomson C 188 WattiezA 136 Yovich J M 164, 168Tolks T B 155 Webb GC 73 Zaitseva M 92194195