Infertility is estimated to affect approximately one in six people around the world at some point in their lives [1]. In vitro fertilization-embryo transfer (IVF-ET) and related techniques are among the most effective treatments for infertility. Patients undergoing fresh cycles may exhibit endometrial abnormalities, high progesterone, pelvic infection after oocyte retrieval, and other complications, which can result in poor clinical outcomes [2]. On the other hand, frozen-thawed embryo transfer (FET) is performed to provide a better implantation environment and to avoid wasting embryos. In addition, whole embryo freezing plays a crucial role in reducing the risk of ovarian hyperstimulation syndrome, ensuring the safety of IVF [3]. Therefore, FET has become an essential treatment option for assisted reproductive technology (ART).

The success of FET is determined by three key factors: embryo quality, endometrial receptivity, and embryo-endometrium synchronization [4]. With improvements in the technique of frozen-thawed embryos, damage to the embryos caused by freezing and thawing has been reduced. Therefore, endometrium receptivity has become a hot topic in the study of frozen embryos.

The endometrial preparation protocols for the FET cycles may affect endometrial receptivity. Commonly used methods to prepare the endometrium include nature cycle (NC), hormone replacement treatment (HRT), ovarian stimulation, and hormone replacement treatment combined with GnRH pretreatment (GnRHa-HRT). HRT has become the most popular method of endometrial preparation in reproductive centers due to its low time restriction and cycle cancellation rate. Gonadotropin-releasing hormone agonist (GnRH-a) exhibits therapeutic effects on endometriosis by inhibiting the hypothalamic-pituitary-ovarian axis so it is considered to improve the clinical outcomes of frozen embryo transfer in patients with endometriosis theoretically, which has been proved in some clinical studies [5, 6]. However, the role that GnRH-a plays in patients undergoing FET without endometriosis, is still controversial.

Some studies on FET revealed that GnRHa-HRT promoted endometrial receptivity. A recent retrospective cohort study [7] indicated that the GnRHa pretreatment can increase live birth, ongoing pregnancy, and clinical pregnancy rates in patients with advanced age and recurrent implantation failure. A single-blind randomized controlled trial (RCT) by Aghahoseini M et al. [8] in patients with hyperandrogenic polycystic ovary syndrome (PCOS) found a higher rate of ongoing pregnancy and a lower rate of miscarriage in the GnRHa downgrading group in the frozen embryo cycles.

But an RCT study [9] that included 188 patients with PCOS showed that there were no significant differences in pregnancy outcomes, incidence of obstetric complications and neonatal malformations between the GnRHa-HRT and HRT groups. Another RCT study [10] included 236 frozen-thawed embryo transfer cycles, randomized to the modified natural group and the GnRHa-HRT group, and all the patients tranferred only one euploid blastocyst. The results of the study showed that the clinical pregnancy, miscarriage, and live birth rates of the two groups were comparable.

The number of participants in the above RCT studies was small. In this study, 2178 FET cycles of GnRHa-HRT or HRT were retrospectively analyzed to investigate the clinical outcome of patients treated with two different endometrial preparation protocols. All patients were diagnosed as non-endometriotic.

FET has become an essential aspect of ART. A successful frozen embryo transfer is determined by three key factors: embryo quality, endometrial receptivity, and embryo-endometrium synchronization [4]. As the methods of endometrial preparation are crucial to endometrial receptivity, it is a fundamental step of the FET.

The nature cycle (NC), the hormone replacement treatment (HRT), the ovarian stimulation, and hormone replacement treatment with the gonadotrophin-releasing hormone agonist pretreatment (GnRHa-HRT) are commonly used to prepare the endometrium [11]. The nature cycle (NC) is the most physiological method and is liable to be performed with fewer costs, but the cycle cancellation rate is high [12]. Women suffering from ovulatory dysfunction with insensitivity to orally administrated estrogens or contraindication of estrogens might benefit from ovarian stimulation. The hormone replacement treatment is the most popular FET method of preparing the endometrium due to its extensive clinical application and time flexibility of transfer scheduling [13]. The hormone replacement treatment is categorized into GnRHa-HRT (Pituitary down-regulation by injecting GnRHa) and HRT. The GnRHa protocol is known to improve the clinical outcome in patients with endometriosis due to the therapeutic effects of GnRHa on endometriosis, but the most effective endometrial preparation protocol remains controversial.

A Cochrane review [14] findings showed that the GnRHa-HRT protocol resulted in a higher live birth rate than the HRT protocol, but neither miscarriage nor ongoing pregnancy. A study [15] showed that GnRHa downregulation is associated with higher rates of clinical pregnancy and live birth rates in FETs with male-factor infertility. Another RCT [16] showed that the GnRHa-HRT group and the HRT group did not differ in clinical pregnancy rates, implantation rates, early pregnancy loss rates, or live birth rates.

However, these two RCT studies had small sample sizes. In this study, it was revealed that there was a significant increase in live birth rate with the GnRHa-HRT protocol, but the clinical pregnancy rate was comparable between the two groups. According to previous studies, the possible reasons for the increased pregnancy outcome of FET when using GnRHa-HRT are as follows: Hormone replacement therapies may still result in the development of follicles that produce endogenous luteinising hormone (LH), which could lead to an earlier implantation window. However, the administration of GnRHa prevented follicular growth by suppressing the pituitary [17]. It was found that GnRHa downregulation significantly increased mRNA and protein expression of HOXA10, MEIS1 and LIF in endometrium [18]. HOXA10, MEIS1 and LIF are marks of endometrial receptivity. There has been research showing that GnRHa increases cytokinesis and integrin expression in endometrium. Neukomm et al. [19] revealed that the increased number of cytokinesis on endometrial cells and up-regulated integrins of endometrium were proved to enhance endometrial receptivity. Research [20] showed that GnRH-a could directly bind to its receptors on the endometrium to regulate decidual endometrial stromal cell motility, thereby improving embryo implantation. Studies showed that endometrial cells expressed higher levels of adhesion molecules when GnRHa bound to its receptors on the endometrium, to inhibit the production of embryotoxic autoantibodies, and increase the implantation rate [21, 22]. The results of a recent retrospective study [23] showed that GnRHa pretreatment can improve the clinical pregnancy and live birth rates in FET cycles. In addition, the study examined the mRNA expression of cytokines associated with endometrial receptivity in the endometrium of patients whose tansfer was canceled on the day of FET. The findings of the study suggest that pretreatment with GnRHa significantly increases the expression of IL-6, IL-11, LIF and integrin $\alpha$v$\beta$3 mRNAs in the endometrium. Compared to the group without GnRHa pretreatment, the expression of these mRNAs and markers was significantly higher in the groups with GnRHa pretreatment. These results suggest the potential utility of GnRHa pretreatment in the regulation of endometrial receptivity.

Although several RCTs have investigated whether GnRHa downregulation affects treatment outcomes in FET cycles for patients without endometriosis, results have varied, even for patients with the same infertility etiology, creating confusion in the clinical setting for clinicians. Additionally, these previous RCTs have suffered from small sample sizes. Therefore, it is necessary to conduct more research to clarify the confusion surrounding how GnRHa downregulation may affect the outcomes of FET. The greatest strength of our study is its large sample size. Using a larger sample size is more accurate because it captures a broader range of patients and can better represent the entire patient population. Our study revealed that in the FET cycles, a significantly increase in live birth rate was observed among GnRHa-HRT among GnRHa-HRT patients without endometriosis when compared with HRT patients. However, these findings need to be confirmed by large-scale prospective randomized controlled trials in the future. It is also necessary to analyse patients in groups according to different infertility etiologies.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

QL: design research and revision of paper. YW: collate original date and write paper, and was a major contributor in writing the manuscript. HXX: collate original date. PS: application of statistical to analyze study data. All authors contributed to editorial changes in the manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work. All authors read and approved the final manuscript.

Informed consent was obtained from all subjects prior to their participation in the study, in accordance with the Declaration of Helsinki. The research protocol was approved by the Reproductive Ethics Committee of Sichuan Provincial People’s Hospital (NO. 202209).

The authors thank colleagues in the embryology laboratory for providing the embryo freezing and thawing process. The authors thank the nursing team for following up on the clinical outcomes of the patients.

This work was supported by the Clinical and Translation Research of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, China [grant number 2021LY19].

The authors declare no conflict of interest.