Correlation between different endometrial preparation protocols and pregnancy outcome of frozen embryo transfer in patients with polycystic ovary syndrome: a retrospective study

Abstract

Objective

We retrospectively analyzed the correlation between different endometrial preparation protocols and pregnancy outcomes in patients with polycystic ovary syndrome (PCOS) who underwent frozen embryo transfer (FET).

Methods

A total of 200 PCOS patients who underwent FET were divided into HRT group (n = 65), LE group (n = 65), GnRHa + HRT group (n = 70) according to different endometrial preparation protocols. The endometrial thickness on the day of endometrial transformation, the number of embryos transferred, and the number of high-quality embryos transferred were compared among the three groups. The pregnancy outcomes of FET in the three groups were compared and analyzed, and a further multivariate logistic regression model was used to analyze the factors influencing FET pregnancy outcomes in PCOS patients.

Results

Endometrial thickness on the day of endometrial transformation, clinical pregnancy rate and live birth rate in GnRHa + HRT group were higher than those in the HRT group and LE group. The results of multivariate regression analysis showed that the pregnancy outcome of PCOS patients undergoing FET was significantly associated with the patient’s age, endometrial preparation protocols, number of embryos transferred, endometrial thickness, and duration of infertility.

Conclusion

Compared with HRT or LE alone, GnRHa + HRT protocol results in higher levels of endometrial thickness on the day of endometrial transformation, clinical pregnancy rate, and live birth rate. Female age, endometrial preparation protocols, number of embryos transferred, endometrial thickness, and duration of infertility are determined as factors influencing pregnancy outcomes in PCOS patients undergoing FET.

Keywords:

• Polycystic ovary syndrome
• different endometrial preparation protocols
• frozen embryo transfer
• pregnancy outcome
• correlation analysis

Introduction

Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder attributed to both genetic and environmental factors and is a main cause of infertility in women of childbearing age [1]. Its prevalence in women of reproductive age in China has been reported to be 5.6%, with a high incidence in the age group of 20–35 years [2]. PCOS is clinically characterized by ovulatory dysfunction and hyperandrogenism [3]. PCOS patients currently receive symptomatic treatment, but spontaneous recovery or recovery after taking medicine cannot be achieved. Assisted reproductive techniques have offered hope to infertile PCOS patients, but also brought many negative effects to patients [4]. The incidence of ovarian hyperstimulation syndrome and the cancelation rate of fresh embryo transfer are relatively high in PCOS patients receiving in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI), increasing the pain and economic burden of patients [5–7].

With the development of science and technology, frozen embryo transfer (FET) has gradually become an important complementary technique to IVF/ICSI [8], especially for PCOS patients with high ovarian response, high number of retrieved oocytes, and prone to ovarian hyperstimulation syndrome. Increasing PCOS patients are successfully pregnant and delivered by frozen-thawed embryo transfer [9]. The thaw survival rate can reach 19% − 99% [10] as embryo freezing technology develops [10]. Reasonable endometrial preparation before FET is the key to successful pregnancy of PCOS patients [11], and the commonly used FET endometrial preparation protocols are natural cycle, ovulation induction (OI), hormone replacement treatment (HRT), and gonadotropin-releasing hormone agonist (GnRHa) for pituitary down-regulation combined with HRT (GnRHa + HRT) [12]. For the characteristics of oligo-ovulation or anovulation, obesity, insulin resistance, and specific endocrine and metabolic disorders in PCOS patients, HRT is preferred by most reproductive centers. OI is a choice for patients with poor response to HRT or contraindications to estrogen use, and GnRHa + HRT is selected for some patients with repeated implantation failure, or with comorbidities of endometriosis or adenomyosis [13, 14]. However, the clinical outcomes of these preparation protocols have been variable due to the patient’s age, embryo quality, and endometrial factors. Therefore, this study compared the effect of HRT, OI using letrozole (LE), GnRHa + HRT on the pregnancy outcome of FET in PCOS patients and explored influencing factors of pregnancy outcome, providing a reference for clinical application of different FET endometrial preparation protocols.

Materials and methods

Baseline information of patients

The clinical data of PCOS patients who underwent IVF/ICSI-FET in our hospital were collected. Two hundred eligible patients were included in this study according to the inclusion and exclusion criteria. According to different endometrial preparation protocols, the patients were divided into HRT group (n = 65), LE group (n = 65), and GnRHa + HRT group (n = 70). Their general clinical data including age, duration of infertility, type of infertility, body mass index, hormone levels including luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol (E2), and quality of transferred frozen embryos were obtained. This study had been reviewed and approved by the Ethics Committee of Subei People’s Hospital of Jiangsu Province (2021ky068).

Inclusion criteria of subjects included [15]: (1) patients aged < 35 years; (2) after excluding other endocrine diseases, a diagnosis of PCOS was made based on 2 of 3 Rotterdam ESHRE/ASR criteria [16]: polycystic ovaries; hyperandrogenism and/or elevated androgen levels; anovulation or oligo-ovulation; (3) patients showed indications for IVF/ICSI-FET surgery; (4) patients with complete medical records; (5) patients and their families voluntarily participated in this study, understood the related rights and risks, and signed informed consent. Exclusion criteria included: (1) patients with significant hydrosalpinx, intrauterine adhesions, uterine malformations, endometriosis; (2) abnormal chromosome in one partner; (3) recurrent miscarriage and repeated implantation failure (defined as 3 or more spontaneous abortions and embryo implantation failure).

Preparation protocols

In the HRT group, routine vaginal ultrasonography and basic sex hormone tests were performed on the 2nd to 3rd day of the menstrual cycle. Estradiol valerate tablets (manufacturer: Bayer HealthCare Co., Ltd. Guangzhou Branch, China; NMPA approval number: J20130009; strength: 1 mg) were given orally at 2–3 mg/time, twice daily. The dosage was adjusted according to endometrial thickness, with a maximum dose of 4 mg. When estradiol valerate tablets were orally administered for > 10 days and endometrial thickness was > 7 mm, dydrogesterone tablets (manufacturer: Solvay Pharmaceuticals, Netherlands; approval number: JX20010415; strength: 10 mg) were added (20 mg/time orally, once daily) combined with 8% progesterone vaginal sustained-release gel (manufacturer: Merck Serono Limited; approval number: H20140552; strength: 90 mg) (90 mg vaginally, once daily) for endometrial transformation. Cleavage-stage embryos were transferred 4 days later, and medication for luteal support was continued until 10 weeks after pregnancy and gradually reduced.

In the LE group, LE tablets (manufacturer: Jiangsu Hengrui Pharmaceuticals Co., Ltd.; NMPA approval number: H19991356; strength: 2.5 mg) were orally administered at 2.5 mg/d for 5 consecutive days from the 3rd to 5th day of menstruation, and endometrial thickness and follicle size were monitored by ultrasound from the 6th day. If the diameter of the dominant follicle was less than 10 mm, 50 to 75 units of low-dose menotrophins (manufacturer: Livzon (Group) Pharmaceutical Factory; NMPA approval number: H10940097; strength: 75 IU) were added. If the follicle diameter was ≥ 10 mm, ultrasound monitoring was continued until the follicle diameter was ≥ 17 mm and the endometrial thickness was ≥ 8 mm and then 10,000 IU of chorionic gonadotropin (manufacturer: Livzon (Group) Pharmaceutical Factory; NMPA approval number: S20210010; strength: 6500 IU) was intramuscularly injected at 9 o‘clock that night. D3 frozen-thawed embryo transfer was performed 4 days later.

In the GnRHa + HRT group, routine vaginal ultrasonography and basal sex hormone tests were performed on the 2nd to 3rd day of the menstrual cycle, and long-acting GnRHa (manufacturer: IPSEN France; approval number: H20090274; strength: 3.75 mg) was intramuscularly injected at 3. 75 mg once daily, and vaginal ultrasound and sex hormones were reexamined 14 days later. After achieving E2 < 50 pg/mL and progesterone < 0.9 ng/mL, estradiol valerate tablets 4 mg (manufacturer: Bayer HealthCare Co., Ltd. Guangzhou Branch, China; NMPA approval number: J20130009; strength: 1 mg) was given orally. When estradiol valerate tablets were applied for > 10 days and endometrial thickness > 7 mm, dydrogesterone tablets (manufacturer: Solvay Pharmaceuticals, Netherlands; approval number: JX20010415, strength: 10 mg) was added (oral route, twice daily) combined with 8% progesterone vaginal sustained-release gel (manufacturer: Merck Serono Limited; approval number: H20140552; strength: 90 mg) (90 mg vaginally, once daily) for endometrial transformation. Cleavage-stage embryos were transferred 4 days later, and medication for luteal support was continued until 10 weeks after pregnancy and gradually reduced.

Outcome measures

Outcome measures included endometrial thickness on the day of endometrial transformation, number of embryos transferred, number of high-quality embryos transferred, live birth rate, embryo implantation rate, biochemical pregnancy rate, clinical pregnancy rate, ectopic pregnancy rate, and abortion rate.

Statistical analysis

SPSS 22.0 statistical software was applied for statistical analysis. Measurement data were expressed as mean ± standard deviation (SD); t-test was used for comparison between the two groups, while one-way analysis of variance was for comparison between multiple groups. Enumeration data were expressed as rate (%) or constituent ratio, and the χ² test or Fisher’s exact test was used for comparison between groups. Additionally, logistic regression models were employed for analyzing factors associated with pregnancy outcomes. p < 0.05 indicated a statistically significant difference.

Results

Baseline data of patients

The three groups showed no significant differences in age, duration of infertility, type of infertility, body mass index, quality of frozen embryos transferred, basal LH level, basal FSH level and basal E2 level (p > 0.05) (Table 1).

Table 1. Comparison of baseline data of patients.

	HRT group (n = 65)	LE group (n = 65)	GnRHa + HRT group (n = 70)	χ^2/F/t	P
Age				5.182	0.075
<28 years	25	28	40
≥28 years	40	37	30
Duration of infertility (year)	5.51 ± 2.04	5.03 ± 2.02	4.80 ± 2.33	1.907	0.151
Type of infertility				0.071	0.965
Primary	50	50	55
Secondary	15	15	15
Body mass index	22.42 ± 1.22	22.24 ± 1.30	22.54 ± 1.15	1.039	0.356
Quality of frozen embryos transferred				0.897	0.639
All high-quality embryos	58	59	60
Mix of high- and low-quality embryos	7	6	10
Basal LH (IU/L)	9.21 ± 2.03	8.98 ± 2.23	9.26 ± 2.28	0.302	0.740
Basal FSH (IU/L)	6.29 ± 1.13	5.90 ± 1.07	5.96 ± 1.25	2.189	0.115
Basal E2 (pg/mL)	45.30 ± 10.00	45.54 ± 8.98	47.30 ± 9.89	0.874	0.419

Measurement data were expressed as mean ± standard deviation, and enumeration data were expressed as number of cases. LH, luteinizing hormone; FSH, follicle-stimulating hormone; E2, estradiol. HRT, hormone replacement treatment; LE, ovulation induction using letrozole; GnRHa + HRT, gonadotropin-releasing hormone agonist for pituitary down-regulation combined with HRT.

Comparison of endometrial thickness on the day of endometrial transformation, number of embryos transferred, and number of high-quality embryos transferred among the three groups

Endometrial thickness on the day of endometrial transformation was (0.89 ± 0.09) cm in the HRT group, (0.88 ± 0.12) cm in the LE group, and (0.93 ± 0.11) cm in the GnRHa + HRT group, with the thickest endometrium in the GnRHa + HRT group. There were significant differences in endometrial thickness on the day of transformation among the three groups (p < 0.05); however, no significant differences were identified in the number of embryos transferred and the number of high-quality embryos transferred among the three groups (p > 0.05) (Table 2).

Table 2. Comparison of endometrial thickness on the day of endometrial transformation, number of embryos transferred and number of high-quality embryos transferred among the three groups.

	HRT group (n = 65)	LE group (n = 65)	GnRHa + HRT group (n = 70)	χ^2/t	P
Endometrial thickness on the day of endometrial transformation (cm)	0.89 ± 0.09	0.88 ± 0.12	0.93 ± 0.11	4.273	0.015
Number of embryos transferred (n)	1.95 ± 0.78	1.84 ± 0.69	2.10 ± 0.84	1.840	0.162
Number of high-quality embryos transferred (n)	1.04 ± 0.21	1.03 ± 0.17	1.04 ± 0.20	0.110	0.896

Measurement data were presented as mean ± standard deviation (SD). HRT, hormone replacement treatment; LE, ovulation induction using letrozole; GnRHa + HRT, gonadotropin-releasing hormone agonist for pituitary down-regulation combined with HRT.

Comparison of pregnancy outcomes of three endometrial preparation protocols

The GnRHa + HRT group showed a higher clinical pregnancy rate (67.10%) and live birth rate (60.00%) compared with the LE group (clinical pregnancy rate: 41.50%; live birth rate: 33.85%) and the HRT group (clinical pregnancy rate: 38.50%; live birth rate: 30.77%), and there were significant differences in the clinical pregnancy rate and live birth rate among the three groups (p < 0.05). However, no significant differences were found in embryo implantation rate, biochemical pregnancy rate, ectopic pregnancy rate, and abortion rate among the three groups (p > 0.05) (Table 3).

Table 3. Comparison of pregnancy outcomes of three endometrial preparation protocols.

	HRT group (n = 65)	LE group (n = 65)	GnRHa + HRT group (n = 70)	χ^2	P
Clinical pregnancy rate (%)	38.50 (25)	41.50 (27)	67.10 (47)	13.533	0.001
Live birth rate (%)	30.77 (20)	33.85 (22)	60.00 (42)	14.450	0.001
Embryo implantation rate (%)	76.92 (50)	78.46 (51)	84.29 (59)	1.284	0.526
Biochemical pregnancy rate (%)	3.08 (2)	3.08 (2)	4.29 (3)	0.070	0.966
Ectopic pregnancy rate (%)	3.08 (2)	1.54 (1)	2.86 (2)	0.339	0.844
Abortion rate (%)	15.38 (10)	15.38 (10)	17.14 (12)	0.105	0.949

Enumeration data were presented as n (%). HRT, hormone replacement treatment; LE, ovulation induction using letrozole; GnRHa + HRT, gonadotropin-releasing hormone agonist for pituitary down-regulation combined with HRT.

Factors influencing clinical pregnancy in frozen embryo transfer

According to success or failure of pregnancy, all patients were divided into a successful pregnancy group (n = 99) and a failed pregnancy group (n = 101). The two groups showed significant differences in age, endometrial preparation protocols, number of embryos transferred, endometrial thickness, and duration of infertility (p < 0.05), but not in basal LH level, basal FSH level, basal E2 level, and type of infertility (p > 0.05) (Table 4).

Table 4. Factors influencing clinical pregnancy in frozen embryo transfer.

	Successful pregnancy group (n = 99)	Failed pregnancy group (n = 101)	χ^2/t	P
Age			72.193	<0.001
<28 years	76	17
≥28 years	23	84
Endometrial preparation protocol			13.533	<0.001
HRT	25	40
LE	27	38
GnRHa + HRT	47	23
Number of embryos transferred (n)	2.23 ± 0.71	1.71 ± 0.75	5.012	<0.001
Endometrial thickness(cm)	0.95 ± 0.10	0.84 ± 0.08	8.464	<0.001
Duration of infertility (year)	3.48 ± 1.05	6.70 ± 1.69	−16.145	<0.001
Basal LH (IU/L)	9.40 ± 2.22	8.91 ± 2.12	1.618	0.107
Basal FSH (IU/L)	6.06 ± 1.17	6.04 ± 1.16	0.104	0.917
Basal E2 (pg/mL)	44.93 ± 9.69	47.20 ± 9.50	−1.655	0.098
Type of infertility			0.852	0.356
Primary	74	81
Secondary	25	20

Measurement data were expressed as mean ± standard deviation, and enumeration data were expressed as number of cases. HRT, hormone replacement treatment; LE, ovulation induction using letrozole; GnRHa + HRT, gonadotropin-releasing hormone agonist for pituitary down-regulation combined with HRT; LH, luteinizing hormone; FSH, follicle-stimulating hormone; E2, estradiol.

The five factors with significant differences between the two groups were included for logistic regression analysis. As shown in Table 5, patient age (OR = 0.129, 95% CI = 0.036 − 0.459, p = 0.002), number of embryos transferred (OR = 3.686, 95% CI = 1.478 − 9.192, p = 0.005), endometrial preparation protocols (p = 0.014), duration of infertility (OR = 0.173, 95% CI = 0.088 − 0.342, p < 0.001), and endometrial thickness (OR = 3.676, 95% CI = 1.760 − 7.680, p = 0.001) were independent factors for clinical pregnancy in PCOS patients undergoing FET. Further analysis of endometrial preparation protocols showed that with HRT protocol as a reference, the OR of LE protocol was 0.307 (95% CI 0.070-1.341, p > 0.05) and that of GnRHa + HRT protocol was 5.731 (95% CI 0.952-34.498, p < 0.05). This result suggested that the GnRHa + HRT protocol was an important factor in promoting the clinical pregnancy of FET in PCOS patients.

Table 5. Multivariate analysis of frozen embryo transfer clinical pregnancy in PCOS patients.

Variables	B	S.E,	Wals	P	OR	95%CI
Age	−2.045	0.646	10.030	0.002	0.129	0.036–0.459
Number of embryos transferred	1.305	0.466	7.829	0.005	3.686	1.478–9.192
Duration of infertility	−1.753	0.346	25.621	<0.001	0.173	0.088–0.342
Endometrial thickness	1.302	0.376	11.993	0.001	3.676	1.760–7.680
Endometrial preparation protocols			8.533	0.014
HRT (A reference)
LE	−1.182	0.753	2.466	0.116	0.307	0.070–1.341
GnRHa + HRT	1.746	0.916	3.635	0.047	5.731	0.952–34.498

HRT, hormone replacement treatment; LE, ovulation induction using letrozole; GnRHa + HRT, gonadotropin-releasing hormone agonist for pituitary down-regulation combined with HRT.

Discussion

PCOS is a common endocrine disorder that can affect endometrial receptivity through a variety of ways, consequently leading to low pregnancy rate and high abortion rate in patients [3]. Importantly, endometrial receptivity and embryo quality are two main factors affecting the success rate of FET in PCOS patients [12, 13]. There are increasing studies on the influencing factors of pregnancy outcomes in patients with FET cycles, but no consensus has been obtained. Therefore, this study retrospectively analyzed the clinical data of 200 patients undergoing FET, and explored the correlation between different endometrial preparation protocols and the clinical pregnancy outcomes of FET cycles.

The GnRHa + HRT protocol refers to the use of GnRH-a for pituitary down-regulation followed by HRT in FET cycles, which can reduce the effect of LH surge on endometrial receptivity [19]. Gonadotropin-releasing hormone is a neuroendocrine substance synthesized and secreted by the hypothalamus and acts on the pituitary gland [20]. Gonadotropin-releasing hormone can also act on the endometrium to increase the expression of integrin αvβ3 involved in blastocyst adhesion, promote embryo to enter the endometrium, and thus improve pregnancy success [20]. GnRHa is a synthetic peptide compound that has similar effects to gonadotropin-releasing hormone but is more active [21]. The endometrial thickness on the day of endometrial transformation, clinical pregnancy rate, and live birth rate in the GnRHa + HRT group were significantly higher than those in the HRT group and LE group in this study, while the three groups had no marked differences in the number of embryos transferred, the number of high-quality embryos transferred, embryo implantation rate, biochemical pregnancy rate, ectopic pregnancy rate, and abortion rate. Our findings are consistent with those of El-Toukhy [17] and Hill [18] studies. The reason for the better effect of the GnRHa + HRT protocol is presumably because GnRH-a down-regulation with HRT increased the expression of various favorable factors on the endometrial surface during the implantation window, thus improving the local microenvironment of the pelvic cavity and endometrium [22]. As a result, the endometrium in patients receiving GnRHa + HRT was more susceptible to exogenous hormone regulation and more synchronous with embryonic development, so the pregnancy outcome of patients was improved [22].

Further multivariate logistic regression analysis showed that patient age, number of embryos transferred, endometrial preparation protocols, duration of infertility, and endometrial thickness were independent factors for clinical pregnancy in FET in PCOS patients. Some studies have also suggested that factors such as the woman’s age, the type and quality of embryos transferred, the thickness and morphology of the endometrium and its synchrony with embryonic development can significantly affect the clinical outcome of FET cycles [9, 23]. Age affects the pregnancy outcome of FET because the patient’s egg quality decreases and the aneuploidy rate of the formed embryo increases with age, resulting in lower embryo quality [24]. Meanwhile, the probability of external influence on the uterus and possibility of decreased endometrial receptivity rises with age, finally causing lower clinical pregnancy rate [24]. Therefore, PCOS patients need to be treated as early as possible once fertility problems are detected. In addition, as the duration of infertility increases, the cause of infertility may gradually develop from a single factor to multiple factors, and infertility becomes more severe [25]. The results of this study showed that thinner endometrial thickness led to lower clinical pregnancy rates, which may be due to the fact that when thick endometrium provides relatively rich blood supply and substance metabolism and exchange can be accelerated, contributing to better embryo implantation and higher clinical pregnancy rate [26]. Increasing the number of embryos transferred also improves the clinical pregnancy rate, but the risk of multiple pregnancy, abortion, and preterm birth increases correspondingly [27]. Therefore, the number of transferred embryos needs to be strictly controlled when performing FET. Choosing the appropriate endometrial preparation protocol is a key link affecting the outcome of FET cycles [11]. Using the HRT protocol as a reference, further analysis of endometrial preparation protocols in this study suggested that GnRHa + HRT protocol was more likely to obtain clinical pregnancy. Collectively, for PCOS patients presenting with a complex endocrine environment, GnRHa + HRT is an ideal choice to restore partial ovulation and menstrual cycles and improve endometrial thickness on the day of embryo transformation, clinical pregnancy rate, and live birth rate compared with the other two preparation protocols. However, the data obtained may be biased due to the small sample size in this study. Therefore, a prospective study with a large sample is required to investigate the application of GnRHa + HRT regimen for FET in PCOS patients in the future.

Conclusion

Compared with LRT or LE alone, the GnRHa + HRT protocol improves pregnancy outcomes by improving endometrial thickness on the day of transformation, clinical pregnancy rate, and live birth rate. Therefore, this combination is an ideal endometrial preparation regimen for FET in PCOS patients, and can be preferred in clinical practice according to the patient’s condition. In addition, women’s age, endometrial preparation protocol, number of embryos transferred, endometrial thickness and duration of infertility are significantly correlated with pregnancy outcomes in PCOS patients undergoing FET, and during the evaluation of pregnancy outcomes of patients, more attention should be paid to the above factors.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.