LIN Si-ting, XIE Bao-guo

1. Hainan Medical University, Haikou 570100, China

2. Department of Reproduction, The First Affiliated Hospital of Hainan Medical College, Haikou 570100, China

3. Hainan Maternal and Child Health Hospital, Haikou 570100, China

Keywords:Hormone replacement therapythawed embryo transfer Endometrial typing Estradiol level Pregnancy outcome

ABSTRACT Objective: To investigate the influence of serum E2 level on pregnancy outcome on endometrial transformation day in hormone replacement therapy-thawed embryo transfer(HRT-FET) cycle. Methods: Totally 834 embryo transfer cycles of cleavage stage treated by HRT-FET in Reproductive Center of First Affiliated Hospital of Hainan Medical College from December 2014 to December 2019 were analyzed retrospectively. Results: The number of transplanted embryos in group A was higher than that in group B, and the difference was statistically significant (P<0.05);The thickness and serum P level of endometrial transformation day in Group B were significantly higher than those in Group A(P<0.05);There was no significant difference in clinical pregnancy rate, implantation rate, abortion rate,ectopic pregnancy rate, biochemical pregnancy rate and live birth rate between Group A and Group B(P>0.05);There was no statistical difference in general data between group A1 and group A2,group B1 and group B2(P>0.05);There was no significant difference in clinical pregnancy rate, implantation rate, abortion rate, ectopic pregnancy rate, biochemical pregnancy rate and live birth rate between group B1 and group B2(P>0.05);The clinical pregnancy rate, implantation rate and live birth rate in group A2 were significantly higher than those in group A1(P<0.05).Conclusions: Endometrial type A or B has no significant effect on pregnancy outcome in hormone replacement therapy-thawed embryo transfer cycle.;In type A endometrium, the serum E2>827 pmol/L on the day of endometrial transformation is helpful to improve the pregnancy outcome of frozen-thawed embryo transfer.

1. Introduction

Since the first report of successful pregnancy with frozen-thaw embryo transfer (FET) in 1983 [1], FET has been widely used in reproductive clinics. With the development of frozen embryo technology and the limitation of the number of embryos transferred,FET has become one of the important tools in the field of assisted reproductive technology, which has the advantages of avoiding repeated egg retrieval by patients, reducing the waste of embryos,increasing the cumulative pregnancy rate, and preventing the occurrence of ovarian hyperstimulation syndrome (OHSS) [2].Hormone replacement (HRT) regimen mainly uses exogenous estrogen to promote endometrial growth and has the advantages of controlled dosing regimen and low risk of cycle rate cancellation[3], therefore, hormone replacement therapy (HRT) has become the endometrial preparation regimen for frozen-thaw embryo transfer (FET) in most fertility centers.In recent years, with the rapid development of assisted reproductive technologies, numerous publications have shown [4] that high levels of serum E2 on the trigger day of a fresh cycle can have toxic effects on embryos, affect the window period for embryo implantation, and increase the risk of adverse obstetric clinical outcomes such as preterm birth and low birth weight infants.Liping Zong et al[5] included 6886 patients in a study of three endometrial preparation protocols for frozenthawed embryo transfer and showed that supplementation with excess estrogen has potentially deleterious effects on maternal and neonatal complications in late pregnancy, but did not address the effects of estrogen in early pregnancy.Based on current studies, the main factors affecting pregnancy outcome are age, embryo quality and endometrial tolerance, but it is controversial whether HRT-FET endometrial transformation day E2 level has an effect on pregnancy outcome, so this study focuses on the effect of endometrial transformation day serum E2 level on pregnancy outcome of high quality frozen-thawed cleavage embryo transfer in patients aged less than 35 years with different endometrial staging in hormone replacement cycles, and provides a theoretical basis for future clinical monitoring indicators.

2. Data and Methods

2.1 Object of the study

Retrospective analysis of 834 cycles of first-time oogenesis embryo transfer by HRT-FET treatment at the reproductive center of the First Affiliated Hospital of Hainan Medical College from December 2014 to December 2019, with all transfer cycles being high-quality embryo transfer cycles.Inclusion criteria: age <35 years; HRT-FET protocol was used; all transferred high-quality oogenesis embryos.Exclusion criteria: artificial cycles with down regulation treatment;endometrial transformation day with dominant follicular growth;genetic disorders such as chromosomal abnormalities in either sex;comorbidities such as genital malformations, endometrial polyps,submucosal fibroids, uterine adhesions,etc.

2.2 Research Methodology

2.2.1 Endothelial preparation

All patients underwent endometrial preparation using the measured HRT protocol. Estradiol valerate tablets were started on day 3-5 of menstruation if ultrasound indicated no abnormality,and estradiol valerate tablets (Tegretol) 4-6 mg were given orally daily, and endometrial morphology, thickness and hormone levels were monitored by regular ultrasound for 10-20 days. If the endometrial thickness is ≥7mm and ultrasound indicates no quality follicle growth, oral progesterone capsules 300mg/d and vaginal progesterone gel 90mg will be started to transform the endometrium.On the 4th day of endometrial transformation, the embryos at the cleavage stage will be thawed and transferred, and luteal support will be continued after the transfer, and if pregnancy continues until 8-10 weeks of gestation.

2.2.2 Hormone testing and endometrial staging monitoring

Blood was routinely drawn for E2 (pmol/L) and P (nmol/L) on the day of endometrial transformation, while ultrasound monitored endometrial thickness and typing.The reference endometrial staging criteria under ultrasound [6]: type A is a typical trilinear type with strong echogenic lines in the outer layer and center and hypoechoic or dark areas in the area between. type B is a homogeneous moderate echogenic type with intermittent strong echogenic midline in the uterine cavity. The endometrial morphology selected for this study was either type A or B endometrium with an endometrial thickness of ≥7 mm.

2.2.3 Oogenesis embryo score determinationThe embryos were classified into four grades of early embryos,namely grade I, II, III and IV, according to the number of oocytes,uniformity of cell size, regularity of shape, granular phenomenon of cytoplasm, and proportion of fragments. High-quality embryos are grade I and II embryos, all of which are treated with vitrification freezing and thawing method, and when resuscitated, embryo cell count >50% is considered successful for resuscitation and feasible for transfer.

2.2.4 Determination of pregnancy

Biochemical pregnancy was confirmed with a blood β-HCG test 12 days after transplantation. If serum β-HCG levels are elevated,transvaginal ultrasonography will be performed 28-35 days after transplantation to confirm clinical pregnancy (intrauterine or ectopic gestational sac or primitive ventricular pulsation on ultrasound).After confirmation of intrauterine pregnancy, luteal support is continued until 8-10 weeks of gestation. Fetal loss before 28 weeks of gestation is diagnosed as miscarriage.

2.2.5 Grouping

Patients were first divided into 236 cycles in group A (type A endometrium) and 598 cycles in group B (type B endometrium)according to endometrial progesterone conversion day endometrial typing, and then group A and group B were each divided into 2 subgroups according to median endometrial conversion day serum E2 levels, group A1 E2≤827 pmol/L (118 cycles); group A2 E2>827 pmol/L (118 cycles); group B1 E2≤867 pmol/L (303 cycles); group B2 E2>867 pmol/L (295 cycles).

2.2.6 Statistical analysisSPSS 23.0 statistical software was used for analysis, and t-test was used for measurement data and x2 test was used for count data. The results were statistically significant at P < 0.05.

3. Results

3.1 Comparison of patients' general information

There were statistically significant differences between groups A and B in endometrial thickness, number of embryos transferred,and P level of endometrial transformation day (P<0.05), and the number of embryos transferred was higher in group A than in group B (P<0.05), and endometrial thickness, endometrial transformation day, and years of infertility were significantly higher in group B than in group A (P<0.05); the remaining indexes, such as age, body mass index, and transformation endometrial day E2, were not statistically(P>0.05), see Table 1.There were no statistical differences (P>0.05)between groups A1 and A2 and B1 and B2 in terms of patient age,years of infertility, body mass index, number of embryos transferred,and daily P levels of transformed endometrium, as shown in Table 2.

Tab 1 General information of patients with different endometrial types(±s)

Tab 1 General information of patients with different endometrial types(±s)

Note: a is the difference is statistically significant. (P<0.05)

Group Group A (n=236) Group AB(n=598) t P Age 29.9±3.0 29.9±3.0 -0.23 0.82 Years of infertility 5.2±2.9 5.7±3.2 -2.1 0.04a Body mass index 21.0±2.6 21.0±2.8 -0.40 0.70 Endometrial thickness 9.5±1.5 9.8±1.6 -2.22 0.027a Number of embryos transferred 2.0±0.5 1.9±0.4 4.93 0.00a Endometrial transformation day P(nmol/L) 2.2±1.5 2.2±4.1 0.003 0.03a Endothelial transformation day E2(pmol/L) 1145.4±1290.0 1218.6±1449.0 -0.68 0.50

Tab 2 General data of patients with different serum E2 levels in type A and type B endometrium(±s)

Tab 2 General data of patients with different serum E2 levels in type A and type B endometrium(±s)

Group Group A1(n=118) Group A2(n=118) t P Group B1(n=303) Group B2(n=295) t P Age 30.0±3.0 30.1±3.0 -1.23 0.22 30.0±3.0 30.0±3.1 -0.10 0.90 Years of infertility 5.0±2.7 5.3±3.0 -0.78 0.44 5.8±3.4 5.6±3.1 0.85 0.40 Body mass index 21.2±2.7 20.8±2.6 1.25 0.21 21.2±2.8 21.0±2.7 1.24 0.22 Endometrial thickness 9.5±1.5 9.5±1.5 0.02 0.98 9.8±1.5 9.7±1.6 0.27 0.79 Number of embryos transferred 2.05±0.5 2.0±0.5 0.88 0.38 1.9±0.4 1.8±0.4 0.59 0.56 Endometrial transformation day P(nmol/L) 2.0±1.4 2.3±1.6 -1.56 0.12 2.4±5.6 1.9±1.3 1.68 0.09

3.2 Comparison of pregnancy outcomes between groups

There was no statistical difference in clinical pregnancy rate,implantation rate, miscarriage rate, ectopic pregnancy rate,biochemical pregnancy rate, and live birth rate between group A and group B, and between group B1 and group B2 (P>0.05), see Tables 3 and 4; clinical pregnancy rate, implantation rate, and live birth rate were significantly higher in group A2 than in group A1 (P<0.05), see Table 4.

Tab 3 Comparison of pregnancy outcomes of patients with different endometrial types(%)

Tab 4 Comparison of pregnancy outcomes of patients with different serum E2 levels in type A and B endometrium(%)

4 Discussion

A hot topic of research in recent years in hormone replacement frozen-thawed embryo transfer cycles has been the optimization of laboratory procedures for freezing and thawing embryos, leading to a significant increase in embryo survival, while little attention has been paid to targeting optimal endometrial preparation prior to transfer [7].The endometrium is an extremely important influence on embryo implantation, and sex hormones lay the foundation for implantation and placentation by regulating endometrial stromal cell (ESC) metamorphosis, while high levels of steroid hormones may interfere with endometrial stromal cell metamorphosis and formation of the endometrium at the maternal-fetal interface,affecting embryo implantation and placentation[8].In clinical work,despite the selection of quality embryos for transfer in the transfer cycle, repeated transfer failures remain a current challenge in the field of reproductive medicine, suggesting that ovarian sex hormone action makes differentiation of the endometrium in the receptive state problematic [9].A large body of literature suggests that high estradiol levels during controlled ovarian stimulation (COH) in the pre-in vitro fertilization-embryo transfer (IVF-ET) period may affect embryo implantation and placenta formation, increasing the risk of obstetric complications [8, 10, 11].However, there is considerable debate as to whether high E2 levels in HRT-FET cycles have an impact on pregnancy outcomes.Endometrial tolerance refers to the ability to allow embryo adhesion, implantation, and implantation,and good endometrial tolerance can improve the clinical pregnancy rate of embryo transfer. In the past, endometrial tolerance could be accurately evaluated by invasive methods such as endometrial histopathological biopsy and hysteroscopy, but the timing of embryo transfer was often not the cycle examined, so there was no guidance for evaluating endometrial tolerance in the proposed transfer cycle.Because of its non-invasive, simple and reproducible nature [12],ultrasonography has become a valuable tool to assess endometrial tolerance prior to embryo transfer, and the parameters commonly evaluated in clinical practice include endometrial thickness, staging and blood flow Doppler index.A number of scholars in the industry have explored the correlation between endometrial thickness,fractionation and embryo transfer outcome, and there is no uniform conclusion yet. Rona et al [13] found that endometrial staging was one of the factors affecting pregnancy outcome when endometrial thickness reached 7-12 mm and that type A endometrium helped to improve the pregnancy rate of embryo transfer by multifactorial logistic regression analysis.However, it has also been shown that endometrial thickness and fractionation do not affect pregnancy outcome, Lina et al [14] included data from 182 patients undergoing in vitro fertilization-embryo transfer in a prospective study, where endometrial thickness, fractionation, and blood flow were recorded by ultrasonography performed 8 hours prior to HCG administration,and patients were grouped according to the endometrial blood flow monitored by ultrasound, where patients with endometrial thickness<7 mm did not receive embryo transfer, and it was found that clinical pregnancy and embryo implantation rates were higher in patients who could monitor endometrial blood flow, and there was no correlation between endometrial thickness and morphology and pregnancy outcome.Tang C.H. et al. also concluded that there is no direct correlation between endometrial thickness and staging and pregnancy rate [15], but the aforementioned studies were fresh cycles with multiple ovulation protocols to stimulate follicular growth, and high levels of estrogen and progesterone may affect endometrial tolerance, causing bias in the findings.Ma Ling et al [16] grouped patients with frozen-thawed embryo transfer cycles according to endometrial morphology and endometrial thickness, respectively,and found that endometrial morphology and thickness did not predict pregnancy outcome by ROC curves and stepwise regression analysis.The mean endometrial thickness of A and B endometrium in this study was 9-12 mm; the thickness of endometrium on the day of transformation was significantly greater in patients with B endometrium than in those with A endometrium (P<0.05), and the number of embryos transferred was higher in group A than in group B, with a statistically significant difference (P<0.05); the clinical pregnancy rate was higher in A endometrium than in B endometrium,but the difference was not statistically significant (P>0.05),considering that the A The higher pregnancy rate of endometrium may be related to the number of embryos transferred.The results of this study demonstrate that when the endometrium reaches a certain thickness, endometrial morphology and thickness have no significant effect on pregnancy rate, which is consistent with the above study.Therefore, the correct assessment of endometrial tolerance in clinical work is a key issue, and the timing of embryo transfer should not be pursued as a matter of endometrial typing.

In recent years, the influence of serum E2 levels on endometrial tolerance on the day of endometrial transformation in artificial cycles has been controversial. The key to the HRT protocol is the regulation of sex hormones, mainly through exogenous supplementation of estrogen and progesterone sequential therapy to mimic the natural cycle, which causes the endometrium to show changes in the proliferative and secretory phases and can artificially regulate the window of embryo implantation. If the "implantation window" is missed, the endometrium no longer accepts embryo implantation,resulting in infertility or pregnancy failure [17].Simon C et al [18]scholars concluded that in hormone replacement therapy thawed embryo transfer, E2 is within a wide range allowing the endometrium to accept implanted embryos, but the exact E2 cut-off value is not known.Ma et al [19] concluded that fluctuations in estrogen within a certain range have no effect on endometrial tolerance, and that only levels below or above a certain level can affect the "timing of the implantation window" of the embryo.Most of the literature has shown that supplementation with supraphysiologic doses of E2 concentrations in HRT cycles adversely affects endometrial tolerance in terms of the expression of relevant biomolecules, thus affecting embryo implantation [20].In the present study, for patients with endometrium in type A, the clinical pregnancy rate, implantation rate, live birth rate, and ectopic pregnancy rate were higher in group A2 (E2>827 pmol/L) than in group A1 (E2≤827 pmol/L), and the difference was statistically significant (P<0.05).The results of this study demonstrate that pregnancy outcomes in HRT-FET cycles can be improved when E2 > 827 pmol/L on the day of endometrial transformation.However, due to inter-individual differences in drug metabolism and variability in endometrial sensitivity to estrogen,there is no uniform cut-off value for serum E2 levels on the day of endometrial conversion in artificial cycles to date.A retrospective analysis of 1502 patients treated with HRT-FET cycles in 2020 in China [21], grouped according to the characteristics of the distribution of E2 levels in the included sample data, showed that when serum E2 > 1101 pmom/L on the day of endometrial transformation would reduce the clinical pregnancy rate of thawed embryo transfer at the cleavage stage, suggesting that excessive serum E2 levels may be detrimental to embryo implantation.Fritz et al [22] analyzed the data of 110 patients treated with HRT-FET cycles and divided into three groups according to peak serum E2 levels: the 10th percentile with the lowest peak E2 (495pmol/L ≤ E2 ≤ 785pmol/L), the 11th-90th percentile with the highest peak E2 (785pmol/L ≤ E2 ≤2528pmol/L) The OP/LB rates (continuation rate/live birth rate) in the three groups were 54.6%, 32.9%, and 9.1%, respectively, and the Pearson correlation analysis showed that the increase in mean serum E2 levels was associated with the decrease in OP/LB rates, and it was concluded that high E2 levels had a negative impact.The above studies all suggest that high E2 levels affect pregnancy outcome in frozen-thawed embryo transfers, but the exact threshold remains controversial.In the data included in the current study, the mean E2 on the day of endometrial transformation was 1145 pmom/L, and E2> 2000 pmom/L was found in only 17 cases, which only confirms the effect of a small range of E2 levels on pregnancy outcome,and it is difficult to determine whether pregnancy outcome is also negatively affected at higher levels of E2.Liu et al [23] included data from 426 HRT-FET cycles treated with 1101 pmom/L, 2202 pmom/L, 3303 pmom/L, and 4404 pmom/L as cut-off groups,respectively, and compared the clinical pregnancy rate and embryo implantation rate of each group by chi-square test without statistical differences, suggesting that high E2 levels on the day of endometrial transformation may not be associated with embryo transfer pregnancy outcome; however, the small sample size in this study and the use of vaginal administration of estrogen supplementation in some patients during endometrial preparation may have caused bias in the results of this study, as it has been studied that oral combined with vaginal administration of estrogen supplementation improves endometrial tolerance and increases the chance of embryo implantation.

Recent studies have concluded that endometrial perfusion can better predict endometrial receptivity to embryos and is a predictor of good pregnancy outcome [24-27], and that the richer the endometrial blood flow and the better the uterine environment on the day of embryo transfer, the higher the clinical pregnancy rate [28].The limitation of this study is that the endometrial blood flow was not involved, and the effect of endometrial blood perfusion on pregnancy outcome was not studied; more study indicators need to be included in the follow-up study, and for patients with poor endometrial blood flow,the patient's condition should be comprehensively assessed, and the timing of embryo transfer should be carefully considered to avoid wasting embryos; in addition, the high level E2 data in the included sample size were too small to further analyze the effect of higher level E2 values on pregnancy outcome, and it is necessary to expand the sample size in the follow-up study and find E2 values that affect a certain range of pregnancy outcome to provide a basis for future clinical monitoring.In addition, due to the limited sample size of the study and the absence of some data to count the risk of developing obstetric comorbidities during pregnancy, it is necessary to collect multiple centers to expand the amount of study data for further deeper investigation.

In conclusion, there was no significant effect of endometrial type A or B on pregnancy outcome in hormone replacement therapythawed embryo transfer cycles, and endometrial perfusion better predicted endometrial receptivity to embryos and was a predictor of good pregnancy outcome; in type A endometrium, when serum E2 >827 pmol/L on the day of endometrial transformation contributed to improved pregnancy outcome in frozen-thawed embryo transfer.

Authors' contributions: Si-Ting Lin designed the article, collected and analyzed the data, and wrote the paper; Bao-Guo Xie was responsible for the quality control of the article.

There is no conflict of interest in this article.