The Role Of Frozen Embryo Transfer On The Risk Of Pre-eclampsia

Chapter I The clinical study of the increased risk of pre-eclampsia from frozen embryo transferObjective:Infertility affects 10%-18%of reproductive-aged women as estimated in our country,which brings a profound impact on the family and society.Assisted reproductive technology(ART)has been established as a standard treatment for infertility and has resulted in over 8 million babies all over the world.However,the safety of assisted reproductive technology and its maternal and infant outcomes have also been concerned.Previous studies show that pregnancies from ART are associated with increased risks of maternal and neonatal complications compared with spontaneous conceptions,including the risk of preeclampsiaAlthough the exact mechanism of preeclampsia was still unclear,a number of factors were reported to be associated with the risk of preeclampsia in natural conceptions,mainly including diabetes(type 1 or type 2),autoimmune disorders,nulliparity,advanced maternal age,a pregnancy interval greater than 10 years,body mass index(BMI)≥35 kg/m2,and multiple gestations.However,in patients who underwent ART,which step(s)further increased their risk of preeclampsia was still not well known We recently completed a series of multicenter randomized trials comparing elective frozen embryo transfer with fresh embryo transfer.The results showed that frozen embryo transfer resulted in a higher risk of preeclampsia compared with fresh embryo transfer.And recently,increasing evidences proved the same result.However,whether other factors also contributed the risk of preeclampsia in pregnancies after IVF and which components increased the raised risk of preeclampsia after frozen embryo transfer was still unclear.In this study,we performed a secondary analysis of the data collected during our previous three multicenter randomized trials to evaluate the predisposing factors of preeclampsia in assisted reproductive technology using autologous oocytesMethods:A nested case-control study from the combined cohort of three multi-center randomized trials comparing fresh to frozen embryo transfer,including women who achieved clinical pregnancy after the first embryo transfer.Multivariable logistic regression was used to assess the effect of baseline characteristics,ovarian response parameters,type of fertilization,type of embryo transfer,and number of gestational sacs on the risk of pre-eclampsia.This was a nested case control study from the combined cohort of three multicenter randomized trials comparing fresh vs.frozen embryo transfer.In this study,we included patients who achieved clinical pregnancy after the first embryo transfer.We used multivariable logistic regression to assess the effect of baseline characteristics(age,body mass index(BMI),mean arterial pressure,parity,duration of attempt to conceive,etiology of infertility,sex hormone parameters,endometrial thickness),the ovarian response parameters,the type of fertilization(IVF vs.ICSI),the type of embryo transfer(frozen vs.fresh embryo transfer,cleavage-stage vs.blastocyst-stage embryo),and the number of fetus(twin vs.singleton pregnancy)on the risk of preeclampsia.Results:There were 2965 clinical pregnancies and 90 women were diagnosed with pre-eclampsia.The BMI(P=0.003),systolic(P<0.001)and diastolic(P=0.005)blood pressure,the percentage of twin pregnancies(P<0.001)and the percentage of frozen embryo transfer(P=0.002)were higher in patients with preeclampsia compared with women without preeclampsia.Serum progesterone level on the day of hCG trigger(P=0.016)and the percentage of cleavage-stage embryo(P=0.049)were higher in preeclampsia patients.Twin gestations(odds ratio[OR]2.34,95%confidence interval[CI]1.50-3.66),mean arterial pressure(OR 1.04,95%CI 1.01-1.07),frozen embryo transfer(OR 2.06,95%CI 1.27-3.35),body mass index(BMI)(OR 1.10,95%CI 1.02-1.18),progesterone level on the day of human chorionic gonadotrophin trigger(OR 1.53,95%CI 1.07-2.20),and the total dose of gonadotrophin(OR 0.999,95%CI 0.999-1.000,P=0.037)were associated with the risk of pre-eclampsia.When the analysis was confined to women who underwent frozen embryo transfer,twin gestations(OR 2.44,95%CI 1.43-4.18),BMI(OR 1.13,95%CI 1.03-1.23)and the total dose of gonadotrophin(OR 0.999,95%CI 0.999-1.000,P=0.014)were still related to the risk of pre-eclampsia.The embryo stage at transfer was not included in the final models.Conclusions:Frozen embryo transfer was an independent risk factor of pre-eclampsia in assisted reproductive technology.The high ovarian response may also increase the risk of pre-eclampsia.The embryo stage at transfer was not related to the risk of pre-eclampsia.Chapter Ⅱ The mRNA-Seq and DNA methylation BeadChip analysis of placenta after frozen embryo transferObjective:Increasing evidences showed that frozen embryo transfer resulted in a higher risk of preeclampsia,however,the underlying mechanism was unknow.Compared with fresh embryo transfer,frozen-embryo transfer was performed after ovarian recovery following the controlled ovarian stimulation,which prevented the embryo from high estradiol levels in the uterus and provided closely physiological conditions for embryo implantation and placentogenesis.Theoretically,it was more profitable for the foundation and maintenance of pregnancy.Furthermore,the embryo cryopreservation was performed at the early stage of embryogenesis from cleavage to blastocyst stage,when the genome was sensitive to the external change and the methylation underwent large-scale reprogramming.It has been proved that the embryo vitrification could affect the epigenetic regulation of imprinted genes and microRNAs,leading to abnormal gene expression and placenta derived pregnancy complications.In our study,mRNA sequencing and DNA methylation BeadChip analysis were performed on placental tissues from different sources,and the differentially expressed genes and differentially methylated genes were analyzed with bioinformatics technology to screen the therapeutic targets for the risk of preeclampsia induced by frozen embryo transfer.Methods:The placenta tissues from different sources were collected for mRNA sequencing(mRNA-Seq)and DNA methylation BeadChip analysis(850k BeadChip),including normal pregnancy from fresh embryo transfer,normal pregnancy from frozen embryo transfer,preeclampsia from frozen embryo transfer,four cases for each group.The differential genes were screened by bioinformatics analysis.Then,the validation of selected genes was performed in expanding placenta samples,including 12 normal pregnancy placentas from fresh embryo transfer and 12 normal pregnancy placentas from frozen embryo transfer,respectively.qRT-PCR was used to measure the mRNA levels of differentially expressed genes and Bisulfite sequencing PCR(BSP)was used to verify the methylation level of differentially methylated genes.Results:According to the gene function and the expression abundance in placenta,27 differentially expressed genes were selected from the mRNA-Seq to perform expanding validation.The qRT-PCR results showed that the expression of STEAP3 and CL-P1 decreased significantly in the normal placenta from frozen embryo transfer.Following the DNA methylation analysis,11 differential methylated genes were selected to perform expanding validation with BSP.The results showed that the promoter region of PROK1 was significantly hypermethylated in the normal placenta from frozen embryo transfer.In functional enrichment analysis,the GO terms and KEGG pathways related to regulation of cell growth and differentiation,female pregnancy,cytokine receptor binding,extracellular matrix and inflammatory pathway were significantly enriched.Conclusion:The expressions of mRNA and DNA methylation status were changed significantly in placentas after frozen embryo transfer.The abnormal expression levels of STEAP3 and CL-P1 and the hypermethylation change of PROK1 may be involved in the increased risk of preeclampsia after frozen embryo transfer.Chapter Ⅲ The function study of STEAP3 gene in HTR-8/Svneo trophoblastObjective:The results from Chapter II showed that the expression of STEAP3 mRNA level in placenta after frozen embryo transfer was significantly decreased than that in fresh embryo transfer group.Whether the altered STEAP3 expression was related to the pathogenesis of preeclampsia was unclear.The pathogenesis of preeclampsia has not been fully elucidated yet.It has been well known that the dysfunction of uterine spiral artery remodeling induced by insufficient invasion of trophoblast was the key of preeclampsia.The normal differentiation of extravillous trophoblast cells(EVT cells)plays an critical role in the process of the remodeling of uterine spiral arteries.EVT cells eventually differentiate into interstitial EVT cells and vascular endothelial EVT cells during the invasion into maternal decidua.The interstitial EVT cells infiltrate the maternal decidual tissue and make the placenta implant in the muscle layer of the decidua,while vascular endothelial EVT cells penetrate the vascular lumen into the deep part of the decidua,and invade and replace the uterine spiral artery cells to obtain the vascular endothelial phenotype.The normal remodeling process of the uterine spiral artery ensures the full exchange of nutrients between the maternal and fetal circulation,which is beneficial and vital to the normal embryogenesis.On the contrary,the abnormal differentiation of EVT cells leads to the damaged uterine spiral artery remodeling,which results in pregnancy complications such as preeclampsia.STEAP3(Six-Transmembrane Epithelial Antigen of Prostate 3),belonging to the metal reductase family,can reduce Fe3+to Fe2+and play an important role in iron metabolism.Irons participate in the process of DNA and ATP synthesis and is essential for cell proliferation and survival.STEAP3 can promote the iron uptake,maintain the iron reserve,and maintain the rapid proliferation of tumor cells under hypoferric state.As a downstream regulator of TP53 activation,STEAP3 plays an important role in tumor cell apoptosis and proliferation.STEAP3 is highly expressed in placenta tissue,but there is a lack of study in the field of reproduction.Therefore,we performed the function study of STEAP3 in trophoblasts to determine whether the abnormal expression of STEAP3 was involved in the regulation of increased risk of preeclampsia after frozen embryo transferMethods:Western blot was used to detect the protein expression levels of STEAP3 in the placentas from normal pregnancy after frozen and fresh embryo transfer and preeclampsia after frozen embryo transfer.The expression levels of STEAP3 in HTR-8/Svneo trophoblast was down-regulated by small interfering RNA(siRNA).Then the proliferation ability of trophoblast was detected by CCK8,the migration and invasion ability of trophoblast was detected by transwell chamber experiments,and the trophoblast endothelial-like tube formation ability was detected by endothelial-like tube formation assayResults:Western blot found that the protein expression levels of STEAP3 in pre-eclampsia group was significantly decreased than that in normal pregnancy after fresh or frozen embryo transfer,and that in frozen embryo group was significantly decreased than that in fresh embryo group.When the expression of STEAP3 was down-regulated in trophoblast,the cell proliferation activity decreased,cell migration and invasion ability decreased,and endothelial-like tube formation ability also decreased.Conclusion:Down regulation of STEAP3 could inhibit the cell proliferation activity,reduce the ability of cell migration and invasion,and affect the ability of endothelial-like tube formation of trophoblast.The decreased expression of STEAP3 in placenta may be involved in the underlying mechanism of the increased risk of preeclampsia after frozen embryo transfer.