| Part I The study of polyspermy outcome and effects of cumulus cells on that in short gamete co-incubationObjective:To investigate the polyspermy outcome and effects of cumulus cells on that in short gamete co-incubation during in vitro fertilization.Methods:Retrospectively analyze data of patients undergoing short gamete co-incubation an long gamete co-incubation in vitro fertilization from January2008to December2010i our reproductive medical center.1104cycles were performed with long game co-incubation,1199cycles were performed with short gamete co-incubation.791oocyte from the41patients performed short co-incubation were divided into2groups:one grou in which the oocytes were surrounded with cumulus cells, the other one was not.T compare normal fertilization rate, polyspermy rate, good quality embryo rate and clinic pregnancy rate of all the groups. Results:1There was no significant difference between the long gamete co-incubation group and short gamete co-incubation group in patients’ age, cycles, infertility years, number of oocytes and normal fertilization rate respectively(P＞0.05).The OPN rate,1PN rate of long gamete co-incubation during IVF were increased significantly compared with the short gamete co-incubation. But the3PN rate of short gamete co-incubation was up to7.9%and more than that of long gamete co-incubation.2The oocytes utilization rate (59.3%VS54.6%), good embryo rate (70.5%VS65.2%) and embryo utilization rate (84.9%VS79.2%) of short gamete co-incubation were significantly more than that of long gamete co-incubation during in vitro fertilization. But no statistic significance were found in clinical pregnancy rates between the two groups.3In short gamete co-incubation group, polyspermy rate of naked oocytes (0.96±1.14) was significantly higher than that of oocytes surrounded with cumulus cells (0.47±0.72)(P＜0.05).But2PN rate,1PN rate,0PN rate, cleavage rate, good quality embryo rate and embryo utility rate were found no statistically difference (P＞0.05).Conclusions:1The polyspermy rate was up to7.9%in short gamete co-incubation, and significantly higher than that in long gamete co-incubation.2Short gamete co-incubation during IVF can improve the rate of oocytes utilization, improve embryo quality and the rate of embryo utilization without changing clinical pregnant outcome. Short gamete co-incubation is a safe and effective fertilization method.3Polyspermy rate can be reduced by delaying the denuding time of cumulus cells of oocytes. Part Ⅱ Average diameter of the pronucleuses of tripronuclear zygote and effect of microsurgical epronucleation on the developmental potential of corrected polyspermy embryoObjective:To measure the diameters and investigate the effect of microsurgical epronucleation on the developmental potential of corrected tripronuclear embryo.Methods:Zygotes of couples performed with short-time incubation in our reproductive medical center from January2011to February2012were collected.338tripronuclear embryos were diploidized by microsurgical removal of the pronucleus.381tripronuclear embryos with no treatment served as a negative control group.1797normal fertilization embryos were defined as a positive control group. To measure and compare the diameter of individual pronucleaus of tripronuclear zygotes. To compare the cleavage rates, formation rates of6-8-cell embryo, good quality embryo and blastocyst.Results:1Average diameter of all3PN zygotes was22.95±1.80μm.95%diameters were from22.84-23.06μm.Average diameters of the largest, medium, smallest pronucleus of3PN zygotes were24.06±1.58μm,22.97±1.38μm,21.82±1.69μm respectively. There were no statistic significance (P＜0.01)2314of338tripronuclear zygotes were successfully epronucleauted and the success rate took92.9%.3Compared with negative control group,the formation rate of2-cell-embryo in epronucleation group significantly increased(74.3%VS36.4%)(P＜0.05). But the rate of3-cell-embryo in epronucleation group was significantly lower (25.7%VS63.6%).Compared with positive control group, the formation rate of2-cell-embryo in epronucleation group was significantly lower (74.3%VS86.0%)(P＜0.01). But the rate of3-cell-embryo in epronucleation group was dramatically higher (25.7%VS14.0%). Moreover,the formation rate of2-cell-embryo in negative control group (36.4%)was significantly lower than that in positive control group (86.0%)(P<0.01), however that rate of3-cell-embryo was63.6%which was dramatically higher comparing with positive control group (14.0%).4The formation rate of6-8-cell embryo in negative control group (88.1%) was similar to that in epronucleation group(87.6%)(P＞0.05).But it was significantly lower in positive control group (65.5%)(P＜0.01). The formation rate of blastocyst in positive control group(58.7%) was the highest (P＜0.01),while that in negative control group(58.7%)was significantly higher than in epronucleation group(21.5%)and negative control group(5.6%)(P＜0.01). The formation rates of D5blastocyst were8.9%in epronucleation group and1.9%in negative control group. The rates of both group were significantly lower than that of positive control group (53.6%)(P＜0.01) The formation rate of D6blastocyst in epronucleation group (12.5%) was significantly higher than those both in positive control group (5.1%) and negative control group (3.7%)(P＜0.01).However no statistic significance was found between the latter2(P＞0.05)Conclusions:1Average diameters of all3PN zygotes was22.95±1.80μm.The mixmum and maximum were21.81μm and24.13μm individually.95%diameters were from22.84～23.06μm. Significant difference can be found among3pronuleauses in tripronulear zygotes.2Most of embryos cleaved to2-cell-embryo for the first time after microsurgical enucleated correction.The correction affected partly on the improvement of embryonic cleavage level and farly lagged from that of2PN zygotes.3Microsurgical enucleation can correct tripronuclear embryos and improve their embryonic developmental potential. But the effect was limited. The embryonic viability of he enucleated embryos was still lower than that of normal fertilized embryos. Part III The study of aneuploidy and heteroparental of microsurgi-cally epronucleated and corrected tripronulear embryosObjective:To detect aneuploidy and heteroparental of blastocysts derived from microsurgically epronucleated tripronuclear embryos for providing theoretical support for embryo transfer and more sources for stem cells research.Methods:60microsurgically epronucleated zygotes were cultured for5-6days.30of60zygotes developed to blastocysts were defined as post-correction blasocyst group. The other30were named as post-correction non-blasocyst group with none blastocyst formation.Besides,30blastocyst derived from tripronuclear zygotes were collected to tripronuclear blastocyst group.57blastocysts from10cycles of patients performed with PGD were collected and created as the fourth normal blastocyst group in our reproductive medical center, from January to March,2013. Samples were performed with whole-pair chromosoms screening by SNP microarray chips and compare the aneulpoidy rates of the four groups. Embryos (35) and parental blood were collected to extract DNAs for heteroparental detection by PCR.Results:1Diploidy of blastocyst from microsurgically pronuleated zygotes(44.4%)was significantly higher than post-correction non-blastocyst group (19.2%) and tripronuclear blastocyst group(0.91%)(P＜0.05).No difference was found comparing with normal blastocyst group(56.9%)(P>0.05).2Aneuploidy of blastocyst from microsurgically pronuleated zygote(55.5%)was significantly lower than post-correction non-blastocyst group (80.8%) and tripronuclear blastocyst group(90.9%)(P＜0.05).No difference was found comparing with normal blastocyst group(P>0.05).3Blastocysts from epronucleated corected zygotes were performed with heteroparental dectcetion.DNA from single blastocyst were amplied and4failed.The results show that25blastocysts were both parental and maternal inheritance,taking account78.1%,6blastocysts were parental(18.8%),1blastocysts was maternal(3.1%).Conclusions:1Microsurgically epronucleated correction is beneficial for restoring diploidy of tripronuclear zygotes and lower the aneuploidy rate.2The developmental arrest of microsurgically epronucleated tripronuclear zygotes was partly correleated with the severe chromosome abnormality.The aeuploidy was not significantly improved although with the capability of self-correction.3Tripronuclear zygotes were corrected to restore diploidy by microsurgically epronucleation.The combination of PGD and heteroparental detection will help to determine whether these embryos can be used for transfer or not. |
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