Effect Of Supra-physiological Level Of Estradiol On The Pregnancy Outcomes Of In-vitro Fertilization-embryo Transfer In Different Population And The Potential Mechanism

Chapter 1 The Effect of Supraphysiological Estradiol on Pregnancy Outcomes in Women with PCOS and Ovulatory Women:a clinical studyBackground:Controlled ovarian stimulation is an integrate part of in vitro fertilization and embryo transfer technique(IVF-ET).It promotes a synchronous development of multiple follicles via administration of exogenous gonadotropin.Compared with previous natural ovulation cycle,controlled ovarian stimulation has greatly increased the number of oocytes and embryos,and consequently raised the success rate of IVF.However,while it increases the number of follicles and oocytes,controlled ovarian stimulation also dramatically elevates the level of serum estradiol that is secreted by the developing follicles.The peak estradiol level of controlled ovarian stimulation can reach more than 10 times of the estradiol level of natural ovulation cycle.Recent researches suggested the supra-physiological level of estradiol may alter the expression level of endometrial genes that are related with implantation,may advance the window of receptivity,and consequently compromise the pregnancy rate of fresh embryo transfer in women with a high ovarian response.Focusing on this problem,freeze-all strategy has been proposed in recent years,which aims to avoid the adverse effect of supra-physiological level of estradiol on embryo implantation by cryopreserving all embryos and performing a deferred frozen embryo transfer.Our team has completed two large-sample multicenter randomized controlled trials respectively in 1508 women with polycystic ovary syndrome(PCOS)and in 2157 ovulatory women comparing fresh cleavage-stage embryo transfer with frozen cleavage-stage embryo transfer.The results of these two studies were not consistent.The trial in women with PCOS showed frozen embryo transfer resulted in a lower rate of pregnancy loss,a higher rate of live birth,and a higher birth weight of singleton,whereas the trial in ovulatory women demonstrated similar rates of pregnancy,pregnancy loss,and live birth as well as a comparable birth weight between the frozen and fresh embryo transfer groups.Aim:To assess the variance in the effect of supra-physiological level of estradiol on pregnancy outcomes between women with PCOS and ovulatory women,to determine the threshold of estradiol level that related to a decrease of pregnancy rate after fresh embryo transfer,to evaluate whether a freeze-all strategy could improve the pregnancy outcomes in women with excess level of estradiolMethods:We performed a secondary analysis in data from our two randomized trials First,logistic regression models were built in women with PCOS and in ovulatory women respectively,including age,body mass index(BMI),duration of infertility,previous pregnancy,serum estradiol level on the day of hCG administration(as a continuous data)or number of oocytes(as a continuous data),groups of intervention(i.e.frozen vs.fresh embryo transfer),and the interaction between groups of intervention and serum estradiol level on the day of hCG administration or the interaction between groups of intervention and number of oocytes.Then,the pregnancy outcomes after frozen or fresh embryo transfer were graphed according to the levels of estradiol or the number of oocytes to provide a visual evidence for the relationship between them.Finally,the cutoff values for the subgroups of estradiol or oocytes number were determined by combining the results of logistic regression and the graphs.The between-group difference in the rates of clinical pregnancy,clinical pregnancy loss,and live birth between the frozen and fresh embryo transfer groups was compared among each subgroup.Results:In women with PCOS,there were statistically significant interactions between the estradiol level on the day of hCG administration and groups of intervention on the rates of clinical pregnancy loss and live birth,indicating the between-group difference in the rates of clinical pregnancy loss and live birth after frozen and fresh embryo transfer varied with the levels of estradiol.When estradiol level>3000pg/ml,compared with fresh embryo transfer,frozen embryo transfer resulted in a lower rate of clinical pregnancy loss(1 1.6%vs.26.3%,OR:0.37,95%CI:0.23-0.57)and a higher rate of live birth(51.9%vs.40.7%,OR:1.57,95%Cl:1.22-2.03).However.when estradiol level ?3000pg/ml,the rates of clinical pregnancy loss(21.5%vs.22.8%,OR:0.93,95%CI:0.53-1.62)and live birth(45.1%vs.44.4%,OR:1.03,95%CI:0.72-1.47)were all comparable between the frozen and fresh embryo transfer groups.In ovulatory women,there was no statistically significant interaction between estradiol level and groups of intervention on any outcomes,indicating that no evidence supported the between-group difference in pregnancy outcomes after frozen and fresh embryo transfer varied with the levels of estradiol.Whatever estradiol level ?3000pg/ml or>3000pg/ml.the rates of clinical pregnancy,clinical pregnancy loss,and live birth were similar after frozen and fresh embryo transfer.Conclusions:The impact of supra-physiological level of estradiol on pregnancy outcomes varied between women with PCOS and ovulatory women.In women with PCOS,excess estradiol was associated with an increased risk of clinical pregnancy loss and a decreased rate of live birth after fresh embryo transfer compared with frozen embryo transfer.However,in ovulatory women,there was no relationship between estradiol level and the between-group difference in pregnancy outcomes after fresh and frozen embryo transfer.In women with PCOS,regarding to the pregnancy outcomes after fresh embryo transfer,the cutoff value for diagnosis of excess estradiol was determined at 3000pg/ml.For women with estradiol level>3000pg/ml,a freeze-all strategy and a deferred frozen embryo transfer could improve the rate of live birth.Nonetheless,the mechanism underlying the associations between excess estradiol and increased risk of pregnancy loss was still unclear and warranted more clinical studies and basic researchesChapter 2 The effect and mechanism of excess estradiol together with hyperandrogenism or/and hyperinsulinism on the biological behavior of extra villous trophoblastBackground:From the clinical study in chapter 1,we found excess level of estradiol>3000pg/ml resulted from controlled ovarian stimulation was closely associated with an increased risk of clinical pregnancy loss after fresh embryo transfer in women with PCOS.However,such association was not observed in ovulatory women.However,the mechanism underlying the inconsistent findings between the two different population was unclear.Observational studies suggested the hyperandrogenic subtypes of PCOS had a higher risk of obstetric complications than the other subtype;and had increased incidences of macroscopic placental lesions and microscopic placental lesions compared with the non-hyperandrogenic subtype.Furthermore,It was shown that women with PCOS who had concomitant insulin resistance suffered from a higher risk of pregnancy loss than those with a normal level of insulin.Thus,we speculated that the featured abnormalities of endocrine and metabolism in women with PCOS may have combined effect with the supra-physiological level of estradiol on the adverse obstetric outcomes after fresh embryo transferThe majority of previous studies mainly focused on the effect of supra-physiological level of estradiol on endometrial receptivity.Abnormal endometrial receptivity usually leads to implantation failure,which mostly manifests as negative pregnancy test or biochemical miscarriage.However,in women with PCOS,we found that estradiol level>3000pg/ml was not associated with a difference in the rate of clinical pregnancy,but was related to a higher risk of clinical pregnancy loss after fresh embryo transfer compared with frozen embryo transfer.These findings suggested that suboptimal maternal internal environment during fresh embryo transfer cycle,i.e.hyperandrogenism and insulin resistance together with excess estradiol,may also have adverse effect on the implanting embryo(s).After embryo implantation,extra villous trophoblasts(EVTs)migrate into maternal endometrium and induce remodeling of uterine spiral arterioles,which is critical for the normal placentation and for the maintenance of pregnancy.The process of proliferation,migration,and invasion of EVTs are regulated by multiple factors.There are still controversies on the impact of excess estradiol on the biological behavior of EVTs.Moreover,few studies have investigated the combined effect of excess estradiol with hyperandrogenism or/and hyperinsulinism on the biological behavior of EVTsAims:To assess the effect of 17? estradiol at different concentrations alone or together with testosterone or/and insulin at varied concentration on proliferation and migration of HTR8/SVneo cells,which is a cell line of EVTs.Afterwards,the underlying mechanism was preliminarily explored with proteomics.Methods:HTR-8/SVneo cells were infected with lentivirus that carried fluorescent protein carrier,which enabled the cells to express green fluorescent protein.The green fluorescent protein in cells allowed us to use the celigo system with automatic image acquisition and analysis for performing the experiments of proliferation and scratch migration.The lentivirus infected cells were cultured with 17? estradiol at different concentrations or 17? estradiol together with testosterone or insulin with varied concentration.Cell counting was performed daily for 5 days after interventions.We made scratches on 96-well plate with a special scratcher,and then culture the cells with the same medicines as above.The area of plate with cells were recorded at baseline and after 24h,and the rate of migration was calculated.Then,the interventions with significant between-group differences in cell proliferation and/or migration were picked out for further mechanism experiments.HTR-8/SVneo cells were cultured with the picked interventions.Proteomics was performed with tandem mass tag method.The differentially expressed proteins were selected by the fold threshold of 1.2 and above or 0.83 and below.The selected proteins with different expression were further verified by parallel reaction monitoring technique for protein quantificationResults:Compared with blank control,estradiol 10 nM alone could decrease the mean proliferation fold(4.23 vs.4.80,P=0.022),and displayed dose-effect relationship.Estradiol alone at any concentration did not significantly change the migration rate.Testosterone 100nM alone could reduce the mean proliferation fold(4.31 vs.4.80,P=0.022),but testosterone at lower concentrations could not.Testosterone alone had no statistically significant effect on the migration rate.Insulin alone at any concentration did not alter the proliferation fold.However,insulin at 5nM or more could decrease the migration rate(42.01%vs.56.72%,P=0.009)with dose-effect relationship.Compared with estradiol 10nM alone,estradiol 10nM together with testosterone 100nM resulted in similar proliferation fold(4.00 vs.4.23,P=0.079),but yielded decreased migration rate(41.11%vs.57.16%,P=0.003).When compared with estradiol 10nM alone,estradiol 10nM together with insulin 5nM also leaded to comparable proliferation fold(4.03 vs.4.23,P=0.132)and lowered migration rate(36.85%vs.57.16%,P=0.002).Whereas,when compared with the group of insulin 5nM alone,the migration rate in the group of estradiol 10nM together with insulin 5nM was not statistically different(36.85%vs.42.01%,P=0.1 70).The results of proteomics showed,compared with estradiol 10nM alone,estradiol 10nM together with testosterone 100nM could up regulate the expression level of TPM1 and CALD1,which were cytoskeletal proteins and were closely related with the function of cell migration.Conclusions:There appeared to be a combined effect between excess estradiol and hyperandrogenism,which resulted in a decrease in EVTs migration.This effect was probably mediated by an impact on cytoskeletal proteins.Further studies are warranted to elucidate the exact machnism.