The efficacy of tamoxifen in the treatment of thin endometrium in frozen-thawed embryo transfer cycle

Background: Thin endometrium is known to adversely affect reproductive performance. There is no agreement about a consensus treatment on thin endometrium. Tamoxifen(TAM) has a positive effect on endometrium when used as ovulation induction agent. Little information is available regarding its use in patients with thin endometrium during frozen-thaw embryo transfer (FET) cycles. This study was designed to evaluate the effectiveness of TAM on women with thin endometrium in frozen-thaw embryo transfer cycles. Methods: A total of 345 thin endometrium women were retrospectively analyzed during their FET cycles. Among them 190 received TAM protocol (TAM 20 mg per day for 5 days) and 155 hormone replacement therapy (HRT) protocol (estradiol val erate 6 mg/d for 14 to 21days). Endometrial thickness and pregnancy outcome were compared between the two groups. Result(s): The endometrial thickness in TAM group was significantly higher compared with HRT group. The clinical pregnancy rate, implantation rate, ongoing pregnancy rate and live birth rate were significantly higher in TAM group than HRT group. Conclusion(s): In patients of recurrent thin endometrium, tamoxifen treatment in endometrium preparation may be a successful alternative approach

regenerative medicine 7-11 . Among them, exogenous estrogen treatment is the most commonly used method 12 . Many factors may influence the effectiveness of estrogen treatment and the subsequent reproductive outcomes 12 13,14 . A high dose and a longer duration of estrogen administration may be effective for some of these patients 12 . However, supraphysiologic estrogen levels resulted from this treatment before FET may have a negative impact on embryo-endometrium asynchrony and consequently reduce implantation rates 15,16,17,18 . Moreover, a high rate of breakthrough bleeding (19), increased risk of some types of tumor such as endometrial cancer (20), deep venous thrombosis (21) and decreased downstream consequences on the quality of placentation and perinatal outcome result from supraphysiologic estrogen levels affect its use in endometrial preparation for FET (22). In addition, some patients were insensitive to estrogen treatment. Alternative methods should be considered for endometrium preparation in these women during their FET cycles.
Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) with chemical properties similar to clomiphene citrate(CC) used in clinic as a highly effective agent for the prevention and treatment of breast cancer 23 . TAM has also been used in ovulation induction 24-26 . In contrast to CC, whose anti-estrogenic effects at the level of the endometrium sometimes result in thin endometrium (27), ovulation induction with tamoxifen improves endometrial thickness in women with a history of thin endometrium (28). However, little information is available regarding its use during FET cycles in that kind of patients. The aim of this study is to evaluate the efficacy of TAM in improving endometrial thickness, optimizing clinical outcome in frozen-thaw embryo transfer cycles. We propose that the result of our study may contribute to the treatment of infertility patients with thin endometrium.

Study design
This retrospective study was performed during the period from January 2016 till August 2017 at the Reproductive Medical Center of Anhui Provincial Hospital. The study was approved by the institutional ethics committee of Anhui Provincial Hospital.
A total of 345 frozen-thaw embryo transfer cycles were included for this analysis. In our center, thin endometrium is diagnosed when the maximal endometrial thickness(EMT) is ˂7 mm, dominant follicles are 18 mm in diameter in ovulatory cycles, or after 12 to 16 days of estradiol (E2) replacement(4-6 mg, Progynova; Bayer Schering Pharma, Roubaix, France). Eligible patients had previously demonstrated a thin endometrium during their 1-2 previous cycles at our center. All women underwent diagnostic hysteroscopy prior to this FET cycle, and intrauterine synechiae were absent in all women. Thus, no participants had uterine or endometrial abnormalities except for a thin endometrium at the time of the FET cycles. Also, patients with repeated implantation failures were excluded. All the patients were between 20-40 years old.
At the discretion of physicians and/or patients' preference, endometrium was prepared with TAM or HRT as described below. Patients in TAM group were fully counseled regarding the novel use of TAM.
Women with multiple types of FETs following the same fresh IVF/ICSI cycle were excluded from the analysis. This study was completely anonymous, obviating the need for informed consent. Otherwise eligible patients with incomplete clinical data were excluded.

The FET cycle
In the group of TAM, 20 mg per day was giving from day 5 of the menstrual cycle for 5 days. Vaginal ultrasound examinations were performed on day 10 of the cycle to monitor the number and size of developing follicles and endometrial thickness. Ovulation was induced with 10,000 I.U. Hcg when the leading follicle reached 18-22 mm and the endometrium thickness reached 7 mm. FET was performed four days later. If no dominant follicle developed, human menopausal gonadotropin was given from day 10 up to Hcg injection. In the group of hormone replacement treatment cycle, oral estradiol valerate (progynova,Schering, German) was taken 6 mg/d from menstrual cycle day 2-3. An ultrasound assessment was done 12 to 14 days later to assess endometrium thickness. Progesterone 40 mg/d which would be changed to 60 mg/d 2 days later, was given to transform the endometrium, provided the endometrial thickness exceeded 7 mm. If the endometrium thickness is not adequate, endometrial preparation continued with step-up dose of E2 or adding vaginal estradiol (Femoston, Solvay pharmaceuticals B.V.) 1-2 mg/d till the endometrium thickness reaching 7 mm. Cycles were canceled in patients whose endometrial thickness remained <7 mm after 21 days of continuous estradiol administration. FET was performed 4 days later.

Embryo vitrification and thawing
After fresh embryo transfer, surplus Day 3 or blastocyst embryos underwent vitrification. For Day 3 embryos, our laboratory procedure of vitrification and warming was the same as the method used for human oocytes as reported by Tong et al. previously (29). For blastocysts, a glass micro-needle was used to collapse the blastocyst before vitrification. The following steps were the same as for the Day 3 embryos. Embryo quality was graded as 'good', 'reasonable','moderate' or 'poor' according to the number of cells, degree of fragmentation and renewed development of the embryo. This standard was based on the ESHRE Istanbul consensus on embryo assessment (30). A score was given to each embryo from 3 (good) to 1(moderate).

Definition of pregnancy
A serum β-hCG assay 11-14 days after ET. Clinical pregnancy was defined as the presence of a gestational sac on transvaginal ultrasound.
Clinical miscarriage was defined if the pregnancy terminated before 12 weeks of gestational age.
Implantation rate was defined as the number of intrauterine sacs divided by the number of embryos transferred.
Ongoing pregnancy was defined as gestations that reached 20 weeks or more. Live birth was defined as give birth to an infant ≥24 weeks' gestation.

Statistical analysis
All analyses have been performed using IBM Spss statistics 21.For continuous variables, Student's ttest and Mann-Whitney test were used for data with homogeneous variance and heterogeneous variance respectively. The x 2 test was used for categorical variables. The variable with greater clinical importance and larger variance was selected for multivariate assessment. Logistic regression analyses were conducted to identify independent correlates between each possible confounding factor, especially protocols for endometrium preparation and pregnancy outcome after adjusting for other confounders that were identified in our univariate analysis. A p-value <0.05 was considered statistically significant.

Results
We analyzed FET cycles of 345women from our unit and 160 of these women (46.37%) achieved clinical pregnancy. Women who achieved a clinical pregnancy were younger and had a shorter duration of infertility. Also, they achieved more oocytes and had a thicker endometrium than their no clinical pregnancy counterparts. There were no significant differences in the other characteristics such as BMI (body mass index), type of infertility, diagnosis of infertility duration of endometrium preparation, embryo score. The demographic and clinical characteristics of these cycles are summarized in Table I. 190 women received TAM and 155 received HRT. A summary of important aspects of the treatment outcome is presented in Table II. There were no significant differences in age, duration of infertility, BMI, type of infertility, the major indication for IVF, duration of stimulation and the number of oocytes retrieved. Statistically significant differences between the two groups were noted in terms of duration of endometrium preparation, embryos transferred, endometrial thickness and its increase in the research cycle compared with the previous cycle as well as embryo score. The clinical pregnancy rate, implantation rate, ongoing pregnancy rate and live birth rate were significantly higher in TAM group than HRT group.

Discussion
The aim of our study was to evaluate the effect of TAM in patients with a thin endometrium in FET cycles. By univariate and multivariate analysis, our results found a superior pregnancy outcome following FET in a TAM ovarian stimulation cycle compared to HRT cycle in women with thin endometrium.
In our study we observed that the durations of endometrium preparation were 4 day shorter in the TAM FET group (10 days) than in the HRT FET group (14 days). This phenomenon is interesting but hard to explain. We hypothesize that ovarian stimulation with TAM may stimulate a faster development of the dominant follicle(s). More prospective studies are needed to explore its clinical relevance.
Our results showed that endometrium thickness was much more improved in TAM group than HRT group. TAM has been reported to improve endometrium thickness while induce ovulation. Kasey Reynolds switched infertile women undergoing OI (ovulation induction) with CC who have an endometrial thickness of <7 mm to TAM for OI in a subsequent cycle and found that endometrial thickness improved (31) .Wang et al compared TAM and CC in ovarian stimulation cycle in combination with HMG (human menopausal gonadotrophin) and found that TAM treated patients had a significantly increased endometrial thickness (28).
The literature on the treatment of thin endometrium with TAM is rather sparse. Chen X et al used TAM in 3 women who showed a repeated unresponsive thin endometrium and resulted in endometrium expansion to at least 7.7mm and conception 32 . Soon after, another study by the same group showed TAM increased the endometrial thickness from 6.5 to 8.8mm in women with thin endometrium undergoing FET 33 . Ke H et al explore the effect of TAM in patients with a thin endometrium in FET cycle. Their study included 226 women who had an EMT of less than 7.5mm in their previous cycles. After the use of TAM, EMTs were significantly improved. When stratified by different previous endometrium preparation protocols, EMT increased from 6.11±0.98mm to 7.87±1.48 in NC group, from 6.24±1.01 to 8.22±1.67 in HRT group and from 6.34±1.03 to 8.05±1.58mm in OI group 34 . In line with the previous data, our result showed that the EMT in TAM group was9.011 5.9-16.4 mm which was significantly thicker than the EMT in HRT groups. In addition, TAM group showed more improvement of EMT in the research cycle compared with previous cycles than HRT group. Some authors found that TAM exposure promotes endometrial cell proliferation through estrogen and non-estrogen pathway. In estrogen signaling pathway, TAM stimulate the GPER (GPR30 Estrogen Receptor), which in turn, activates the SF1 (Steroidogenic Factor 1), which is a transcription factor that induce aromatase expression in endometrial cells (35). In none-estrogen signaling pathway, a study found that TAM up-regulates the expression of Ki67 and IGF-1,markers of proliferation 36 .
Our study focus on demonstrating the clinical effect of TAM in FET cycles either. Different from the previous study, our study compared the effect of TAM with the mostly commonly used protocol-HRT protocol rather than describe the reproductive outcome only so that this effect can be analyzed precisely. Not only the clinical pregnancy rate and the implantation rate but also the ongoing pregnancy rate and live birth rate of TAM group were significantly higher than HRT group. Logistic regression analysis showed that after adjusting for the confounders that were identified in our univariate analysis, protocols for endometrium preparation is independent factors correlate with

Conclusions
The results of this study showed that beyond endometrial expansion, TAM protocol beneficially affect pregnancy outcome in women with thin endometrium undergoing FET.  Table Ⅲ

Declarations
Multivariable analysis of variables for clinical pregnancy and ongoing pregnancy