Application Analysis Of Different Sequencing Platforms In Preimplantation Genetic Testing And Study On Pregnancy Outcome Of Mosaicism Embryos

Chapter 1 Correlation analysis between two sequencing platforms and pregnancy outcomes in preimplantation genetic testing cyclesObjectivesTo compare clinical pregnancy outcomes of patients undergoing preimplantation genetic testing（PGT）using the Ion Torrent^TM sequencing platform and Miseq^DX sequencing platform.MethodsThis was a retrospective cohort study,including couples who underwent PGT cycles in the Reproductive Hospital of Shandong University from December 2017 to November 2019.1.To explore the effect of different platforms on the pregnancy outcomes of patients.The patients were divided into Ion Torrent^TM group and Miseq^DX group according to the different detection platforms used in clinical trials.2.To analyze the influence of different sequencing platforms on the pregnancy outcomes of patients with different indications,the patients were divided into two groups according to whether the patients had chromosomal abnormalities:PGT-SR group and PGT-A group.3.In order to analyze the pregnancy outcome of the platform test results.We divided patients into euploid embryo group and chimeric embryo group.4.To test the mosaic embryos by different platforms,the other platform of the amplified products was performed.5.The primary outcomes were the live birth rate in the transfer cycles.The secondary outcomes were the embryo aneuploidy rate,clinical pregnancy rate,ongoing pregnancy rate and miscarriage rate.Result:1.A total of 3471 embryos from 865 patient couples from 886 PGT cycles were included in this study.According to their different sequencing platforms,they were divided into two groups:445 couples in Ion Torrent^TM group（1836 embryos in 452 egg retrieval cycles）;Miseq^DX group with 420 couples（1635 embryos in 434 egg retrieval cycles）.2.There was no significant difference of the basic data between two groups.including maternal age,body mass index（BMI）,the number of pregnancy,the number of births,basal estradiol（E2）,follicle-stimulating hormone（FSH）,anti-Müllerian hormone（AMH）,the male sperm concentration,and sperm motility.The number of eggs retrieved,and the number of final blastocysts were not significantly different.The live birth rate was 53.91%in the Ion Torrent^TM group and 58.67%in the Miseq^DX group.There was no significant difference between the two groups（P=0.163）.In the PGT-A group,the live birth rate of the Ion Torrent^TM group was 48.43%and that of the Miseq^DX group was 53.37%.Among the PGT-A patients,there was no significant difference in the live birth rate between two groups（P=0.291）.In the PGT-SR group,the live birth rate of the Ion Torrent^TM group was 60.68%,and that of the Miseq^DX group was 64.67%.In the PGT-SR patients,there was no significant difference in the live birth rate between the two groups（P=0.416）.Among the patients with euploid embryos,the live birth rate of the Ion Torrent^TM group was 54.48%and that of the Miseq^DX group was 58.20%.There was no significant difference in the live birth rate between the two groups of patients with euploid embryos（P=0.291）.Among the patients who transferred mosaicism embryos,the live birth rate of the Ion Torrent^TM group was 43.48%,and that of the Miseq^DX group was 68,00%（P=0.087）.The overall live birth rate of euploid embryos was 56.18%,and the overall live birth rate of mosaicism embryos was 56.25%,and there was no significant difference（P=0.992）.Among patients who transferred mosaicism embryos,the live birth rate of patients with low proportion of chimeric embryos was significantly higher than that of patients with high proportion of mosaicism embryos（72.00%vs.39.13%,P=0.022）.The aneuploidy rate of embryos in the Ion Torrent^TM group was 56.46%and the aneuploidy rate in the Miseq^DX group was 46.03%（P=0.392）.30.44%of the 23 mosaicism embryos transplanted in the Ion Torrent^TM group were inconsistent with the original test results after the Miseq^DX sequencing platform,while 24.00%of the 25 mosaicism embryos transplanted in the Miseq^DX group were tested by the Ion Torrent^TM sequencing platform.The sequencing results were inconsistent,but there was no significant difference（P=0.445）.ConclusionDuring PGT cycles,the pregnancy outcome of the patient was not related to the sequencing platform.The Ion Torrent^TM sequencing platform and Miseq^DX sequencing platform had the same ability to detect embryos.There were false positives in the detection of mosaicism.In the absence of euploid embryo transfer,chimera could be considered for transfer.The live birth rate was lower after a high proportion of mosaicism embryo transfers.Chapter 2 Correlation Analysis of Mosaicism Embryos Rating and Pregnancy Outcomes in preimplantation genetic testing cyclesObjectivesA retrospective analysis of mosaicism embryos in the preimplantation aneuploidy detection cycle was conducted.To explore whether the chimeric proportion of mosaicism embryos and the grading of mosaicism embryos were related to the pregnancy outcome of mosaicism embryo transfer.This study provides a theoretical basis for the transfer selection priority of mosaicism embryos.MethodsA retrospective cohort study enrolled patients treated with PGT-A in our hospital from October 2017 to May 2020.The embryos were divided into euploid group and mosaicism group according to the PGT detection.In order to analyze the effect of different mosaicism ratios and embryo grades on the pregnancy outcome after mosaicism embryo transfer.According to the embryo mosaicism ratio,if the mosaicism ratio of the transferred embryos is≤40%（low ratio group）;if the mosaicism ratio of the transferred embryos>40%（high proportion group）.We analyzed the relationship between different mosaicism ratio embryos and pregnancy outcomes.Result1.A total of 2100 cycles of 1902 couples were included in this study（7673 blastocysts were biopsied,including 2880 euploid embryos and 982 mosaicism embryos）.1595 euploid embryos and 129 mosaicism embryos were transferred.2.The live birth rate after euploid embryo transfer was 56.74%,and the live birth rate after mosaicism embryo transfer was 50.39%.There was no significant difference between two groups（P=0.164）.The live birth rate of transplantation in the low proportion mosaicism group was 55.68%,and the live birth rate in the high proportion mosaicism group was 39.02%.Compared with the euploid embryos,the low proportion mosaicism group had no significant difference（P=0.846）.The high proportion of mosaicism group was significantly lower than the live birth rate of euploid embryos（P=0.024）.3.In the euploid group,the live birth rate of embryo transfer with trophectoderm score A was 62.46%.The live birth rate of embryo transfer with score B was 57.05%.The live birth rate of embryo transfer with score C was 45.41%.The live birth rate of embryos graded A was higher than that of embryos graded B.There was no significant difference between two groups（P=0.078）.Embryos graded A（P<0.001）and grade B（P=0.002）had higher live births rates than those of embryos graded C.The embryo transfer live birth rate was 66.67%when the inner cell mass score was A.The embryo transfer live birth rate was 53.44%when the score was B.The live birth rate of embryos with a score of A was significantly higher than that of embryos with a score of B（P<0.001）.4.In the low proportion group,the live birth rate of embryo transfer with trophectoderm score A was 38.46%,the live birth rate of embryo transfer with score B was 62.71%,and the live birth rate of embryo transfer with score C was 43.75%.When the inner cell mass score was A,the live birth rate of embryo transfer was 58.33%,and when the score was B,the live birth rate of embryo transfer was 55.26%,and there was no significant difference between score A and score B（P=0.842）.5.In the high proportion group,the live birth rate of embryo transfer with trophectoderm score A was 33.33%,the live birth rate of embryo transfer with score B was 36.67%,and the live birth rate of embryo transfer with score C was 60.00%.When the inner cell mass score was A,the live birth rate of embryo transfer was 20.00%,and when the score was B,the live birth rate of embryo transfer was 41.67%,and there was no significant difference between the two groups（P=0.352）.Conclusion1.During PGT cycles,the pregnancy outcome of low level mosaicism embryo was no related to the morphological grade embryos;2.During PGT cycles,the mosaic result of PGT may be false positive,especially in embryos with low level mosaicism or low morphological grade;3.If there is no euploid embryo transfer,choosing chimeras for transfer might be optional.When selecting chimeric embryos for transfer,it is preferred to select a low proportion of chimeric embryos for transfer.4.When a high level mosaicism embryos must be transferred,priority may be given to the transfer of low morphological grade,which may have a higher live birth rate.