| Part 1 Analysis about the influence of different embryo transfer stategies on the efficiency of embryo transferPart 1 Analysis about the influence of different embryo transfer stategies on the efficiency of embryo transferObjective The aim of this retrospective study was to compare the clinical outcome between high responders and normal responders when using different transfer strategies; ultimately, to find out optimal transfer strategy that could decline multiple pregnancies and increase the chance of pregnancy for different subgroups of patients.Methods Cycles simulated by standard long protocol were selected to avoid possible bias from inconsistent protocols. Inclusion criteria were primary infertility, female age ^35 years, FSH level on female cycle day 2-3≤12mIU/ml, at least 6 good quality embryos available on day 3. Patients classified as normal responders when the number of oocytes retrieved was between 6 and 15, or high responders when more than 15 oocytes were retrieved. Three different transfer strategies were used for these patients: day 3 double embryo transfer (DET), day 5 elective single blastocyst transfer (eSBT) and the frozen-thawed embryo transfer (FET) instead of fresh embryo transfer. Retrospectiveanalysis was performed to find the optimized transfer strategy for different kinds of patients.Results 1.For the normal responders, the clinical pregnancy rate of day 3 double-embryo transfer (DET) was comparable to that of day 5 elective single blastocyst transfer(eSBT) (64.04% vs.60.33%, p>0.05), but the multiple pregnancy rates of eSBT group was significantly lower than DET group (4.11% vs.35.62%, p<0.01) 2. When eSBT was used, the clinical pregnancy rate of high responders was significantly lower than normal responders group (43.35% vs.60.33%, p<0.05) 3. For the high responders, the rates of clinical pregnancy and implantation in frozen-thawed embryo transfer (FET) cycles were notably higher than that in eSBT cycles (60.60% vs.43.35% and 60.60% vs.43.35%, respectively, p<0.05).Conclusions For normal responders, eSBT might be a better strategy to reduce multiple pregnancy rates while maintaining ideal pregnancy rates. In order to decline multiple pregnancies and increase the rate of pregnancy for high ovary responders, FET may be a preferable strategy.Part 2 The effect of blastocyst culture on embryo sex ratio bias during IVF-ET Objective To investigate the influence of blastocyst culture technology on sex ratio bias of fresh blastocyst transfer.Methods For patients applied ART at Reproductive Hospital affiliated to Shandong University between March 2013 and August 2014, the remaining embryos after transfer and frozen were collected separately, and testified the sex chromosome specific sequence of each embryos using PCR technology. The sex ratio of the remaining embryos was compared with the sex ratio of babies born from procedures of IVF/ICSI technology in China according to multi-center study, and babies born from vitrification-thawed blastocyst transfer among 2012 to 2013 in our center. The sperm was also tested by droplet digital PCR to compare the Y/X ratio before and after sperm collection.Results 1. Through the X/Y ratio analysis before insemination, the proportion of Y sperm compared to X sperm in sample after sperm collection was significantly lower than the theoretical value (0.68:1 vs.0.83:1, P<0.01), and also significantly lower than the sperm before washing (0.68:1 vs.0.76:1, PX<0.01).2.1n IVF cycles, there was also no significant difference in male ratio between remaining embryos and babies born from multi-center in China (52.94%vs.52.33%, P>0.05), in ICSI cycles, there was no significant difference in male ratio between remaining embryos and babies born from multi-center (47.59% vs.49.73%, P>0.05).3. There was no significant difference in male ratio between babies born from vitrification-thawed blastocyst transfer cycles and babies born from day 3 embryo transfer cycles (53.70%vs.52.33%, P>0.05). Vitrfication-thawed blastocyst transfer did not lead to significant sex ratio bias testified that the blastocyst culture itself would not cause the sex ratio bias. Other reason such as blastocyst selection time may cause the little bias in fresh blastocyst transfer cycles. Conclusions Blastocyst culture technology itself could not lead to more male blastocysts survival. Through the analysis of sex ratio in babies born from vitrification-thawed blastocyst transfer, the sex ratio bias in fresh blastocyst transfer cycle may be associated with the blastocyst selection time point. There was a trend of male ratio bias in IVF cycles compared to ICSI cycles, but this trend was not caused by the number preponderance of Y sperm.Part 3 Impact of modified blastomere fixation method on the blastomere fixation rate and the effect of probe hybridization during the precdure of fluorescence in situ hybridizationObjective Fluorescence in situ hybridization (FISH) has been widely used for genetic analysis in preimplantation genetic diagnosis (PGD), the reliability of FISH depends largely on the effect of blastomere fixation and probe hybridization. The effect of blastomere fixation depends on many factors, this part of the study was mainly to evaluate effect of modified blastomere fixation method to improve the result of probe hybridization, and to analysis the relationship between day 3 embryo quality and nucleus spreading rate/signal resolution rate in Fluorescence in situ hybridization (FISH) during the PGD procedure.Method For patients that applied Preimplantation Genetic Diagnosis (PGD) using Fluorescence in situ hybridization (FISH) tehnology at Reproductive Hospital affiliated to Shandong University between February 2012 and January 2015, normal blastomere fixation method and modified blastomere fixation method were used during blastomere fixation. Day-3 embryos were classified based on morphological scoring:grade 1 to grade 4 was defined from worse to better embryo quality. Day 3 embryos were classified as good quality when the number of blastomeres was between 6 and 10 and grade better than 2. Nucleus spreading, signal and the full signal rates were compared between embryos with different morphological scoring.Result 2939 embryos were included in this study.1. There was no difference in nucleus spreading rate between modified fixation method and normal blastomere fixation method (92.02%vs.91.17%, p>0.05), and the signal rate was significantly higher in modified fixation procedure group than in normal fixation procedure group (99.02% vs.85.16%, p< 0.01), and the full signal rate was also significantly higher in modified fixation procedure group than in normal fixation procedure group (91.79%vs. 77.80%, p< 0.01).2.When using normal fixation method, nucleus spreading rate of the embryos was higher in morphological good quality group when compared with the 2’group (92.52%VS.87.62%, p=< 0.01). The full signal rate of the embryos was higher in morphological good quality group than bad quality group (79.32% VS.65.05%, p=0.0000), but there was no significant difference in the signal rate (85.60 vs.83.94%, p>0.05).3.When using modified fixation method, the nucleus spreading rate of the embryos was also higher in morphological good quality group when compared with the bad quality group (93.61% VS.87.90%, p<0.05), but there was no difference in signal rate and full signal rate between morphological good quality group when compared with the bad quality group(99.56%vs.97.57%, P=0.0066; 91.96%vs.91.29%, P>0.05).Conclusions Modified blastomere fixation method could effectively improve the probe hybridization effect after blastmere fixation, which is useful for interpreting the hybridization result, and reduce the chances of misdiagnosis. No matter using normal blastomere fixation method or modified blastomere fixation method, the nucleus spreading rate of blastomeres from day 3 embryos with better morphological quality was higher than that from embyo with poor morphological quality, suggesting that we can choose morphological good quality embryos to biopsy when a patient has enough embryos. This strategy can effectively reduce the cost of the PGD. |
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