Effect of human chorionic gonadotropin on pregnancy outcomes in patients with polycystic ovary syndrome undergoing frozen-thaw embryo transfer in hormone replacement therapy cycles: A retrospective cohort study

doi:10.21203/rs.3.rs-3759651/v1

Background

Endometrial preparation protocols for frozen-thawed embryo transfer (FET) and hormone replacement therapy (HRT) lack luteinizing hormone (LH) peaks and luteal function due to anovulation. Human chorionic gonadotropin (hCG) supplementation improves endometrial receptivity and promotes embryo implantation. Therefore, we aimed to compare whether there was a difference in pregnancy outcomes with hCG supplementation prior to secretory transformation in patients with polycystic ovary syndrome (PCOS).

Methods

Patients undergoing their first FET cycle between 2018 and 2022 at the university-affiliated reproductive and genetic centre participated in this retrospective cohort study. After standard screening and propensity score matching, patients were assigned to the HRT (n = 343) and HRT + hCG groups (n = 195). The primary outcomes were implantation rate (IR) and live birth rate (LBR), and the secondary outcomes included other pregnancy-related issues.

Results

There were no significant differences in IR or LBR between the two groups. However, compared to the HRT group, the HRT + hCG group had a significantly higher biochemical pregnancy rate (BPR) (75.9% vs. 64.7%, P = 0.007); the clinical pregnancy rate was also higher, but the difference was not significant (58.5% vs. 50.4%, P = 0.073). Ectopic pregnancy, ongoing pregnancy, miscarriage, and preterm birth rates were similar in both groups. After adjusting for possible confounders, logistic regression analyses showed that the effect of hCG addition on BPR (OR 0.585, 95% CI 0.393–0.872, P = 0.009) remained significant. Subsequent subgroup analyses showed a statistically significant improvement in BPR after hCG supplementation in cleavage-stage embryo transfer cycles (75.3% vs. 64.7%, P = 0.020).

Conclusions

In patients with PCOS, intramuscular hCG injection before secretory transformation had no impact on pregnancy outcomes, except for BPR. Notably, the addition of hCG did not show a significant trend towards the increased BPR observed in patients undergoing blastocyst transfer. Future prospective randomised controlled studies are required to confirm our findings.

human chorionic gonadotropin

polycystic ovary syndrome

frozen-thaw embryo transfer

hormone replacement therapy

pregnancy outcomes

propensity score matching

Polycystic ovary syndrome (PCOS), a disease involving endocrine and metabolic disorders, is one of the most common causes of infertility in reproductive-age women (1). The number of patients with PCOS undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment for assisted conception show an increasing trend. With the development of vitrification cryopreservation technology and the diversification of controlled ovarian hyperstimulation (COH) protocols, the number of cycles of frozen-thawed embryo transfer (FET), an important aspect of assisted reproductive technology (ART), has significantly increased (2). Compared with fresh embryo transfer, FET is characterised by a reduced incidence of ovarian hyperstimulation syndrome (OHSS), improved endometrial tolerance, and increased flexibility in the ART process (3, 4). In addition to the abovementioned characteristics, a randomised controlled trial (RCT) found that FET has the advantage of significantly increasing the live birth rate (LBR) in patients with PCOS, a population with a high ovarian response.

There are multiple endometrial preparation protocols in the FET cycle, with the common clinical protocols including natural cycle, hormone replacement therapy (HRT) cycle, and stimulation cycle. However, there is no definitive conclusion as to which endometrial preparation protocol is most beneficial for patients with PCOS. A meta-analysis found that all endometrial preparation protocols appeared to be equally effective in terms of clinical pregnancy rate (CPR), ongoing pregnancy rate (OPR), and LBR (5, 6). Due to ovulation disorders, the most commonly used endometrial preparation protocol for PCOS patients is HRT, which regulates endometrial growth and transformation through the use of exogenous estrogen and progesterone (P) and has been favoured by clinicians for its greater control and flexibility over the timing of embryo transfer (7). In comparison to natural and stimulation cycles, HRT is effective in reducing cycle cancellation rates (8). However, the application of exogenous hormones, which inhibit hypothalamic pituitary function through negative feedback, differs from anovulation, resulting in a lack of luteinizing hormone (LH) peak and luteal function, which have been found to produce worse obstetric and perinatal outcomes (9).

Recently, studies have been conducted to determine whether human chorionic gonadotropin (hCG) injections before P supplementation in FET cycles under HRT can improve pregnancy outcomes. Human luteinizing hormone chorionic gonadotropin receptor (LHCGR), a G protein-coupled receptor, is present in human endometrium. Studies have shown that hCG and LH activate LHCGR expression and mediate its intracellular functions (10, 11). A growing number of clinical trials have demonstrated that hCG supplementation in FET cycles with endometrial preparation using HRT may positively impact pregnancy outcomes such as CPR and LBR (12). Some studies have also found no significant benefit of hCG addition on clinical indicators, such as implantation rate (IR) and CPR (13–15). The variability in the above findings may be related to the timing of hCG addition and the population observed.

Based on these data, we concluded that detailed clinical research is needed to guide future clinical treatment protocols. The aim of current study was to investigate the effect of intramuscular injection of hCG before secretory transformation on clinical pregnancy outcomes in patients with PCOS undergoing their first FET with HRT for endometrial preparation. In addition, we applied propensity score matching (PSM) and multivariate logistic regression analysis to minimise potential bias and reduce the effects of confounding factors on the results.

Study design and participants

In this retrospective cohort study, relevant data were retrieved from patients who underwent their first FET cycle under HRT between January 2018 and November 2022 at the Department of Reproduction and Genetics, Affiliated Hospital of Shandong University of Traditional Chinese Medicine. All patients signed an informed consent form before undergoing FET, and this retrospective, single-centre cohort study was approved by the Ethics Committee of Reproductive Medicine of this hospital (Approval No.: SZ202309003).

Patients who met the inclusion criteria were: age < 43 years, body mass index (BMI) between 18–30 kg/m², meeting the Rotterdam diagnostic criteria for PCOS (16): oligo-/anovulation, clinical or biochemical hyperandrogenism, and ultrasonographic evidence of polycystic ovarian morphology (defined as ≥ 12 follicles of 2–9 mm diameter on the same section and/or ovarian volume > 10 cm³). The exclusion criteria were as follows: endometriosis, untreated hydrosalpinx, abnormal uterine anatomy (uterine malformations, submucosal fibroids, and uterine adhesions), a history of recurrent spontaneous abortion, chromosomal abnormalities, endocrine disorders (diabetes mellitus, thyroid diseases, and hyperprolactinemia), and cancellation of the cycle due to embryo thawing and survival failure. Most of the data were obtained from patients’ ART electronic medical records, and the remaining missing data were obtained by querying patients’ paper medical records and telephone follow-ups.

Controlled ovarian hyperstimulation and laboratory procedures

All patients were treated with IVF/ICSI as clinically indicated; that is, clinicians performed COH using flexible gonadotropin-releasing hormone (GnRH) antagonist (GnRH-ant) protocol (Cetrorelix; Merck Serono, Darmstadt, Germany) or GnRH agonist long (GnRH-a) protocol (Triptorelin, Decapeptyl, Ipsen, France), depending on the patient's underlying characteristics. The COH protocols and laboratory techniques did not change during the study period. Patients were injected with recombinant human follicle-stimulating hormone (rFSH, GONAL-F, Merck Serono, Darmstadt, Germany) at 150–225 IU/d, with or without human menopausal gonadotropin (HMG, Lizhu Pharma, Shaoguan, China). The gonadotropin (Gn) dosage was adjusted according to the patient’s individual situation, follicular growth, and serum sex hormone levels during COH. When at least two follicles reached 18 mm in diameter or three follicles reached 17 mm in diameter, a trigger injection of 250 µg of recombinant hCG (Ovidrel, Merck Serono S.p.A, Modugno, Italy) was administered. Oocytes were retrieved by transvaginal ultrasonography 36–38 h later. Fertilisation was conducted by IVF or ICSI, depending on the results of semen analysis or previous fertilisation.

Normal fertilisation was confirmed when two pronuclei and two polar bodies were observed 16–18 h after insemination. Amphicytula was cultured in a cleavage medium (Cook Medical, Bloomington, IN, USA). Embryo development, i.e., number, shape, evenness, and fragmentation rate of the embryos, was assessed daily. Three days after oocyte retrieval, patients either underwent fresh embryo transfer and vitrification of the remaining available embryos (17) or used a freeze-all strategy according to clinical criteria. Embryos that did not meet the freezing criteria were transferred to a blastocyst medium (Cook Medical) for culturing to the blastocyst stage, followed by vitrification and freezing. Embryos at the cleavage stage were graded according to the following three criteria determined by the Istanbul Consensus Workshop (18): (i) ≥ 6 cells 3 days after fertilisation, (ii) < 10% fragmentation, and (iii) symmetric blastomeres. Blastocysts were graded according to the Gardner classification (19). Blastocysts were considered to be of good quality if the following criteria were met: (i) complete expansion of blastocyst and the cavity completely occupied the total volume of the embryo (grade 3); (ii) the inner cell mass was composed of several loosely grouped cells (grade B); and (iii) the trophectoderm contained few cells that formed a loose epithelium (grade B). Depending on the quality and stage of embryos and the patient’s age, the number of embryos transferred ranged from 1 to 2.

Endometrial preparation protocols

All patients underwent pre-FET endometrial preparation using HRT (Fig. 1). On days 2–4 of menstruation, basal endocrine levels, including follicle-stimulating hormone (FSH), LH, estradiol (E₂), P, and testosterone (T) levels, were measured, and transvaginal ultrasound was performed to assess baseline endometrial conditions. If the baseline conditions were appropriate, E₂ tablets (Progynova, Bayer, Germany) were administered orally at doses of 4–8 mg/day. After 9–12 days of continuous Progynova supplementation, transvaginal ultrasound was performed to measure the endometrial thickness determining whether it was in a proper state for embryo transfer (usually at least 7 mm). If endometrial thickness was inadequate (< 7 mm), the oral Progynova dosage was increased or vaginal E₂ tablets (Femoston, Abbott, Netherlands) were added, with a maximum E₂ dosage of 10 mg/d, until the endometrial thickness reached 7 mm. If endometrial thickness did not reach the desired thickness after 20 days of E₂ supplementation, cycle cancellation was advised, and the final decision on whether to transfer was made by the patient (who normally has a thin endometrium). Serum E₂ and P levels were measured when endometrium reached the desired thickness (cycles were cancelled if P > 1.6 ng/mL). The decision to use hCG before endometrial secretory transformation was made by a treating physician. In the HRT + hCG group, 2000 IU of hCG (Lizhu Pharma, China) was administered intramuscularly on the day of endometrial attainment, and 20 mg of P (Xianju, China) was injected the next morning. No hCG was administered to the HRT group and 20 mg of P was injected on the same day to enable endometrial transformation. On the second day after starting P injections, their dosage was increased to 40 mg/day in both groups. Embryos were transferred according to their developmental stage at the time of freezing. FET was performed 3 days (embryos at the cleavage stage) or 5–6 days (embryos at the blastocyst stage) after the P injection of 20 mg.

E₂ and P supplementation were continued after FET. Serum β-hCG levels were measured 14 days after FET. A positive pregnancy test was identified if the serum β-hCG level was ≥10 mIU/mL (20). Transvaginal ultrasonography was performed at 5-6 weeks of gestation to confirm intrauterine pregnancy and determine the number of gestational sacs. Supplementation was discontinued in cases of negative pregnancy test result or pregnancy loss.

Outcome measures

The primary outcomes were LBR, calculated as live births at gestational age > 26 weeks per FET cycle (21), and IR, calculated by dividing the number of gestational sacs by the total number of embryos transferred. Biochemical pregnancy rate (BPR), CPR, ectopic pregnancy rate (EPR), OPR, miscarriage rate (MR), and preterm birth rate (PBR) were secondary outcomes. Biochemical pregnancy was defined as a positive pregnancy test result with no intrauterine or extrauterine gestational sacs detected on vaginal ultrasound. Clinical pregnancy is defined as the appearance of a gestational sac with a fetal heart at 5–6 weeks of gestation. Ectopic pregnancy was diagnosed when an extrauterine gestational sac was detected laparoscopically or ultrasonographically. Ongoing pregnancy was defined as a clinical pregnancy lasting beyond 12 weeks (22). Miscarriage was defined as pregnancy loss at less than 28 weeks of gestation and fetal weighting less than 1000 g. Preterm birth was defined as birth before 37 weeks of gestational age.

Bias and confounding

In this observational study, we used a PSM method based on a "counterfactual inference model" to balance the differences in baseline characteristics between two groups to reduce confounding bias. The propensity score for endometrial preparation was estimated using a multivariate logistic regression model based on the following 18 covariates: female age, BMI, infertility duration, infertility type (primary or secondary), gravidity, parity, basal sex hormone levels (FSH, LH, E₂, P, and T), protocol of COH (GnRH-a long protocol or GnRH-ant protocol), initial treatment (IVF or ICSI), protocol in fresh cycle (fresh embryo transfer or freeze-all strategy), duration of E₂ supplementation, number of embryos transferred, number of high-quality embryos transferred, and stage of embryo transferred (cleavage or blastocyst). A new database containing only matched cases after PSM was created using nearest-neighbour random matching without replacement based on the closest propensity score of each subject, with 1:2 matching at a caliper size of 0.02.

Statistical analysis

Histograms and Shapiro-Wilk tests were used to assess the normal distribution of continuous data. Student's t-test was taken if the data conformed to normal distribution and expressed as mean ± standard deviation (SD); otherwise, Mann-Whitney U-test was used and expressed as median and interquartile range (IQR). Categorical data were expressed as frequencies (percentages) and analysed using the χ² test. We also used binary logistic regression analysis to assess the association between endometrial preparation regimen and other potential factors with pregnancy outcomes. We calculated crude odds ratios (ORs) and adjusted ORs with 95% confidence intervals (CI). P < 0.05 was considered statistically significant. Data were analysed using SPSS (version 23.0; IBM Corp., Armonk, NY, USA).

From January 2018 to November 2022, 2,182 patients underwent their first FET under the HRT protocol, according to the ART system of the Department of Reproduction and Genetics, Affiliated Hospital of Shandong University of Traditional Chinese Medicine. After excluding 1, 333 patients based on the eligibility criteria, the study population consisted of 849 patients (626 in the HRT group and 223; HRT + hCG group). After performing a 1:2 PSM, 343 patients in the HRT group and 195 patients in the HRT + hCG group constituted the matched study cohort (Fig. 2). The balance assessment before and after matching is shown in Fig. 3.

Baseline and IVF/ICSI cycle characteristics of patients

The baseline and IVF/ICSI cycle characteristics of the patients in the HRT and HRT + hCG groups are shown in Table 1. Overall, no significant differences were observed between the two groups in terms of the statistics before and after PSM (P > 0.05). In particular, the covariates of patient age and fresh cycle protocol were effectively balanced after matching.

Table 1

The baseline and IVF/ICSI cycle characteristics of two groups before and after PSM
Variables	Before PSM			After PSM
Variables	HRT (n = 626)	HRT + hCG (n = 223)	P-value	HRT (n = 343)	HRT + hCG (n = 195)	P-value
Patient age (years)	30.53 ± 3.75	31.13 ± 4.21	0.061^a	30.76 ± 3.78	30.81 ± 4.10	0.888^a
Infertility duration (years)	3 (2, 4)	3 (2, 5)	0.102^b	3 (2, 4)	3 (2, 4)	0.498^b
Infertility type (%)			0.350^c			0.467^c
Primary	300/626 (47.9)	115/223 (51.6)		170/343 (49.6)	103/195 (52.8)
Secondary	326/626 (52.1)	108/223 (48.4)		173/343 (50.4)	92/195 (47.2)
Gravidity (n)	1 (0, 1)	0 (0, 1)	0.714^b	1 (0, 2)	0 (0, 1)	0.258^b
Parity (n)	0 (0, 0)	0 (0, 0)	0.414^b	0 (0, 0)	0 (0, 0)	0.927^b
BMI (kg/m²)	24.35 ± 3.05	24.62 ± 3.08	0.265^a	24.51 ± 3.02	24.60 ± 3.12	0.757^a
Basal FSH (mIU/mL)	6.17 ± 1.65	5.97 ± 1.95	0.185^a	6.12 ± 1.65	6.10 ± 1.83	0.891^a
Basal LH (mIU/mL)	6.82 ± 2.81	6.49 ± 2.83	0.137^a	6.49 ± 2.72	6.72 ± 2.80	0.353^a
Basal E₂ (pg/mL)	37.39 ± 12.14	38.30 ± 11.62	0.332^a	37.00 ± 11.85	38.37 ± 11.26	0.190^a
Basal P (ng/mL)	0.55 ± 0.31	0.54 ± 0.31	0.796^a	0.53 ± 0.30	0.53 ± 0.31	0.753^a
Basal T (ng/mL)	1.20 ± 0.80	1.26 ± 0.83	0.384^a	1.29 ± 0.84	1.29 ± 0.84	0.854^a
Protocol of COH (%)			0.435^c			0.974^c
GnRH-a long protocol	133/626 (21.2)	53/223 (23.8)		84/343 (24.5)	48/195 (24.6)
GnRH-ant protocol	493/626 (78.8)	170/223 (76.2)		259/343 (75.5)	147/195 (75.4)
Initial treatment (%)			0.535^c			0.866^c
IVF	468/626 (74.8)	162/223 (72.6)		251/343 (73.2)	144/195 (73.8)
ICSI	158/626 (25.2)	61/223 (27.4)		92/343 (26.8)	51/195 (26.2)
Protocol in fresh cycle (%)			0.081^c			0.928^c
Fresh embryo transfer	57/626 (9.1)	12/223 (5.4)		20/343 (5.8)	11/195 (5.6)
Freeze-all strategy	569/626 (90.9)	211/223 (94.6)		323/343 (94.2)	184/195 (94.4)

Data are presented as mean±SD, n (%), or median (IQR).

^a Student's t-test, ^b Mann-Whitney U-Test, ^c c²-test.

Abbreviations: BMI Body mass index, COH Controlled ovarian hyperstimulation, E₂ Estradiol, FSH Follicle-stimulating hormone, GnRH-a Gonadotropin releasing hormone agonist, GnRH-ant Gonadotropin releasing hormone antagonist, ICSI Intracytoplasmic sperm injection, IVF In vitro fertilization, LH Luteinizing hormone, P Progesterone, T Testosterone.

Characteristics of the FET cycles

Table 2 shows characteristics of the FET cycles in the two groups of patients before and after PSM. Before matching, there were significant differences between several covariates such as the duration of E₂ administration (P=0.010), P level on the transfer day (P=0.001), number of embryos transferred (P<0.001), and stage of embryos transferred (P<0.001). After matching, the P level on transfer day was lower in the HRT+hCG group than that in the HRT group (18.19±10.55 vs 20.37±10.57, P=0.021), and other demographic characteristics were similar.

Table 2

Characteristics of FET cycles in two groups before and after PSM
Variables	Before PSM			After PSM
Variables	HRT (n = 626)	HRT + hCG (n = 223)	P-value	HRT (n = 343)	HRT + hCG (n = 195)	P-value
Duration of E₂ administration (days)	12.47 ± 2.43	12.09 ± 1.68	0.010^a	12.30 ± 2.36	12.17 ± 1.69	0.446^a
E₂ levels (pg/mL)*	338.62 ± 346.21	389.72 ± 370.85	0.064^a	363.38 ± 375.24	388.31 ± 359.28	0.452^a
P levels (ng/mL)*	0.74 ± 0.43	0.68 ± 0.42	0.073^a	0.72 ± 0.43	0.68 ± 0.42	0.249^a
Endometrial thickness (mm)*	10.77 ± 3.14	11.07 ± 3.21	0.233^a	10.83 ± 3.08	11.11 ± 3.26	0.319^a
E₂ on transfer day (pg/mL)	799.11 ± 524.04	736.83 ± 530.51	0.129^a	789.78 ± 525.18	769.22 ± 527.80	0.663^a
P on transfer day (ng/mL)	20.51 ± 10.48	17.78 ± 10.46	0.001^a	20.37 ± 10.57	18.19 ± 10.55	0.021^a
No. of embryos transferred (n)	1.83 ± 0.38	1.69 ± 0.46	< 0.001^a	1.80 ± 0.40	1.78 ± 0.42	0.596^a
No. of high-quality embryos transferred (n)	0.66 ± 0.64	0.62 ± 0.62	0.408^a	0.64 ± 0.63	0.67 ± 0.65	0.586^a
Single embryo FET cycles (%)	106/626 (16.9)	69/223 (30.9)	< 0.001^b	69/343 (20.1)	43/195 (22.1)	0.595^b
Single cleavage-stage embryo	35/548 (6.4)	13/166 (7.8)	0.515^b	24/292 (8.2)	11/162 (6.8)	0.584^b
Single blastocyst	71/78 (91.0)	56/57 (98.2)	0.166^b	45/51 (88.2)	32/33 (97.0)	0.312^b
Stage of embryo transferred (%)			< 0.001^b			0.528^b
Cleavage stage	548/626 (87.5)	166/223 (74.44)		292/343 (85.1)	162/195 (83.1)
Blastocyst stage	78/626 (12.5)	57/223 (25.6)		51/343 (14.9)	33/195 (16.9)
Data are presented as mean ± SD, n (%).
^a Student's t-test, ^b χ²-test.
* hCG injection day for HRT + hCG group and progesterone injection day for HRT group.
Abbreviations: E₂ Estradiol, FET frozen-thawed embryo transfer, P Progesterone.

Pregnancy outcomes

Before performing PSM, the differences in pregnancy outcomes between the two groups were not statistically significant. However, after PSM, some of the intergroup differences changed (Table 3). Specifically, the primary outcomes of LBR and IR were higher in the HRT+hCG group than in the HRT group (43.6% vs. 38.5% and 38.6% vs. 36.0%, respectively); however, these differences were not statistically significant (P>0.05). Notably, compared with the HRT group, the HRT+hCG group showed a significant increase in BPR (75.9% vs. 64.7%, P=0.007) and a slight increase in CPR (58.5% vs. 50.4%); however, this difference was not statistically significant (P=0.073). The differences in the EPR, OPR, MR, and PBR between the two groups before and after PSM were not statistically significant (P>0.05).

As covariate imbalance remained after PSM; we used a binary logistic regression model to adjust for residual confounding and screening for confounders with a criterion of P <0.2(23) to further assess the relationship between endometrial preparation protocols and pregnancy outcomes (Table 4). In both the crude and adjusted models, the addition of hCG improved the BPR, whereas it was comparable to the HRT group in terms of the LBR, CPR, EPR, OPR, MR, and PBR. For patients with PCOS, the use of hCG or not in the first FET cycle under HRT was not a significant independent factor influencing the above pregnancy outcomes, except for BPR.

Table 3

Pregnancy outcomes of FET cycles before and after PSM
Outcomes	Before PSM			After PSM
Outcomes	HRT (n = 626)	HRT + hCG (n = 223)	P-value^a	HRT (n = 343)	HRT + hCG (n = 195)	P-value^a
Implantation rate (%)	409/1146 (35.7)	144/377 (38.2)	0.380	222/617 (36.0)	134/347(38.6)	0.416
Biochemical pregnancy rate (%)	422/626 (67.4)	163/223 (73.1)	0.115	222/343 (64.7)	148/195 (75.9)	0.007
Clinical pregnancy rate (%)	325/626 (51.9)	124/223 (55.6)	0.343	173/343 (50.4)	114/195 (58.5)	0.073
Ectopic pregnancy rate (%)	5/325 (1.5)	4/124 (3.2)	0.445	2/173 (1.2)	4/114 (3.5)	0.346
Ongoing pregnancy rate (%)	274/325 (84.3)	97/124 (78.2)	0.128	146/173 (84.4)	92/114 (80.7)	0.416
Miscarriage rate (%)	74/325(22.8)	30/124 (24.2)	0.749	39/173 (22.5)	25/114 (21.9)	0.903
Preterm birth rate (%)	18/325 (5.5)	4/124 (3.2)	0.310	11/173 (6.4)	4/114 (3.5)	0.289
Live birth rate (%)	246/626 (39.3)	90/223 (40.4)	0.781	132/343 (38.5)	85/195 (43.6)	0.246
Data are presented as n (%).
^a χ²-test.

Table 4

Relationship between endometrial preparation and pregnancy outcomes in Different Models
Pregnancy outcomes	Group	Crude model			Adjusted model^a
		OR (95% CI)	P-value	OR (95% CI)		P-value
Biochemical pregnancy	HRT	Reference 0.583 (0.392–0.866)	0.007	Reference 0.585 (0.393–0.872)		0.009
	HRT + hCG
Clinical pregnancy	HRT	Reference 0.723(0.507–1.031)	0.073	Reference 0.744(0.520–1.063)		0.104
	HRT + hCG
Ectopic pregnancy	HRT	Reference 0.322 (0.058–1.786)	0.195	Reference 0.289 (0.051–1.652)		0.163
	HRT + hCG
Ongoing pregnancy	HRT	Reference 1.293(0.695–2.405)	0.417	Reference 1.205(0.639–2.274)		0.564
	HRT + hCG
Miscarriage	HRT	Reference 1.036(0.586–1.831)	0.903	Reference 1.131(0.629–2.034)		0.681
	HRT + hCG
Preterm birth	HRT	Reference 1.867(0.580–6.015)	0.295	Reference 1.898(0.584–6.171)		0.287
	HRT + hCG
Live birth	HRT	Reference 0.810 (0.567–1.157)	0.246	Reference 0.806(0.562–1.156)		0.240
	HRT + hCG
^a Adjusted for basal estradiol and progesterone on transfer day.
Abbreviations: CI Confidence interval, OR Odds ratio.

Subgroup analysis

We performed subgroup analysis according to the stage of embryo transferred (Table 5). In cleavage-stage embryo transfer cycles, hCG supplementation significantly improved the BPR values (75.3% vs. 64.7%, P=0.020); however, the trend of BPR higher in the blastocyst transfer cycles (78.8% vs. 64.7%, P=0.168) was not significant (P>0.05). None of the other pregnancy outcomes differed significantly in the subgroup analysis.

Table 5

Subgroup analysis of pregnancy outcomes of FET cycles
Outcomes	Cleavage-stage embryo			Blastocyst
Outcomes	HRT (n = 292)	HRT + hCG (n = 162)	P-value^a	HRT (n = 51)	HRT + hCG (n = 33)	P-value^a
Implantation rate (%)	194/560 (34.6)	115/313 (36.7)	0.534	28/57 (49.1)	19/34 (55.9)	0.532
Biochemical pregnancy rate (%)	189/292 (64.7)	122/162 (75.3)	0.020	33/51 (64.7)	26/33 (78.8)	0.168
Clinical pregnancy rate (%)	146/292 (50.0)	95/162 (58.6)	0.077	27/51 (52.9)	19/33 (57.6)	0.677
Ectopic pregnancy rate (%)	1/146 (0.7)	4/95 (4.2)	0.157	1/27 (3.7)	0/19(0)	1.000
Ongoing pregnancy rate (%)	127/146 (87.0)	79/95 (83.2)	0.410	19/27 (70.4)	13/19 (68.4)	0.887
Miscarriage rate (%)	31/146 (21.2)	16/95 (16.8)	0.401	8/27 (29.6)	9/19(47.4)	0.220
Preterm birth rate (%)	10/146 (6.8)	2/95 (2.1)	0.177	1/27 (3.7)	2/19 (10.5)	0.752
Live birth rate (%)	114/292 (39.0)	75/162 (46.3)	0.133	18/51 (35.3)	10/33 (30.3)	0.636
Data are presented as n (%).
^a χ²-test.

In recent years, several scholars have investigated whether the addition of hCG to FET cycles under HRT can improve pregnancy outcomes; however, to the best of our knowledge, few studies have focused on patients with PCOS alone. In this retrospective cohort study, we assessed whether intramuscular injection of hCG prior to secretory transformation could have a beneficial effect on pregnancy outcomes in patients with PCOS. The results showed that hCG injection significantly increased the patients’ BPR; CPR was also elevated, however, this difference was not statistically significant. Subsequent logistic regression results showed that the addition of hCG was an independent factor for BPR improvement. However, hCG injections did not significantly change the LBR, IR, or other pregnancy outcomes.

HCG is the first hormone synthesised by the embryo, and β-hCG transcripts could be detected as early as the two-cell stage, where the protein is produced prior to blastocyst implantation (24), and has an important role in the establishment, facilitation and maintenance of pregnancy (25, 26). LH and/or the highly homologous hCG can exert biological effects by binding to LHCGR, which is expressed in the glandular epithelium of the endometrium (10), thereby affecting cellular environment during embryo implantation (27). HCG regulates pregnancy by improving the endometrial tolerance, immunomodulation, and endocrine regulation. It has been shown that hCG, when combined with LHCGR, is able to improve endometrial tolerance in various ways by promoting trophoblast invasion and differentiation, promoting endometrial angiogenesis and remodelling, and maintaining the relative quiescent state of the myometrium (28, 29). Additionally, several studies have elaborated on the modulatory effect of hCG on the immune response during pregnancy, which improves maternal tolerance of the embryo by affecting uterine natural killer (uNK) cells, regulatory T cells (Tregs), and B cells (the three main cell populations that maintain pregnancy) in the endometrium (30-32), thereby promoting embryo implantation. The purpose of applying hCG before the secretory transformation in HRT cycles is to mimic the LH peak during natural ovulation, which is a major factor influencing changes in endometrial type and state (33). However, it has been shown that binding of LH or hCG to LHCGR triggers different intracellular signalling cascades (AKT, extracellular signal-regulated protein kinase 1 and 2 [ERK1/2], MAPK, and β-arrestin 2) and steroidogenesis (34, 35), and that the resulting reproductive outcomes vary, such as pregnancy rates (36). Our study showed that both groups had similar pregnancy outcomes, which partly explains why HRT combined with hCG mimicking LH peaks is not a substitute for the natural cycle, a regimen of endometrial preparation that is close to the physiological state (37).

PCOS is an endocrine disorder and metabolic dysfunction that leads to decreased endometrial tolerance and fertility (ovulatory function and oocyte quality) (38). Compared to that in healthy women, the endometrium of patients with PCOS has dysregulated expression of sex hormone receptors and co-receptors, increased insulin resistance, chronic low-grade inflammation, immune dysfunction, and disturbed angiogenesis (39, 40), and the above pathological features lead to impaired endometrial receptivity and poor pregnancy outcomes. Current studies suggest that the molecular mechanisms by which hCG improves endometrial tolerance in patients with PCOS mainly consist of the following: The level of HOXA10, a key transcription factor for endometrial tolerance (41), is significantly reduced in the endometrium of patients with PCOS (42), and previous study has shown that hCG upregulates the expression of HOXA10 (43). Meanwhile, P resistance in patients with PCOS affects endometrial metamorphosis and tolerance in the secretory phase (44); however, hCG activates the ERK1/2 pathway in human endometrial stromal cells to upregulate P receptor expression (43). Additionally, hCG administration modulates the expression levels of molecules such as insulin-like growth factor binding protein 1 (45), interleukin 6 (46), vascular endothelial growth factor (47), and the proportion of uNK cells (48), which are markers of endometrial abnormalities in patients with PCOS (40, 49), prolonging the implantation window and increasing the possibility of pregnancy by modulating endometrial tolerance/metaplasia, modulating the immune-inflammatory response, and stimulating angiogenesis and growth. In the present study, we found that the addition of hCG prior to secretory transformation did not increase IR in patients, but BPR was significantly higher, which may, to some extent, be related to the above physiological effects of hCG.

In the subgroup analysis in this study, we found a trend of elevated BPR in cleavage-stage embryo transfer cycles, despite of it not being our primary outcome indicator. These findings may be related to the transfer principles consistently adhered to in our IVF/ICSI centre, where single embryo transfers were mostly chosen for blastocyst transfer cycles (77/84, 91.7%) and only a few were for cleavage-stage embryo transfer cycles (35/454, 7.7%). Additionally, this may be related to the insufficient sample size. In the future, we expect obtaining the high credibility results after expanding a group of participants. The reason for the better pregnancy outcomes obtained in the cleavage-stage embryo transfer cycles may be due to the fact that endogenous hCG, compared to blastocysts, is clinically important for embryos at the cleavage stage (50). The longer half-life of hCG coincidentally compensates for the lack of endogenous hCG action in the endometrium during cleavage to the blastocyst stage.

Previous research has focused mostly on the effect of adding hCG to the natural cycle on pregnancy outcomes, ovulation induction, or luteal support, and there is still no consensus. In recent years, an increasing number of studies have reported the effects of hCG on the HRT cycles. In contrast to the findings of our study, intramuscular injection of 10,000 IU of hCG before secretory transformation significantly increased LBR, CPR, and OPR in patients; however, subsequent subgroup analyses showed that the trend for LBR to be elevated in patients undergoing blastocyst transfer was not significant (51). Another RCT showed that transferring embryos at the cleavage stage with an intramuscular injection of 10,000 IU hCG prior to secretory transformation significantly increased CPR, but BPR and MR were similar (52). Several RCTs have investigated the effects of intramuscular hCG injections during the secretory phase on pregnancy outcomes. Notably, all three trials reached the same conclusion that hCG supplementation during the secretory phase did not have any significant effect on FET cycle outcomes under HRT despite the varying timing and dosage of hCG injection (13-15). The results of our study showed that intramuscular injection of hCG prior to secretory transformation did not significantly improve the final pregnancy outcome of FET cycles in patients with PCOS, which may be related to the use of a lower dose of hCG (2,000 IU) in our study and is not excluded from the influence of the study population. Whether different periods, high dosages, and multiple frequencies of hCG injections produce better cycle outcomes in patients with PCOS, requires further investigation.

Most studies have focused on the effects of hCG on pregnancy outcomes in patients with repeated implantation failure (RIF). It has been shown that the endometrial proteome composition during the implantation window in patients with RIF is different from that in healthy women, and that intrauterine infusion of hCG before FET may improve chemokine-triggered embryo-endometrial dialogue and intensify angiogenesis and immune responses, thereby increasing the pregnancy rate (53). Most studies in patients with RIF have shown similar results, with intrauterine infusion of hCG before FET significantly improving CPR and LBR (54-56). It has also been found that intrauterine infusion of hCG did not show a significant improvement in clinical outcomes in patients with or without RIF (57). For patients with endometriosis, Du et al. found that intramuscular injection of hCG before secretory transformation improved CPR but had no significant effect on LBR (12); similarly, Xu et al. found that intrauterine infusion of hCG before FET improved BRP, CPR and birth weight, whereas other pregnancy outcomes were similar (58). No study has yet reported the effect of hCG on patients with PCOS under HRT, and we found that the final pregnancy outcomes were similar between the two groups. Only a minority of patients with PCOS in our study had a history of fresh embryo transfer (31/538, 5.8%) owing to the limitations of the COH regimen and the high risk of ovarian hyperstimulation syndrome, based on which we performed subgroup analyses (Supplemental Table 1). The results showed essentially similar pregnancy outcomes, except that the improved efficacy of BPR was more pronounced in patients without a history of fresh embryo transfer, and this did not exclude the effect of the uneven distribution of sample sizes between the subgroups. However, this finding is only preliminary and exploratory, and a larger multicentre sample with an RCT design is required to validate and confirm our results.

The strengths of this study are the large sample size and the application of PSM to balance the variables that may affect the results to obtain realistic and reliable data. However, this study had some limitations. First, a selection bias was inevitable owing to its retrospective design. Second, other confounding factors associated with pregnancy outcomes were not considered, such as patients' anti-Müllerian hormone levels and other IVF/ICSI cycle characteristics, such as number of oocytes obtained and number of embryos available, and the interval between IVF/ICSI and FET cycles. Additionally, it is worth noting that the P levels on the transfer day were significantly different both before and after PSM and were lower with the addition of hCG, and we were unable to determine whether there was a correlation between them and the potential impact on pregnancy outcomes. Evidence from only one retrospective study is inconclusive regarding whether intramuscular hCG administration prior to secretory transformation improves pregnancy outcomes in patients with PCOS, and further evidence is needed to obtain the accurate evidence-based data.

Our findings suggest that in patients with PCOS undergoing FET for the first time, the addition of hCG before secretory transformation under HRT cycles resulted in a higher BPR, especially in cleavage-stage embryo transfer cycles; however, no significant clinical benefit was observed in terms of LBR, IR, CPR, OPR, MR, EPR, and PBR. However, as this study has some limitations, well-designed prospective clinical trials are needed to further evaluate the beneficial effects of hCG addition in HRT cycles in patients with PCOS, including detailed follow-up information involving obstetrics. These studies will help further explore this relationship and apply in clinical practice.

ART: assisted reproductive technology; BMI: Body mass index; BPR: Biochemical pregnancy rate; CI: Confidence interval; COH: Controlled ovarian hyperstimulation; CPR: Clinical pregnancy rate; E₂: Estradiol; EPR: Ectopic pregnancy rate; ERK1/2: Extracellular signal-regulated protein kinase 1 and 2; FET: Frozen-thawed embryo transfer; FSH: Follicle-stimulating hormone; Gn: Gonadotropin; GnRH: Gonadotropin-releasing hormone; GnRH-a: Gonadotropin-releasing hormone agonist; GnRH-ant: Gonadotropin-releasing hormone antagonist; hCG: Human chorionic gonadotropin; HRT: Hormone replacement therapy; ICSI: Intracytoplasmic sperm injection; IQR: Interquartile range; IR: Implantation rate; IVF: In vitro fertilization; LBR: Live birth rate; LH: Luteinizing hormone; LHCGR: Luteinizing hormone and choriogonadotropin receptor; MR: Miscarriage rate; OHSS: Ovarian hyperstimulation syndrome; OPR: Ongoing pregnancy rate; OR: Odds ratio; P: Progesterone; PBR: Preterm birth rate; PCOS: Polycystic ovary syndrome; PSM: Propensity score matching; RCT: Randomized controlled trial; RIF: Repeated implantation failure; SD: Standard deviation; T: Testosterone.

Ethics approval and consent to participate

The retrospective cohort study was approved by the Ethics Committee of Reproductive Medicine of the Affiliated Hospital of the Shandong University of Traditional Chinese Medicine (approval number: SZ202309003). All patients signed an informed consent form before FET. All methods were conducted in compliance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Availability of data and materials

The datasets generated and/or analysed in this study are not publicly available for legal or ethical reasons, but will be made available by the corresponding author (Pingping Cai: [email protected]) upon a reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study received supports from the National Natural Science Foundation of China (Grant No. 82274573, 82174429); the Natural Science Foundation of Shandong Province (Grant No. ZR2021MH255); the Shandong Province Taishan Scholar Project (Grant No. tsqn202103182).

Authors’ contributions

FL and PC conceived and designed this study. LD and HW contributed to the acquisition and analysis of data, drafting of the manuscript. WC and QL were responsible for data collection. YW contributed to manuscript revision. All authors approved the final submitted version.

Acknowledgments

The authors thank the nurses and laboratory staff of the Department of Assisted Reproduction for their technical assistance.

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No competing interests reported.

supplementtable.docx

Effect of human chorionic gonadotropin on pregnancy outcomes in patients with polycystic ovary syndrome undergoing frozen-thaw embryo transfer in hormone replacement therapy cycles: A retrospective cohort study

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Study design and participants

Controlled ovarian hyperstimulation and laboratory procedures

Endometrial preparation protocols

Outcome measures

Bias and confounding

Statistical analysis

Results

Baseline and IVF/ICSI cycle characteristics of patients

Characteristics of the FET cycles

Pregnancy outcomes

Subgroup analysis

Discussion

Conclusions

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors’ contributions

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1