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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