Inspite of the widespread application of FET cycles, no significant improvement in terms of the pregnancy rate has been observed in comparison to the fresh cycles. The present study found that the GnRHa FET group produced a significantly higher percentage of endometrium with triple line pattern, and an increased implantation and clinical pregnancy rate, as compared to the HRT FET cycles without GnRHa group. Logistic regression analysis showed the GnRHa FET group to be significantly associated with an increased chance of clinical pregnancy compared with HRT FET without GnRHa group. These results demonstrated that the administration of a single dose of long-acting GnRHa in early follicular phase of the same FET cycle can improve the clinical outcome of HRT cycles, possibly by improving the receptivity of the endometrium. The relatively large amount of data provide evidence for the clinical application of this method in FET cycles. However, RCTs will be needed to prove the results presented in this study.
The method using FET with pituitary downregulation has been used before the HRT cycles without GnRHa suppression. There is no consensus on which method is consistently better for the outcome of pregnancy. As compared to a natural or modified natural cycle protocol, data from a retrospective 1391 cycles reported that the synthetic HRT protocol with GnRHa was associated with a higher live birth rate in the blastocyst-stage of the FET cycles . However, there were no differences in the reproductive outcomes between the two methods in majority of studies, which included patients with regular ovulation [15–19]. Similar negative results were observed between the GnRHa HRT and HRT alone cycles in patients with regular menstrual cycles [5, 8] and those with functioning ovaries [7, 9, 11]
It has been reported that pituitary suppression when initiated in the middle luteal phase in HRT cycles results in higher clinical pregnancy and live birth rates than the HRT cycles without prior GnRHa therapy in patients with regular menstrual cycles . Hebisha et al. reported that the administration of a GnRHa for HRT FET during endometrial preparation increased the implantation and pregnancy rates in patients with undefined ovary functions , with a similar pregnancy outcome being observed in other studies [6, 12]. Our study also found that the downregulation of FET produced a higher percentage of endometrium with triple line pattern, and a higher implantation and clinical pregnancy rates as compared to the HRT FET cycles without GnRHa. These patients had abnormal ovulation and irregular menstrual cycles.
Differently, we administered long-acting GnRHa during menstruation in the same FET cycles, while short-acting GnRHa was administered on a daily basis in the middle phase in the three above mentioned studies with positive outcomes [4, 10, 14]. Le et al administered medroxy-progesterone acetate for 10 days in order to induce menstruation and a half dose of long-acting GnRHa on the third day of medroxy-progesterone acetate. The administration of exogenous estradiol was initiated on the third day of menstruation. They found comparable pregnancy outcomes to those observed for modified natural cycles . Nekoo et al and Prato et al administered 3.75 mg of long-acting GnRHa at the mid-luteal phase (day 21) of the previous cycle, resulting in a similar pregnancy rate between the HRT and GnRHa HRT FET cycles [5, 7]. Xie et al. administered 3.75 mg of long-acting GnRHa on day 3 of menstruation. After 28 days, estrogen and progesterone were administered for endometrium preparation. The data showed that the resultant clinical pregnancy and live birth rates were higher in the GnRHa HRT FET cycle than that in the HRT FET cycle. In Qi’s study, 3.75 mg of long-acting GnRHa was injected in day 2 or 3 of menstruation. HRT was initiated 28 days later. They found pregnancy outcomes were improved in patients with endometriosis and PCOS. However, in patients aged 38 years or older, Dong et al failed to find a significant difference in pregnancy and live birth rate between two groups. An RCT reported that in patients with repeated implantation failure, short term GnRHa from 21 day of menstruation did not increase the pregnancy and implantation rates of subsequent HRT cycles. In our study, we did not indicate the patients and there were no difference between the two groups in terms of patients parameters including ages, percentage of PCOS and endometriosis.
A 28 days interval between the administration of GnRHa and estrogen was thought to be sufficient as the pituitary suppression exerted by GnRHa was relieved upon embryo implantation, wherein the GnRHa-HCG system could play an important role in embryo implantation and development . Palmerola et al administered 1000 mg of leuprolide acetate on day 2 of the cycle. Estrogen administration was initiated 14 days later when the rate of suppression was adequate. As a result, the implantation and the clinical pregnancy rates were found to be satisfactory . Our results suggested that an interval of 14 days between the GnRHa and estrogen administration did no harm to the embryo implantation but rather increased the percentage of triple line endometrium, the implantation rate and clinical pregnancy rate. An interval of 14 days reduced the patient waiting time to start endometrial preparation. We also speculated that GnRHa does direct effect in endometrial development to improve receptivity. Maybe shorter interval or administration of GnRHa and HRT together can also improve pregnancy outcome of FET. It deserves to be studied in further.
Additionally, in this study, we found that 14 days after administering a single dose of GnRHa (3.75 mg), dominant follicles developed and the endometrium grew to a normal thickness in 37 patients. This is most likely due to the flare-up effect of GnRHa, which stimulated the development of the follicles. After direct HCG triggering or after necessary HMG stimulation, ET was found to produce 54.1% of CPR, suggesting that the downregulation of GnRHa did not affect embryo implantation at this situation; thus, the “cyst” need not to be punctured and the cycles need not to be cancelled under these conditions. However, the limited data available in our study suggests a tendency for a high early miscarriage rate. We cannot exclude the effect that downregulation by GnRHa could have on luteal function. The effect of an additional estrogen supplementation will need to be investigated in future studies.
The mechanism by which GnRHa improves the outcome of pregnancy in HRT FET cycles remains to be elucidated. The GnRH/ GnRHR system occurs in the endometrium, ovaries, and human preimplantation embryos. The expression of this system supports its physiological regulatory role in the functioning of the corpus luteum, endometrium receptivity, the capability of trophoblast invasion, and the processes related to embryo implantation [25, 26]. Some studies implied that GnRHa facilitates embryo implantation by enhancing luteal secretion. However, the results obtained in our study cannot be explained on that basis. In this study, the exogenous estrogen and progesterone were administered when there was no corpus luteum, except for the 37 patients who were undergoing ovulation. Meanwhile, after 8 weeks of administering long-acting GnRHa, the pituitary was found to begin to recover its functions . In our study, ET was performed at about 31–40 days after administering GnRHa, which is when the pituitary was in a state of suppression and the corpus luteum could not be stimulated.
A meta-analysis  including six RCTs showed that a single or continuous injection of short-acting GnRHa during the luteal phase for luteal support increased the pregnancy and live birth rates in both the agonist and antagonist protocols. Fujii et al continuously administered GnRHa during the luteal phase until 14 days after oocyte retrieval in the long protocol IVF . The serum concentrations of estradiol and progesterone on the day of embryo transfer and 7 days after oocyte retrieval were similar to those obtained using the long protocol alone. However, the implantation and live birth rates of the GnRHa group were significantly higher. A possible explanation could be the direct action of the GnRHa in regulating embryo invasion and development, endometrial receptivity, and cross talk between the embryo and the endometrium. In a murine model, ovarian stimulation decreased the expression of the endometrial expression of integrin beta-3 subunit, leukaemia inhibitory factor, and the implantation rate during the implantation window. These were partially restored by the administration of the GnRHa. These results suggest that the GnRHa plays an important role in improving the endometrial receptivity . An in vitro study  demonstrated that the GnRH-II agonist promoted the cell motility of human decidual endometrial stromal cells by binding to the GnRH-I receptor, leading to the phosphorylation of protein kinase 1/2 and the activation of MMP-2 and MMP-9. These findings suggest that the GnRH-II agonist has a strong effect on the endometrial decidualization and embryo implantation. In this study, an increased percentage of triple line endometrium after GnRHa suppression suggests an improved endometrial receptivity. Meanwhile, GnRHa may improve the invasive and secretary capabilities of the embryonic trophoblast cells, thus facilitating embryo implantation and cross talk in the uterus. After administration of GnRHa during pregnancy, the serum levels of human chorionic gonadotropin (hCG) were found to be significantly elevated. The results indicated that GnRH could stimulate the release of hCG by the trophoblast cells of the placenta . Peng et al showed that GnRH-induced RUNX2 expression could strengthen the invasive capacity of the human extravillous trophoblast cells by modulating the expression of MMP9 and MMP2 .
This study has several limitations. Since it was a retrospective study and not a prospective randomized study, undetected biases may occur. Thus, we performed logistic regression analysis to reduce biases. A relatively large number of patients were included under strict criteria. We did not evaluate some of important data, such as we did not compare the live birth rates between the groups, and we did not conduct routine tests to determine the serum hormone levels on the days of progesterone supplementation, embryo transfer, implantation window, or pregnancy testing. Owing to the small patient sample size, the clinical outcomes between GnRHa + ovulation and GnRHa + HRT were inconclusive. However, the measurements of endometrial thickness and clinical pregnancy rate were satisfactory in these groups of patients. The rate of miscarriage need be evaluated in further studies.
In conclusion, the administration of a single dose of long-acting GnRHa during the early follicular phase and initiating HRT 14 days later can improve the clinical outcome of the FET cycles. The mechanism of this process may rely on the direct effect of the GnRHa on the regulation of embryo invasion and development, endometrial receptivity, and the cross talk between the embryo and the endometrium. However, further RCTs will be needed in order to validate the results presented in this study.