Roles of estradiol levels on the day of human chorionic gonadotrophin administration in the live birth of patients with frozen embryo transfer

Abstract Background Estradiol (E2) is an important hormone in women. Changes of serum E2 levels may affect the endometrial receptivity for embryo implantation and thus affect pregnancy outcomes. This study was to assess the association between serum E2 levels on the day of human chorionic gonadotrophin (HCG) administration and live‐birth rates in patients with frozen embryo transfer (FET). Methods Totally 2071 women receiving long protocols of long‐acting gonadotropin‐releasing hormone (GnRH) agonists were enrolled. According to the E2 levels on the day of HCG administration, these patients were divided into four groups: 676 cases of E2 ≤ 3051 pg/mL in Q1 group, 676 cases of 3051 pg/mL < E2 ≤ 4558 pg/mL in Q2 group, 675 cases of 4558 pg/mL < E2 ≤ 6718 pg/mL in Q3 group, and 674 cases of E2 > 6718 pg/mL in Q4 group. The clinical indicators including female age, body mass index (BMI), duration of infertility, infertility styles, treatment protocols, hormone levels, total antral follicle count, endometrial thickness, top‐level embryos, and live‐birth rates were analyzed, and multivariable logistic model was conducted to select significant variables. Results Significant differences were observed for the female age (OR = 0.965, 95% CI: 0.946‐0.985, P < .001), total antral follicle counts (OR = 1.025, 95% CI: 1.008‐1.043, P = .004), transferring what day of embryos (OR = 1.242, 95% CI: 1.137‐1.356, P < .001), endometrial thickness (OR = 1.058, 95% CI: 1.004‐1.115, P = .035), top‐level embryos (OR = 1.416, 95% CI: 1.157‐1.731, P = .001), and E2 levels on HCG day >6781 pg/mL (OR = 1.344, 95% CI: 1.069‐1.690, P = .011) between live‐birth and non‐live‐birth groups. The area under the curve (AUC) for E2 levels on HCG day was 0.558, the sensitivity was 54.75%, and the specificity was 55.10%. Conclusion Serum E2 level on HCG day was an independent predictor of live‐birth achievement in patients with FET.


| INTRODUC TI ON
Infertility is a highly prevalent problem of global proportions which is estimated to affect as many as 186 million people worldwide.
Although more than half of the world's childless cases are caused by male infertility, the social burden is still on women. The infertility rate of the population at childbearing age is up to 15%-20%, and over 20 million couples have fertility issues in China. 1 Assisted reproductive techniques (ARTs), as an emerging treatment for infertility, are achieved more attention in recent decades. Since the birth of the first test-tube baby in 1978, 2 in vitro fertilization and embryo transfer (IVF-ET) is gradually applied in clinic. The previous study revealed that the successful pregnancy rates after IVF-ET were 39% in America. 3 Frozen embryo transfer (FET) is an assisted reproductive technology procedure in which can reduce the risk of ovarian hyperstimulation syndrome (OHSS), maximize the utilization of embryo in oocyte retrieval per cycle, 4 and provide a choice for patients without conducting the fresh embryo transfer. Evidences showed that FET can obtain better outcomes in perinatal period and live birth, such as ectopic pregnancy, premature birth, low birth weight neonates, and placenta previa. [5][6][7] Implantation is a complex process regulating by multiple factors of which human chorionic gonadotrophin (HCG) is one of the most important factors. 8 HCG, a glycoprotein, serves as a surrogate for luteinizing hormone (LH) used to induce the maturation and ovulation of oocytes in ovarian stimulation cycles, 9 which is an early embryonic signal secreted via the embryo before implantation in primates.
HCG can stimulate the connective tissue of follicle softened to easily separate the oocyte cumulus complex from the follicle wall, and enable to perform the aspiration during oocyte retrieval. Estradiol (E 2 ), one of the three main self-produce estrogens, is an important hormone in women. E 2 has a crucial influence on the growth and function of the female reproductive system and the mammary gland in physiological conditions. Evidences showed that the serum E 2 level is an integral part of evaluating response to controlled ovarian stimulation, and the elevated E 2 levels may affect the endometrial receptivity for embryo implantation owing to controlled ovarian hyperstimulation. 10 In recent decades, the association between serum E 2 levels on the day of HCG administration on pregnancy outcomes in IVF was assessed. 11,12 To the best our knowledge, however, the influence of E 2 levels on live-birth rates in embryo implantation has been rarely reported. Herein, we investigated the effect of E 2 levels on the day of HCG administration on live-birth rates in patients with FET.

| Patients
A total of 2071 patients with FET admitted to Chenggong Hospital Affiliated to Xiamen University were enrolled from 2012 to 2017 in this study. According to the E 2 levels on the day of HCG administration, these patients were divided into four groups, containing 676 cases of E 2 ≤ 3051 pg/mL in Q 1 group, 676 cases of 3051 pg/mL < E 2 ≤ 4558 pg/mL in Q 2 group, 675 cases of 4558 pg/ mL < E 2 ≤ 6718 pg/mL in Q 3 group, and 674 cases of E 2 > 6718 pg/ mL in Q 4 group. A number of baseline clinical indicators, including female age, body mass index (BMI), duration of infertility, infertility styles, treatment protocols, hormone levels, total antral follicle count, endometrial thickness, top-level embryos, and live-birth rates were noted. Endometrial thickness was detected using B-scan ultrasonography. This study was approved by the Institutional Review Board (IRB) of Chenggong Hospital Affiliated to Xiamen University.

| Scoring criteria for embryo selection
On day 3 of cleavage stage, the scoring criteria for embryo selection were based on the previous study. 13 The cell number of toplevel embryos was 7, 8, and 9, respectively. In the merged period (M), the cells presented the uniform size, regular shape, intact zona pellucida, uniform and clear cytoplasms, with no particles, and the debris ranged 0%-5%.
The blastular scoring on day 5 was on the basis of the Gardner score. 14 The top-level blastocysts were defined as fully expanded blastocysts, namely the blastular cavity was full of embryos with larger volume and thinner zona pellucida, or the hatching blastocysts part of which were escaped from the zona pellucida. The number of cells in the inner cell group was large and closely arranged, the upper cells of the trophoblast were composed of more cells, and the structure was tight.

| Ovarian stimulation
The ovarian stimulation was conducted using the long protocols of long-acting GnRH agonists in this study. 15 In the luteal (on day 7 after ovulation) or follicular phases (on day 1 or 2 during menstruation), 1.25-3.75 mg of the long-acting GnRH agonists (Diphereline, Tianjin Yipusheng Pharmaceutical Co., Ltd.) was intramuscularly injected.
The levels of serum E 2 , follicle-stimulating hormone (FSH), LH, and vaginal ultrasound were detected after 14-28 days. After hormone levels were up to the down-regulation standard (E 2 < 50 pg/mL, FSH and LH < 5 IU/mL, endometrial thickness <5 mm), human menopausal gonadotrophin (HMG) (Zhuhai Lizhu Pharmaceutical Co., Ltd.) and/or recombinant human FSH (Gonal-f, Serono Pharmaceutical Co., Ltd.) were injected at 112.5-225 IU/d. According to the development of follicles and the levels of serum sex hormone, the HCG trigger was added at the appropriate time.

| Natural cycle
According to the previous monitoring of ovulation, the vaginal ultrasound was used to monitor the development of follicles on day 9-11 of menstruation, which was suitable for patients with regular ovulation of menstruation. When the diameter of the follicle was about 16 mm, the urine LH was monitored and the E 2 , LH, and P were detected by blood drawing until the day of ovulation. On the day of ovulation, the patients took 20 mg of dydrogesterone (Abbott), twice daily. On day 3 after ovulation, the embryos in the cleavage stage were thawed and transplanted. The blastocysts were thawed and transplanted on day 5 after ovulation.

| Hormone replace treatment (HRT)
On the second day of menstruation, the patients underwent B-scan ultrasonography, which showed no abnormalities in the endometrium, so the interference factors including ovarian cysts, larger follicles, were excluded. The patients orally took 6-8 mg of progynova (Bayer), and the dosage was appropriately adjusted when the endometrial growth was monitored by B-scan ultrasonography after 8-10 days. On day 14-16, when the endometrial thickness was ≥8 mm, 40 mg/d of progesterone (Xianju) was intramuscularly injected, then dydrogesterone (Abbott) was orally taken 20 mg/bid the next day. The embryos in cleavage stage were performed the FET on day 5 of progesterone injection. The blastocysts were conducted the frozen blastocyst transfer on day 7 of progesterone injection. After transplantation, the original dose of hormone replacement and luteum support were continued.
The levels of these hormones including basal FSH, basal LH, basal prolactin (PRL), basal E 2 , basal testosterone (T), basal progesterone (P), and basal gonadotropin (Gn) were detected in this study.

| Statistical analysis
Statistical analysis was performed using SPSS 24.0 (SPSS, Inc).
Continuous data were presented as the median (P 25 , P 75 ) and analyzed by Kruskal-Wallis test. Categorical data were presented as n and analyzed using chi-squared test. One-way analysis of variance (ANOVA) with least significant difference post hoc test was utilized to compare the groups. Multivariable logistic regression analysis was used to identify the variables that may contribute to the livebirth rate. The predictive value of E 2 day HCG on live-birth rates was assessed by receiver operating characteristic (ROC) curve analysis. P < .05 was considered to indicate a statistically significant difference.

| The characteristics of patients with FET
A total of 2071 FET patients receiving the long protocols of long-acting GnRH agonists were enrolled in this study. The characteristics of patients were shown in Table 1. There were significant differences in age, BMI, duration of infertility, treatments (IVF, ICSI, and RICISI), basal FSH, basal LH, basal T, total Gn, total antral follicle counts, transferring what day of embryos, top-level embryos, and live-birth rates among the four groups (P < .05).

| Multivariable logistic analysis of livebirth rates
The live-birth rate as the dependent variable, and the independent variables including female age, total antral follicle counts, transferring what day of embryos, endometrial thickness, toplevel embryos, and E 2 levels on HCG day were analyzed (Table 3).

| D ISCUSS I ON
Previous studies discovered that high serum E 2 level may be not in favor of embryo implantation 16 ; nevertheless, effects of serum E 2 levels on live-birth rates have been not conclusive during the FET cycle. In this present study, we evaluated the effect of serum E 2 levels on the day of HCG administration on live-birth rates in patients with FET. Our findings revealed that the total antral follicle counts, transferring what day of embryos, endometrial thickness, and toplevel embryos were risk factors, and the female age was a protective factor of the live birth in FET cycle. Additionally, serum E 2 level on HCG day was an independent predictor of live-birth achievement.

TA B L E 2
Single-factor analysis of livebirth rates endometria to some extent. 26 Early studies founded that E 2 was essential for the receptive establishment 27 and endometrial proliferation, and played a determinant role in implantation, decidualization, and early embryo development. 28 The concentration dependence of serum E 2 levels on pregnancy rates has been mentioned. 29 Previous studies reported the prognostic value of E 2 level at the time of HCG triggering during the fresh IVF/ICSI cycles, which may have an impact on the number of oocytes and top-level embryos. 30 The finding we discovered that the top-level embryo was a risk factor for the live birth, and as the increased number of the top-level embryos, the live-birth rates were increased. In addition, the endometrial thickness in the live-birth group was higher than that in the nonlive-birth group. Previous studies showed that increased endometrial thickness can significantly improve pregnancy outcomes among women undergoing IVF-ET. 31,32 On the day of HCG administration or embryo transfer, the endometrium pattern and thickness were obviously associated with the rates of clinical pregnancy and embryo implantation. 33 Our results founded that serum E 2 level on the day of HCG administration was an independent predictor of live-birth achievement, and high serum E 2 level on HCG day was associated with decreased live-birth rates in patients with FET. The impaired oocyte maturation and the decreased pregnancy achievement caused by elevated E 2 levels have been mentioned. 34,35 Clinical studies suggested that high E 2 levels could adversely affect perinatal outcomes. The occurrence of complications associated with abnormal placenta was significantly increased when serum E 2 level was at >10 000 pmol/L. 36 Previous studies showed that the alterations of these hormones such as basal LH, basal PRL, and basal P levels were associated with pregnancy in embryo transfer. [37][38][39] In our study, there were no statistical differences in the levels of basal LH, basal PRL, and basal P between the live-birth group and the no-live-birth group. Then, the significant variables with single-factor analysis were further analyzed using stepwise multivariable logistic anal- TA B L E 3 Multivariable logistic analysis of live-birth rates F I G U R E 1 The ROC curve of serum E 2 level on the day of HCG administration on live-birth rates in FET patients E 2 levels to improve the live-birth rates. There was a limitation that should be warranted caution for interpreting the data in this study.
Our investigation was a retrospective study based on a single center, which included only 2071 infertile women. Thus, prospective multicenter studies with larger samples should be needed for further verification of the effect of serum E 2 level on HCG day on live-birth rates in clinic.

| CON CLUS ION
In summary, the total antral follicle counts, transferring what day of embryos, endometrial thickness, and top-level embryos were risk factors, and the female age was a protective factor of the clinical live birth in FET cycle. Our findings demonstrated that high serum E 2 level on HCG day was associated with decreased live-birth rates in patients with FET.

AUTH O R CO NTR I B UTI O N S
All authors read and approved the final manuscript. HXC designed the study, wrote and critically reviewed the manuscript. JLC and LLL involved in the data curation, validation and formal analysis. XHS involved in supervision and validation.

This study was approved by the Institutional Review Board (IRB) of
Chenggong Hospital Affiliated to Xiamen University and had been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

I N FO R M E D CO N S E NT
Informed consent was obtained from all individual participants included in the study.