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

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