The Effect of Intramuscular Human Chorionic Gonadotropin on Endometrial Preparation in Frozen-Thawed Embryo Transfer: A Randomized Clinical Trial.

Successful embryo implantation depends primarily on two factors: embryo quality and endometrial receptivity. Implantation failure accounts for approximately 50-75% of in vitro fertilization (IVF) failures ( 1 ). Evidence suggests that human chorionic gonadotropin (hCG) levels influence the implantation rate ( 2 ). Notably, embryos begin secreting hCG prior to implantation, starting as early as the two-cell stage ( 3 ). Studies have demonstrated that hCG enhances the expression of genes critical for the transformation of endometrial stromal cells into decidual cells and plays a key role in upregulating proteins involved in implantation ( 4 ). During the implantation window, hCG influences both endometrial epithelial and stromal cells, protecting decidual cells from oxidative damage. Additionally, it increases progesterone receptor expression in the endometrium and the production of molecules essential for implantation, such as galectin-3 and homeobox A-10. Furthermore, hCG facilitates maternal immune tolerance toward fetal cells, preventing immune-mediated rejection ( 5 ). The shared receptors for luteinizing hormone (LH) and hCG are present in the endometrium and play critical roles in the final stages of oocyte maturation and corpus luteum formation in the ovaries. The peak activity of LH/hCG receptors in the endometrium occurs at the onset of the secretory phase, coinciding precisely with the implantation window ( 6 ). The effects of hCG on the endometrium are independent of ovarian function. These receptors are expressed in both the epithelial and stromal cells of the endometrium, with the highest expression during the luteal phase. Given that hCG has a higher affinity for LH receptors and a slower clearance rate than LH, a single mid-cycle hCG dose can mimic endogenous LH activity in patients whose LH is suppressed by gonadotropin-releasing hormone (GnRH) agonist ( 5 ). The lack of effect of mid-cycle hCG injection in individuals with persistently elevated serum LH supports the concept that hCG exerts its effects via LH receptors on the endometrium ( 7 ). Additionally, LH receptors are present in the uterine myometrium, with maximal expression in the luteal phase and minimal levels during the follicular phase. These receptors likely contribute to uterine hyperplasia and regulate uterine motility by reducing intracellular calcium in myometrial smooth muscle cells, with high receptor concentrations inducing uterine relaxation and quiescence ( 8 ). Licht et al. ( 9 ) demonstrated that the presence of hCG receptors in the endometrium reduces macrophage colonystimulating factor (MCSF) concentrations while simultaneously stimulating the production of key mediators, such as leukemia inhibitory factor (LIF) and vascular endothelial growth factor (VEGF), which play important roles in embryo implantation. Additionally, hCG enhances endometrial stromal cells’ responsiveness to interleukin-1 (IL-1), thereby promoting uterine vascular endothelial cell proliferation and angiogenesis ( 10 ). Through stimulation of matrix metalloproteinase (MMP) secretion and inhibition of their tissue inhibitors, hCG facilitates remodeling of the endometrium, supporting cytotrophoblast invasion. It also decreases the production of insulin-like growth factor binding protein 1 (IGFBP-1), which increases IGF-2 secretion, contributing to neovascularization ( 11 ). Moreover, hCG inhibits apoptosis of cytotrophoblast cells, further supporting implantation ( 12 ). Based on current evidence, hCG plays a pivotal role in embryo implantation and may improve pregnancy outcomes in women undergoing assisted reproductive technology (ART) cycles. However, robust, clinical trials are necessary to confirm this hypothesis. Mansour et al. ( 13 ) were the first to report that intrauterine administration of 500 units of hCG positively effects pregnancy outcomes. Subsequent studies have investigated intrauterine hCG infusion in both fresh and frozen embryo transfer (ET) cycles, an approach that avoids systemic exposure. Nonetheless, some studies have suggested that the intrauterine hCG injection shortly before ET may cause embryo displacement or expulsion ( 14 ). In contrast, intramuscular hCG administration has been less extensively studied ( 14 - 19 ), with variations in dosage, timing, and frequency across trials. Moreover, most investigations focus primarily on implantation and clinical pregnancy rates, with fewer reporting live birth outcomes. Therefore, the definitive benefits of hCG administration in ART cycles remain uncertain. In this context, the present study evaluated the effects of intramuscular hCG injection during hormonal replacement cycles with GnRH-agonist protocols for frozen ET.

A total of 200 women were enrolled and randomly assigned equally to the intervention and control groups. Twelve participants discontinued the study, resulting in 93 women in the intervention group and 95 in the control group ( Fig .1 ). As presented in Table 1, baseline demographic and clinical characteristics were comparable between the two groups, with no significant differences observed. Hormonal profiles, number of ET, and the proportion of good or excellent-quality embryos transferred were also similar between groups. Baseline demographic and clinical characteristics of study participants All values are presented as mean ± SD for continuous variables and as n (%) for categorical variables. 1; Mixed infertility includes both male and ovulatory factor, *; Basic hormonal profile includes, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, TSH; Thyroid-stimulating hormone, PRL; Prolactin, and AMH; Anti-müllerian hormone. The CONSORT flow diagram of the study. Regarding ART outcomes ( Table 2 ), no significant differences were observed between groups in chemical pregnancy rate (P=0.912), clinical pregnancy rate (P=0.999), or implantation rate (P=0.903). However, the miscarriage rate was lower in the intervention group compared to the control group. This reduction approached statistical significance per cycle (P=0.059) and reached statistical significance per pregnancy (P=0.011). Embryo transfer cycle characteristics and reproductive outcome Values are presented as mean ± SD for continuous variables, and as n (%) for categorical variables. 1 ; Pre-eclampsia and 2 ; Gestational hypertension and Pre-eclampsia

This RCT evaluated the effects of intramuscular administration of 5000 IU hCG on frozen-thawed ET outcomes in hormonal replacement cycles using a GnRH-agonist protocol. The hCG injections, administered 72 hours before ET, on the day of ET, and 72 hours after ET, did not significantly affect the implantation rate, clinical pregnancy rate, live birth rate, or pregnancy complications compared with the control group. The only significant difference observed between groups was a reduction in miscarriage rate in the hCG-treated group. Experimental evidence indicates that the human endometrium expresses LH/hCG receptors, and activation of these receptors may enhance embryo implantation ( 24 ). Several studies have examined the effects of intrauterine hCG infusion on outcomes in both fresh and frozen ET cycles. A 2022 meta-analysis by Conforti et al. ( 13 ) concluded that intrauterine hCG administration may benefit women undergoing cleavage-stage ET; however, these findings should be interpreted cautiously due to limited data on live birth rates. In contrast, the effects of systemic hCG administration, either intramuscular or subcutane ous, have been investigated in relatively fewer studies. Akbari et al. ( 15 ) reported that intramuscular injection of 10,000 IU hCG prior to progesterone initiation improved pregnancy rates in hormonal replacement frozen ET-cleavage cycles. However, no statistically significant difference was observed in miscarriage rates. A limitation of their study was the lack of reporting on ongoing pregnancy and live birth rates. Similarly, Shiotani et al. ( 18 ) administered 3000 IU intramuscular hCG three times (on days 17, 20 , and 23 of the cycle) in patients undergoing frozen ET with hormonal replacement, performing ET on day 17 for cleavage-stage and on day 20 for blastocyst-stage embryos. They found no significant differences in implantation, pregnancy, or miscarriage rates, with the study’s limitation being the absence of ongoing pregnancy and live birth data. Conversely, Ben-Meir et al. ( 25 ) conducted an RCT investigating subcutaneous administration of 0.25 mg recombinant hCG on the day of progesterone initiation, the day of ET, and six days postET in frozen ET-cleavage. They observed no significant difference in implantation or pregnancy rates. A strength of their study was the serial monitoring of serum estradiol and progesterone levels throughout endometrial preparation and their correlation with outcomes. Eftekhar et al. ( 16 ), in a RCT, reported that intramuscular injection of 5000 IU hCG administered twice (on the day of progesterone initiation and on ET day) in hormonal replacement ET-cleavage cycles did not significantly affect pregnancy rate, ongoing pregnancy, or miscarriage rate. Conversely, in a retrospective study, the same group found a significant increase in pregnancy rate but no significant differences in implantation or miscarriage rates following four intramuscular injections of 5000 IU hCG (on ET day and every 72 hours thereafter) in similar cycycles. Xin et al. ( 19 ) retrospectively evaluated the impact of a single 2000 IU intramuscular hCG on ET day in frozen ET cycles (cleavage or blastocyst) with hormonal replacement. They observed a significant improvement in clinical pregnancy rate but no significant difference in miscarriage or live birth rates. While their large sample size strengthens the study, results were not stratified by embryo stage (cleavage versus blastocyst). Similarly, Deng et al. ( 14 ) reported that intramuscular administration of 10,000 IU hCG on the day of progesterone initiation improved implantation, clinical pregnancy, and ongoing pregnancy rates in frozen ET cycles (cleavage or blastocyst), but live birth rate improvements were significant only in cleavage-stage transfers. No significant differences in miscarriage rate were found. The large sample size is a notable strength of this study ( 14 ). In contrast to the aforementioned studies, our study employed an endometrial preparation protocol involving hormonal replacement combined with a GnRH-agonist. Available evidence suggests that, without GnRH-agonist suppression, endogenous basal LH secretion remains sufficient to activate LH/HCG receptors ( 25 ). Consequently, it is hypothesized that hCG supplementation in hormonal replacement cycles utilizing a GnRH-agonist would enhance ET outcomes. However, our findings do not support this hypothesis, as hCG administration was significantly associated only with a reduction in miscarriage rate, without affecting implantation, clinical pregnancy, or live birth rates. In a double-blind randomized controlled trial, Lee et al. ( 26 ) evaluated the effects of intramuscular administration of 1500 IU hCG twice, on the day of ET and six days later, in natural frozen ET-cleavage cycles. Their results demonstrated a significant improvement in the rate of high-quality ET but showed no significant differences in implantation, clinical pregnancy, ongoing pregnancy, live birth, or miscarriage rates. The study’s strengths include its double-blind design, large sample size, and serial measurement of serum estradiol during endometrial preparation, which was correlated with ET outcomes. In a retrospective study, Reichman et al. ( 27 ) assessed the impact of intramuscular hCG injections (2500- 10 ,000 IU, dose adjusted by BMI) administered the day after the LH surge in natural frozen ET cycles. This study included transfers of blastocyst-stage and euploid embryos. While no significant differences were found in pregnancy or miscarriage rates, the hCG-treated group exhibited a significantly higher ongoing pregnancy rate. In a non-randomized clinical trial, Davar et al. ( 28 ) reported that intramuscular injection of 150 IU/day hCG from day 8 of endometrial preparation until the endometrium reached a thickness of at least 7 mm significantly improved pregnancy outcomes in patients with a history of thin endometrium. A limitation of this study was its small sample size. Similarly, Du et al. ( 29 ) conducted a retrospective study investigating the effects of intramuscular hCG injection on frozen ET outcomes of in women with endometriosis. Following administration of 8000 IU hCG prior to progesterone initiation, they observed a significant increase in pregnancy rates but no significant difference in live birth rates. The relatively large sample size is a strength of this study. Variations in hCG dosage, timing and frequency of administration, endometrial preparation protocols (natural versus hormonal replacement), and ET (cleavage-stage versus blastocyst) among existing studies complicate the assessment of hCG efficacy in frozen ET cycles. Our study is distinct in evaluating intramuscular hCG administration during hormonal replacement cycles with GnRH-agonist suppression. Unlike many prior investigations, our study reported outcomes through live birth and included detailed monitoring of fetal, neonatal and pregnancy complications, which represent key strengths of this work. The study period coincided with the COVID-19 pandemic, which restricted patient access and prolonged recruitment. Furthermore, SARS-CoV-2 infection at the time of ET contributed to participant attrition. Without these challenges, a larger sample size may have been achievable.

The study suggests that intramuscular administration of 5000 IU hCG at three times points (72 hours before ET, on the day of transfer, and 72 hours afterward) does not significantly improve outcomes of frozen-thawed cleavage-stage ET in hormonal replacement cycles using a GnRH-agonist protocol. Nonetheless, larger RCTs are warranted to confirm these findings. Additionally, in vitro studies are needed to elucidate the optimal dose, timing, and frequency of hCG administration for enhancing implantation success.

The randomized controlled trial (RCT) was conducted at the ROYAN Institute, Tehran, Iran (ClinicalTrials.gov registration: NCT04855383 ; retrospectively registered April 22, 2021). A total of 200 patients presenting to the infertility clinic between July 2020 and August 2022 were enrolled. Participants were randomized into two groups, with or without intramuscular hCG injection using a computer, generated, unconcealed randomization list. All study procedures were performed in accordance with the ethical standards of the ROYAN Institute and 1964 Helsinki Declaration and its later amendments. Written informed consent was obtained from all participants prior to enrollment. The study protocol was approved by the Institutional Review Board and Ethics Committee of the ROYAN Institute, ACECR, Tehran, Iran on June 30, 2020 (IR.ACECR.ROYAN.REC.1399.015). Inclusion criteria were infertile women who were candidates for frozen ET at the cleavage stage, aged under 40 years, with a body mass index (BMI) below 30 kg/ m2, and possessing at least three frozen embryos of good or excellent quality. Exclusion criteria included autoimmune, endocrine, or hematologic disorders; chromosomal abnormalities or genetic diseases; uterine anomalies, fibroids, sever adenomyosis, endometriosis, or hydrosalpinx; history of recurrent pregnancy loss (RPL) or recurrent implantation failure (RIF); and severe male factor infertility. Additionally, patients with an endometrial thickness below 7 mm at the start of progesterone administration were excluded. Each patient was eligible to participate only once. The sample size was calculated based on the difference in implantation rate between the groups: 17.5% in the intervention group ( 16 ) and 35.8% in the control group ( 20 ), with a 5% margin of error and 80% study power. Considering a 10% attrition rate, a total of 200 patients were required, with 100 in the intervention group and 100 in the control group. Participants were assigned to two parallel groups through a computer-generated blocked randomization process. Allocation concealment was maintained using sealed opaque envelopes. The randomization list was accessible only to the epidemiologist. To ensure concealment, 200 envelopes were prepared, and only the methodologists had access to the randomization sequence. Upon confirming patient eligibility, the epidemiologist provided the corresponding envelope to the treating physician. Ovarian stimulation in IVF and intracytoplasmic sperm injection (ICSI) cycles was conducted using either a standard long GnRH-agonist protocol ( 21 ) or a GnRH -antagonist protocol ( 22 ). Oocyte retrieval was performed 34-36 hours after triggering final oocyte maturation. Embryos were cryopreserved at the cleavage stage, two to three days post-retrieval. Endometrial preparation was performed using a standard GnRH-agonist protocol ( 21 ). Participants in the intervention group received 5000 IU intramuscular hCG (Fulignan, Darupakhsh, Iran). hCG was administered 72 hours before ET, on the day of ET and 72 hours after ET. The control group did not receive hCG. ET was performed 48-72 hours after initiating daily progesterone injections. One to three embryos were transferred into the uterine cavity using a standard ET catheter (Labotect Gmbh, Labor-Technik-Göttingen, Kampweg 12, 37124 Rosdorf, Germany) following established protocols ( 23 ). Luteal-phase support consisted of estradiol valerate 6 mg/day and intramuscular progesterone 50 mg/day for two weeks. Upon a positive β-hCG test, the same doses of estradiol and progesterone were continued until up 12 weeks of gestation. The primary outcome was the implantation rate, defined as the number of gestational sacs per embryo transferred. The secondary outcome was the live birth rate, defined as the delivery of at least one live fetus beyond 24 completed weeks of gestation. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software (version 20.0; IBM Corp., Armonk, NY, USA). The Kolmogorov–Smirnov test was used to assess the normality of continuous variables. Results for continuous variables are presented as mean ± standard deviation (SD). Independent samples t test was used for comparisons of normally distributed continuous variables. Categorical variables were analyzed using the Chi-square (χ 2 ) test or Fisher’s exact test, as appropriate. Statistical significance was defined as a P<0.05.