- Academic Editor
Background: Whether gonadotropin-releasing hormone agonist (GnRH-a)
pituitary suppression improves clinical outcomes in non-endometriosis patients
undergoing frozen embryo transfer remains controversial. The objective of this
study is to investigate whether GnRHa combined with hormone replacement treatment
(HRT) compared to HRT alone can improve the clinical outcomes of frozen-thawed
embryo transfer in patients without endometriosis. Methods: This is a
retrospective cohort study. This study involved 2178 frozen-thawed embryo
transfer (FET) cycles of non-endometriosis patients between January 2018 and
December 2019, of these cycles, 1535 were GnRHa-HRT combined cycles and 643 were
HRT alone cycles. The primary outcomes were the clinical pregnancy and live birth
rates. SPSS software (version 23.0, IBM Corp., Chicago, IL, USA) was
used for data analysis. Results: Single-factor analysis showed
that the live birth and implantation rates -were higher in the GnRHa-HRT group
than those in the HRT group (p
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.
This is a retrospective study included 2178 FET cycles performed between January 2018 and December 2019 at the Center of Assisted Reproductive Medicine Center of Sichuan Provincial People’s Hospital. The inclusion criteria were as follows: (1) patients with endothelial preparation using GnRHa-HRT or HRT, (2) patients under 40 years of age. The exclusion criteria were as follows: (1) patients with endometriosis or/and adenomyosis, (2) patients with a body mass index (BMI) over 30 kilograms per square meter, (3) patients with hydrosalpinx on one or both sides, (4) patients with intrauterine adhesion, (5) patients with congenital malformations of the uterus, (6) patients with uterine fibroids, (7) chromosome abnormalities of either one of the couples, (8) patients who received oocytes. Of these 2178 FET cycles, 1535 were GnRHa-HRT cycles and 643 were HRT cycles. The decision to implement GnRHa-HRT or HRT was based on the physician’s approval of the protocol, the patient’s time and affordability, and previous embryo transfer history. 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).
Daily oral administration of estradiol valeratewas 3 mg (Progynova, Bayer
Schering Pharma, Berlin, Germany) twice a day starting from the 2nd or 3rd day of
menstruation. Additional doses, starting from 2 mg/d were given if the
endometrial thickness was
3.75 mg triptorelin acetate (Diphereline, Ipsen Pharma Biotech, Paris, France) was administrated by injection at a dose of 3.75 mg on the 2nd–5th day of menstruation. Progynova was given in the HRT cycles after 28 days.
In both the two groups, luteal support was started 4 days before the transfer of the split embryo and 5 days before the transfer of the blastocyst. Luteal support was achieved by daily injection of 60 mg progesterone (Zhejiang Xianju Pharmaceutical Co Ltd., Taizhou, Zhejiang, China). Alternatively, a 90 mg daily progesterone sustained-release vaginal gel (Crinone, Fleet Laboratories Ltd., Watford, United Kingdom) vaginally.
All embryos were cryopreserved using the vitrification method.
In the afternoon 1 day prior to transfers, the D3 embryos were thawed. On the morning of the embryo transfer day, the D5/D6 blastocysts were thawed. The survival of more than half of blastomeres was defined as embryonic survival; While the absence of blastomere damage was defined as whole embryonic survival.
Biochemical pregnancy was diagnosed by serum ß-HCG
Clinical pregnancy was defined as observation of a gestational sac via ultrasound after 30 days of transfer. The embryo implantation rate was the ratio of the number of gestational sacs to the number of embryos transferred.
The SPSS software (version 23.0, IBM Corp., Chicago, IL, USA) was used
for data analysis. Continuous variables were described as the
mean
Table 1 provides a description of the patient’s baseline characteristics. The
BMI, mean number of embryos transferred, the ratio of primary infertility to
secondary infertility, and the blastocyst transfer rate between the two groups
were not significant different. There was a significant difference (p
Characteristic | GnRHa-HRT (n = 1535) | HRT (n = 643) | p value | ||
Female age (y) | 30.29 |
29.75 |
–3.052 | 0.002 | |
BMI (kg/m |
22.06 |
22.08 |
0.143 | 0.886 | |
Infertility type (n,%) | 0.947 | 0.330 | |||
Primary infertility | 800 (52.1) | 320 (49.8) | |||
Secondary infertility | 735 (47.9) | 323 (50.2) | |||
The endometrial thickness on the day of transfer (mm) | 10.16 |
9.48 |
–10.164 | ||
The mean number of embryos transferred | 1.76 |
1.73 |
–1.142 | 0.254 | |
Blastocyst transfer rate | 73.50% | 73.30% | 0.013 | 0.910 |
Values represent means
GnRHa, gonadotrophin-releasing hormone agonist; HRT, hormone replacement treatment; BMI, bodymass index.
Table 2 showed the pregnancy outcomes. The HCG positivity, clinical pregnancy,
and first-trimester abortion rates were not significant different between the two
groups. However, there was a statistically significant difference (p
Pregnancy outcomes | GnRHa-HRT (n = 1535) | HRT (n = 643) | p value |
HCG positivity rate (%) | 70.2 (1078/1535) | 66.1 (425/643) | 0.057 |
Clinical pregnancy rate (%) | 60.5 (928/1535) | 56.6 (364/643) | 0.096 |
Embryos implantation rate (%) | 44.1 (1191/1115) | 38.6 (430/2669) | 0.002 |
First-trimester miscarriage rate (%) | 11.0 (102/928) | 12.9 (47/364) | 0.331 |
Mid-to-late-term miscarriage rate (%) | 1.7 (16/928) | 3.6 (364) | 0.044 |
Values represent percentages (no./no.) of the patients.
HCG, human chorionic gonadotropin; GnRHa, gonadotrophin-releasing hormone agonist; HRT, hormone replacement treatment.
Table 3 showed the patients’ obstetrical outcomes. There was a higher rate of
live birth rate in the GnRHa-HRT group (52.3%) compared to the HRT group
(46.3%), showing statistically significant differences (p
Obstetric outcomes | GnRHa-HRT (n = 1535) | HRT (n = 643) | p value |
Multiple pregnancy rate (%) | 26.7 (248/928) | 24.5 (89/364) | 0.402 |
Live birth rate (%) | 52.3 (803/1535) | 46.3 (298/643) | 0.011 |
Preterm delivery rate (%) | 28.0 (225/803) | 24.2 (72/298) | 0.2 |
Values represent percentages (no./no.) of the patients.
GnRHa, gonadotrophin-releasing hormone agonist; HRT, hormone replacement treatment.
Multivariate logistic regression analysis was conducted with clinical pregnancy and live birth rates as dependent variables and endometrial preparation protocol, average age, endometrial thickness on embryo transfer day, number of embryos transferred, BMI and infertility type as independent variables. Table 4 showed the results. Clinical pregnancy and live birth rates were significantly affected by mean age and number of embryos transferred. Endometrial thickness, BMI, and infertility type did not influence live birth and clinical pregnancy rates. Clinical pregnancy rate did not differ significantly between the two methods of endometrial preparation, but the GnRHa-HRT group had a higher live birth rate than the HRT group.
Independent variables | Clinical pregnancy rate | Live birth rates | |||
Value | OR value (95% CI) | p value | OR value (95% CI) | p value | |
Endometrial preparation protocols | |||||
HRT | Ref. | Ref. | |||
GnRHa-HRT | 1.174 (0.968–1.424) | 0.103 | 1.289 (1.065–1.560) | 0.009* | |
Female age (y) | 0.953 (0.931–0.976) | 0.949 (0.928–0.972) | |||
The endometrial thickness on the day of transfer | 1.021 (0.965–1.081) | 0.467 | 1.005 (0.950–1.063) | 0.868 | |
BMI | 1.011 (0.973–1.050) | 0.587 | 1.003 (0.966–1.042) | 0.865 | |
Infertility type | |||||
Primary infertility | Ref. | Ref. | |||
Secondary infertility | 1.166 (0.976–1.393) | 0.091 | 1.106 (0.928–1.318) | 0.258 | |
Number of embryos transferred | 1.491 (1.231–1.805) | 1.503 (1.241–1.820) |
*p
CI, confidence interval; OR odds ratio; BMI, body mass index; GnRHa, gonadotrophin-releasing hormone agonist; HRT, hormone replacement treatment.
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
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.
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