Study On Factors Affecting Monozygotic Polyembryony And Solution Strategy In Assisted Reproductive Technology

The incidence of monozygotic polyembryony(MZP)increases following assisted reproduction technology(ART)compared with natural pregnancies,which greatly increased the risk of pregnancy.At present,studies have confirmed that younger women,ovulation induction drugs and oral contraceptives increase the incidence of MZP,and the risk of MZP is higher with blastocysts transfer than that with cleavage stage embryos,while embryo cryopreservation has no effect on MZP.The effect of insemination methods and assisted hatching(AH)on MZP is still controversial which might be because factors such as maternal age and the embryo stage of using AH have not been considered in most studies.However,most available reports were focused on analysis of factors increasing MZP,but not on how to reduce MZP in human ART.While the patient's own conditions cannot be changed and there are strict restrictions on the selection of insemination methods in China,this study mainly analyzed other factors that may increase MZP and proposed solution stretagy.Main results were as followed:1.Analysis factors of MZPIn this chapter,we analyzed the basic data and the occurrence of MZP after in vitro fertilization-embryo transfer(IVF-ET)treatment in one reproductive medical center from November 2010 to October 2019.After excluding the uncertain factors-AH,logistic regression analysis was used to analyze the possible factors of MZP.The results showed that blastocyst transfer and multiple embryos transfer increased the risk of MZP,while patients with higher GN dosage(?3000 IU)had lower risk of MZP.2.The relationship between AH of D2-D3 embryos and MZPNext,we analyzed the relationship between laser zona pellucida(ZP)thinning of D2-D3 embryos and incidence of MZP and combined with the safety of laser ZP thinning to determine more reasonable indications for AH.In order to reduce the influence of age on the results,the age was divided into two stages: ?38 years(the older patients)and <38 years(the young patients).The results showed that the risk of MZP was 2.6 times higher in the older patients than that in young patients(P < 0.01).The latest research showed that the AH in the older patients could not improve the pregnancy rate and reduced the live birth rate.Therefore,it is necessary to stop AH using for D2-D3 embryos of the older patients.Laser thinning of D2-D3 embryos in young patients can reduce the incidence of MZP(odd ratio = 0.36,P < 0.01)without increasing the risk of pregnancy,so it can be carried out for young patients to reduce the risk of MZP.3.Laser ZP breaching of blastocysts and MZPTo test whether the higher MZP of blastocysts compared to cleavage stage embryos was associated with changes of the ZP such as increasing density caused by prolonged embryo culture in vitro,the blastocyst's ZP was breached with a hole.The results showed that the MZP rate in IVF cycles for the blastocyst ZP breaching group was still significantly higher than that of cleavage stage embryos without AH(odds ratio=2.05,P<0.05),but the MZP rate was not significantly different following ICSI(odds ratio=0.66,P>0.05).Meanwhile,ZP breaching did not significantly increase the risk of pregnancy.Thus,ZP breaching was useful to reduce the incidence of MZP in ICSI-generated blastocysts.4.Screening of marker genes related to blastocyst formation in granulosa cellsZP breaching does not effectively reduce the risk of IVF-derived blastocyst division,and multiple embryos transfer also increases the incidence of MZP.Therefore,RNA sequencing(RNA-Seq)technology was used to screen marker genes of granulosa cells related to high developmental competence of oocyte,so as to select embryos with good development potential by marker genes and perform single embryo transfer at cleavage stage.A total 129 differentially expressed genes were found to be related to blastocyst formation.Through GO enrichment and KEGG enrichment analysis,a total of 16 differentially expressed candidate genes were found which might be used to predict the embryo development competence.Three differentially expressed genes(REN,COL1A1 and COL1A2)were selected and further validated by RT-qPCR.The expression trend was up-regulated,consistent with the RNA-Seq results.The relative expression of REN and COL1A2 in high blastocyst rate group was significantly higher than that in low blastocyst rate group.Therefore,REN and COL1A2 can be used to predict embryo quality.In summary,this study found that the factors of MZP that can be controlled in ART include the number of embryo transfer,AH and the developmental stage of embryo transfer.In order to reduce the impact of multiple embryos transfer and blastocyst transfer on MZP,REN and COL1A2 can be used as marker genes to select high-quality embryos to transfer at cleavage stage,so as to reduce the number of embryo transfer or only transfer single embryo.At the same time,the following measures can be adopted for patients with cleavage stage embryo transfer: ZP laser thinning can be carried out to further decrease MZP in young patients(<38 years),but not for older patients(?38 years).