The Research Of Influencing Factors Of Human Blastocyst Vitrification

Part Ⅰ Extended culture and vitrification of the human blastocysts derived from "discarded" embryosObjectiveTo identify the viability of the "discarded" embryos that grow slowly and have low morphological scores at cleavage stage and the possibility to utilize them for infertility treatment.MethodsThe "discarded" embryos were extended cultured until day6after insemination. The embryos were checked on day5and day6to examine the in vitro blastocyst development. Blastocysts with optimal morphology derived from the discarded embryos were vitrified. After warming, the survived blastocysts were transferred to patients and the clinical outcomes of these cycles were analyzed.ResultsA total of597blastocysts (24.7%) were obtained from2421"discarded" embryos after extended culture. One hundred sixty blastocysts (6.6%) with optimal morphology were vitrified. Embryo utilization rate was increased from30.8%on day3to32.6%on day6. After warming,61out of92blastocysts (66.3%) survived and were transferred to44patients. The clinical pregnancy rate and the implantation rate was40.9%(18/44) and32.8%(20/61) respectively. Thirteen healthy babies were born, and5pregnancies aborted spontaneously.ConclusionBlastocysts derived from "discarded" embryos can be successfully vitrified and give rise to live births. Select and vitrify viable embryos after extended culture of "discarded" embryos can be a proposal to avoid embryo wastage. Part Ⅱ Effects of blastocyst quality and developmental rate of human blastocysts before vitrification on the clinical outcomes after transfer in warming cyclesObjectiveTo assess the influences of morphological score and developmental rate of human blastocysts before vitrification on the clinical outcomes after transfer in warming cycles and to determine the clinical value of the retarded blastocysts in infertility treatment program.MethodsHuman embryos were extended cultured in vitro and the derived blastocysts scored better than3BB on day5and scored better than3BC on day6or day7were vitrified at day5, day6, and day7respectively. Comparisons were made of the clinical outcomes among day5, day6, and day7frozen blastocysts transfer cycles. Comparisons were made of the clinical outcomes among patients who have vitrified blastocysts with different morphology score on day5or day6.Results1. The percentage of good quality blastocysts of day5, day6, and day7frozen blastocysts were significantly different (72.8%,56.7%,32.0%,P<0.05). The survival rate of D5, D6and D7blastocysts were similar (98.1%,96.7%,92.6%,P>0.05). The implantation rate of D5, D6and D7frozen blastocysts were56.4%,45.0%,20.0%; and the clinical pregnancy rate of them were69.6%,57.0%,29.4%respectively. The implantation rate (P<0.001) and clinical pregnancy rate (P<0.01) of D5blastocysts were significantly higher than that of D6or D7. The implantation rate and clinical pregnancy rate of D6blastocysts were significantly higher than that of D7(P<0.05). The abortion rate of D5, D6and D7blastocysts were not different (8.5%,9.2%,20.0%, P>0.05).2. The implantation rate and clinical pregnancy rate of single good quality blastocyst (GQB) transfer cycles were significantly higher than that of single poor quality blastocyst transfer cycles (61.5%vs.24.3%, P<0.001).3. The implantation rate of frozen blastocyst transfer cycles with double GQB was significantly higher than that of cycles with only one GQB (59.7%vs.51.1%, P <0.01), but the clinical pregnancy rate of them were not different (74.8%vs.69.6%, P>0.05). The implantation rate and clinical pregnancy rate of cycles with no GQB (23.8%,35.7%) were significantly lower than that of cycles with1or2GQB (P<0.001). The abortion rate of cycles with no GQB was higher than that of other two groups but not significantly different (16.7%,6.9%,8.3%,P>0.05).4. The implantation rate and clinical pregnancy rate of frozen blastocyst transfer cycles with no GQB were lower than that cycles with at least one GQB(24.0%vs.55.9%, P<0.001;30.4%vs.70.2%,P<0.001)and the abortion rate of cycles with no GQB was higher than that of cycles with at least one GQB (18.8%vs.8.0%,P<0.05).5. For patients who have only GQB, the implantation rate and clinical pregnancy rate were similar no matter the blastocyst were vitrified on day5or day6(61.6%vs.57.5%, P>0.05;73.1%vs.67.1%, P>0.05).Conclusion The morphological score of blastocysts before vitrification can influence the clinical outcomes of warming transfer cycles significantly. Totally, the implantation rate and clinical pregnancy rate of D5frozen blastocysts were higher than that of D6blastocysts. But the development potential of good quality blastocyst vitrified on D6is comparable to those vitrified on D5. The retarded blastocyst can be vitrified as late as day7with lower pregnancy rate. Part Ⅲ Effects of assisted hatching and roles of assisted hatching site of vitrified blastocysts on clinical outcomes after transfer in warming cyclesObjectiveTo assess the effects of assisted hatching (AH) of vitrified blastocysts after warming on the clinical outcomes after transfer and to identify the effects of different assisted hatching site on the clinical outcomes.MethodsHuman blastocysts scored better than3BB on day5and better than3BC on day6were vitrified respectively. Assisted hatching was performed at any site of the zona pellucida immediately after warming, or performed at the certain site near or opposite to the inner cell mass (ICM) after the blastocysts partially re-expanding. Other vitrified blastocysts were transferred without assisted hatching as control. Comparisons were made of the clinical outcomes between cycles with or without AH and among the AH groups performing AH at different sites.Results1. Totally, the percentage of good quality blastocyst (GQB) of AH group was higher than that of control group (68.2%vs.60.9%,P<0.01). The implantation rate and clinical pregnancy rate of AH group were higher that that of control group(57.4% vs.48.8%,P<0.001;70.9%vs.60.7%,P<0.01).2. The implantation rate and clinical pregnancy rate of AH group were higher that that of control group for patients who with only GQB (66.2%vs.55.7%,P<0.01;77.9%vs.67.5%,P<0.05).3. The implantation rate and clinical pregnancy rate of AH group were not significantlly higher that that of control group for patients who with one GQB of two transferred blastocysts (53.7%vs.51.2%,P>0.05;72.5%vs.69.3%,P>0.05)4. The implantation rate and clinical pregnancy rate of AH group were not significantlly higher that that of control group for patients who have no GQB (34.3%vs.27.9%,P>0.05;44.9%vs.34.7%,P>0.05).5. The implantation rate, clinical pregnancy rate and monozygotic twins pregnancy rate after transfer were not significantly different among AH groups performing AH at any site, near to ICM or opposite to ICM (56.4%,58.4%,58.3%,P>0.05;70.4%,71.1%,71.6%,P>0.05;1.9%,5.1%,5.2%,P>0.05).Conclusion Assisted hatching can enhance the implantation rate and pregnancy rate for vitrified blastocysts with good morphology. Effects of different assisted hatching site on clinical outcomes are not found. Part Ⅳ Clinical outcomes of single and double blastocyst(s) transfer in warming cyclesObjectiveTo compare the pregnancy outcome after single or double blastocyst transfer and compare the obstetrical outcomes of singleton or twin pregnancy.MethodsHuman blastocysts scored better than3BB on day5and better than3BC on day6were vitrified respectively. One or two blastocyst(s) were transferred in warming cycles. Comparisons were made of the clinical outcomes between cycles after single or double embryos transfer. Comparisons were made of the obstetrical outcomes between singleton pregnancy and twin pregnancy.Results1. The clinical pregnancy rate after single vitrified blastocyst transfer was lower than double blastocysts transfer significantly (49.8%vs.68.9%, P<0.001) but the implantation rate of these two groups was not different(49.8%vs.52.9%, P>0.05). The multiple pregnancy rate was much lower in single transfer group than that of double transfer (0.7%vs.53.7%,P<0.001). Ectopic pregnancy rate was0.9%in double transfer group but no ectopic pregnancy in single blastocyst transfer.2. For good quality blastocyst with assisted hatching after warming, the clinical pregnancy rate and implantation rate were not different between single or double transfer(78.8%vs.79.1%, P>0.05;78.8%vs.65.9%, P>0.05). The multiple pregnancy rate was66.7%after double transfer but no twin pregnancy in single blastocyst transfer.3. The rate of pre-term birth (11.1%vs.52.5%, P<0.001) and low birth weight (6.3%vs.38.6%, P<0.001) were higher in twins than in singletons. The mean birth weight of twins was lower than that of singletons(2555±520g vs.3335±605g, P<0.001).ConclusionFor good quality blastocyst hatching or hatched before vitrification or with assisted hatching after warming of expanded blastocysts, single blastocyst transfer can achieve similar pregnancy rate with double blastocysts transfer and reduce multiple pregnancy rate significantly. Singleton pregnancies have a higher incidence of term delivery of healthy babies.