Maternal And Neonatal Outcomes And Embryo Transfer Strategies In IVF/ICSI-ET For Patients With Thin Endometrium

A successful pregnancy depends on embryo quality,endometrial receptivity,and the interaction between embryo and maternal endometrium.Ideally,the method of assessment for endometrial receptivity should be easily performed in daily clinical routine and preferably be non-invasive.Endometrial thickness(EMT)can be easily measured by transvaginal ultrasonography.The study focused on endometrium-related infertility,and selected the thin endometrium as the research direction,which still has many clinical difficulties to overcome.There is no consensus on the definition of thin endometrium,and the widely accepted cutoff value is 7～8 mm.Most studies suggest that when EMT<7～8mm,pregnancy rate decreases and obstetric complications increase.At our reproductive center a cut-off of 7.5 mm on human chorionic gonadotrophin(HCG)trigger day is used to diagnose a thin endometrium.Our series of studies also used the 7.5mm cut-off value and came to meaningful conclusions.At present,the study of thin endometria is in the research and exploration stages,and some therapeutic effects have yet to be proven.On the one hand,for patients with thin endometrium,whether to choose direct fresh embryo transfer(Fre-ET)or selective frozen-thawed embryo transfer(FET)is still controversial.On the other hand,for these patients,if they choose an elective FET,it is difficult to estimate the probability of endometrial preparation leading to endometrial growth,which is not conducive to individual treatment.With the worldwide application of assisted reproductive technology(ART),the safety of offspring has become the focus of attention.To solve the above problems,we carried out the following three clinical studies.1.We explored the relationship between EMT on human chorionic gonadotropin(hCG)trigger day and the delivery of small-for-gestational-age(SGA)in in vitro fertilization(IVF)or intracytoplasmic sperm injection(ICSI)treatment.2.We retrospectively analyzed the influence of embryo transfer(ET)type on pregnancy outcomes of patients with thin endometrium during IVF/ICSI cycles.3.For the first time,we developed the nomogram scoring system to predict the probability of an EMT>7.5 mm in the subsequent FET cycle for women with a thin endometrium.Intrauterine adhesion(IUA)is an important and common cause of thin endometrium.IUA is caused by excessive curettage,induced abortion and other intrauterine operations damage to the endometrial basal layer.It is often accompanied by clinical symptoms such as decreased menstruation,periodic abdominal pain and infertility.Endometrial fibrosis is the main pathological feature and final result of uterine adhesion.When large trauma or excessive trauma exceeds the regeneration ability of normal parenchymal cells around the damage,under the regulation of inflammatory cells and cytokines,a large number of extracellular matrix(ECM)will repair the defective tissue and deposit a large amount in the endometrium,while the proliferation of normal endometrium is inhibited,and the endometrial tissue is gradually replaced by fibrous connective tissue,resulting in endometrial fibrosis,scar formation and adhesion.In recent years,some scholars have studied the effects of βklotho(KLB)on the occurrence and development of various human tumors and fibrotic diseases.Studies have confirmed that KLB affects the occurrence and development of liver fibrosis,liver cancer,lung cancer and thyroid cancer.However,there are no reports about the relationship between KLB and endometrial stromal cells and endometrial fibrosis.We proved that KLB protein was significantly overexpressed in endometrial stromal cells of patients with IUA by immunohistochemistry,immunofluorescence and western blotting.At the same time,there was a significant correlation between the expression of KLB protein and fibrosis marker Fibronectin in primary endometrial stromal cells.Therefore,we established IUA cell model with TGF-β1 and carried out further research to explore the potential relationship between KLB and endometrial fibrosis,and further study its mechanism.PART I:Endometrial thickness is associated with incidence of small-for-gestationalage infants in fresh IVF/ICSI-ET CyclesObjective:To investigate whether EMT is associated with adverse maternal and neonatal outcomes in fresh IVF/ICSI-ET cycles.Methods:Women under the age of 42 years who underwent IVF/ICSI treatment and received fresh ET in our unit from January 2017 to December 2018,resulting in a live singleton birth were enrolled.All patients were categorized into three groups by measuring the EMT on hCG trigger day,including 132 women with EMT ≤7.5 mm,2,166 with EMT>7.5～12 mm,and 859 with EMT>12 mm.The primary outcome measures were neonatal outcomes,including gestational age,birth weight,small-for-gestational-age(SGA),larger-for-gestational age,and preterm birth.Given that gestational age at delivery is critical to neonatal birth weight,we believe that SGA is a representative indicator.Therefore,after discovering the relationship between the incidence of SGA and EMT,we first performed univariate and multivariate regression analysis of variables predicting the incidence of SGA.Results:The risk of being born SGA was statistically significantly increased in the EMT ≤7.5 mm group compared with those from the EMT>12 mm group(adjusted odds ratio[aOR]2.391;95%confidence interval[CI],1.155-4.950).Moreover,maternal body mass index,secondary infertility,preterm delivery,and hypertensive disorders were all independent predictors for SGA.The mean birth weights of singletons in women with EMT ≤7.5 mm was lower than in the groups with EMT>7.5-12 mm and EMT>12 mm(3.25±0.56 kg vs.3.38±0.51 kg and 3.39±0.53 kg,respectively).Conclusion:After fresh IVF/ICSI-ET,the risk of SGA was increased twofold in women with EMT ≤7.5 mm compared with women with EMT>12 mm.We suggest that women with a thin EMT after obtaining a pregnancy by IVF should receive improved prenatal care to reduce the risk of delivering a SGA infant.PART Ⅱ:Fresh versus frozen embryo transfer in women with thin endometrium:a retrospective cohort studyObjective:A thin endometrium affects embryo implantation.We designed a retrospective cohort study to analyze the differences of IVF/ICSI outcomes between Fre-ET and FET in patients with a thin endometrium.Methods:The present study comprised of 1664 patients with a thin endometrium undergoing IVF/ICSI between January 2013 and December 2017 in our unit.Propensity score matching(PSM)was used to balance the influence of intergroup confounding factors and to compare the pregnancy outcomes of the matched patients in the two groups.The fresh embryo group and frozen embryo group consisted of 1037 and 627 women,respectively.After PSM,243 patients were included in the two groups,respectively.Results:The frozen embryo group showed a higher live birth rate(32.4%vs.19.2%,P<0.001;32.1%vs.18.9%,P<0.001),clinical pregnancy rate(42.9%vs.29.0%,P<0.001;43.2%vs.29.2%,P<0.001),and biochemical pregnancy rate(48.8%vs.36.1%,P<0.001;48.1%vs.37.9%,P<0.001)than the fresh embryo group before and after PSM.Conclusion:Our results demonstrated that for women with a thin endometrium who were undergoing IVF/ICSI,the live birth rate,clinical pregnancy rate,and biochemical pregnancy rate after FET were significantly higher than in the Fre-ET group.PART Ⅲ:Nomogram to predict an endometrial thickness above 7.5 mm in the frozen embryo transfer cycle of women with a thin endometriumObjective:The present study aimed to identify which characteristics of patients with a thin endometrium suggest the possibility of an EMT>7.5 mm in the subsequent frozen cycle.Methods:Data were collected from the university-affiliated Centre for Reproductive Medicine between January 2013 and September 2019.Multivariable logistic regression was used to generate the final prediction model and construct the nomogram.Model performances were quantified by discrimination and calibration.Results:The predictive variables that entered the final model were:hysteroscopic adhesiolysis history,polycystic ovary syndrome status,application of clomiphene in the ovarian stimulation process,the ovarian stimulation protocol and the endometrial preparation protocol.The receiver operating characteristic(ROC)curve for the final model and validation cohort was 0.760(95%confidence interval[CI]0.722-0.797)and 0.713(95%CI 0.664-0.759),respectively.Discrimination performed well in both the modelling and validation cohorts.Conclusions:In women with a thin endometrium(EMT ≤7.5 mm on HCG trigger day),the absence of a hysteroscopic adhesiolysis history,the presence of polycystic o vary syndrome,the application of clomiphene in the ovarian stimulation process,the application of a gonadotrophin-releasing hormone agonist short protocol,mild stimulation protocol,natural cycle protocol,and natural cycle for endometrial preparation are prognostic for an increased possibility of an EMT>7.5 mm in the subsequent FET cycle.PART Ⅳ:βKlotho promotes the development of intrauterine adhesions via PI3K/AKT signaling pathwayObjective:To investigate the expression difference of βKlotho(KLB)in IUA patients and normal endometrium,and its correlation with Fibronectin,a marker of fibrosis.Further studies were carried out to explore the potential relationship between KLB and endometrial fibrosis and how it functions,using the cell model of IUA established with TGF-β1.Methods:The endometrium of IUA patients and normal persons were collected,and the differential expression of KLB in endometrial stromal cells was detected by immunofluorescence,RT-qPCR and Western Blot.Human Endometrial Stromal Cells(HESC)were stimulated with TGF-β1 to construct IUA cell models and detect KLB protein expression.The molecular mechanism was studied by transfecting KLB plasmid.The effect of KLB on fibrosis was studied by Western Blot to detect Collage I and Fibronectin.The effect of KLB on proliferation was detected by CCK8 and EdU experiments.LY294002,an inhibitor of PI3K/AKT signal pathway,was used in the rescue experiment.Results:1.The expression of KLB protein is increased in endometrial stromal cells of patients with IUA.2.TGF-β1 stimulated HESC to construct IUA cell model and detected the increased expression of KLB protein.3.KLB can promote TGF-β1-induced HESC fibrosis4.KLB can promote HESC proliferation5.KLB activates the PI3K/AKT signaling pathway in HESC6.PI3K/AKT pathway inhibitors reduced the fibrosis and proliferation capacity of HESCConclusion:Our studies have shown that KLB protein is highly expressed in endometrial stromal cells of patients with IUA,which can promote stromal cell proliferation and promote cell fibrosis by activating the PI3K/AKT pathway,thus promoting the development of IUA.