Improved Pregnancy Outcomes with Granulocyte Colony Stimulating Factor and Human Chorionic Gonadotropin Co-Treatment in Recurrent Intracytoplasmic Sperm Injection Failure: A Randomized Clinical Trial.

Despite substantial advances in assisted reproductive technologies (ART), embryo implantation rates (IRs) remain suboptimal. Successful implantation relies on multiple factors, including high-quality embryos, a receptive endometrium, and precise embryo transfer techniques ( 1 ). Endometrial receptivity refers to a well-prepared uterine environment that supports the transformation of endometrial cells into decidual cells, facilitates blastocyst attachment, and promotes placental development ( 2 ). This complex biological process is tightly regulated by immune responses, hormonal fluctuations, cytokines, and various growth factors ( 3 ). In recent years, advancements in ART have markedly improved pregnancy outcomes in both in vitro fertilization (IVF) and ICSI procedures ( 4 ). With improvements in vitrification techniques and preimplantation genetic testing, experts have gained deeper insights into the two principal factors underlying repeated implantation failure (RIF): embryo quality and endometrial receptivity. This evolving understanding has corrected the earlier misconception that a viable embryo alone, irrespective of uterine conditions, is sufficient to achieve successful pregnancy ( 5 , 6 ). A functionally receptive endometrium remains essential for the transformation of endometrial cells into decidual tissue, proper embryo attachment, and timely placental development ( 7 ). Granulocyte colony stimulating factor (G-CSF), a cytokine secreted at the maternal-fetal interface during early pregnancy, plays a key role in regulating decidual and placental functions ( 8 ). Its receptor is increasingly expressed during follicular maturation, within the endometrium, and in luteinized granulosa cells ( 9 ). Beyond stimulating neutrophil production and differentiation, GCSF modulates immune responses by regulating T helper 2 cytokines, activating regulatory T cells, and modulating uterine natural killer (NK) cell activity. Furthermore, it enhances endometrial vascularization, a critical factor for effective embryo-endometrial communication during early gestation ( 9 ). Embryo implantation is a highly complex biological process regulated by multiple factors, with human chorionic gonadotropin (HCG) playing a pivotal role ( 10 ). In primates, HCG serves as one of the earliest embryonic signals, produced by the developing embryo prior to implantation ( 11 ). This hormone facilitates trophoblast invasion during hemochorial placentation and modulates maternal immune tolerance to support embryo survival ( 12 ). The seminal work by Licht et al. ( 13 ) first demonstrated HCG’s direct modulatory effects on the endometrium, showing that in vitro exposure to 500 IU/mL HCG significantly downregulated endometrial secretion of both IG -FBP-1 and M-CSF. This foundational discovery was later translated clinically, with randomized trials indicating that intrauterine instillation of 500 IU HCG immediately prior to embryo transfer significantly improved both IR and clinical pregnancy rates (CPR) in ICSI cycles ( 14 ). Emerging evidence suggests that G-CSF therapy may enhance reproductive outcomes in patients experiencing recurrent pregnancy loss or persistent implantation failure ( 15 - 17 ). Novel delivery methods, including transvaginal administration of G-CSF, have shown particular promise in women with refractory thin endometrium (<7 mm) ( 7 , 18 ). Although some studies report that G-CSF is associated with endometrial thickening ( 19 ), the evidence remains inconsistent. Notably, Eftekhar et al. ( 8 ) observed increased pregnancy rates following intrauterine G-CSF in frozen -thawed embryo transfer (FET) cycles, even in the absence of significant changes in endometrial thickness. The potential synergistic effects of G-CSF and HCG coadministration on endometrial receptivity biomarkers and reproductive outcomes remain poorly understood in the clinical literature. As the first randomized controlled trial to evaluate this combination therapy, the present study addresses a critical knowledge gap by systematically assessing the efficacy of concurrent G-CSF (300 μg subcutaneous) and HCG (2,500 IU intramuscular) administration on the day of embryo transfer in women with ≥1 prior ICSI-embryo transfer failure. Therefore, this randomized clinical trial was designed to evaluate whether combined subcutaneous G-CSF and intramuscular HCG administration on the day of embryo transfer could improve the clinical pregnancy rate as the primary outcome in women with recurrent ICSI failure.

This randomized clinical trial (RCT) was conducted at the Reyhana Infertility Treatment Center, Qom University of Medical Sciences, Qom, Iran, between December 2024 and September 2025. A total of 150 infertile women aged 20-40 years with a history of at least one unsuccessful intracytoplasmic sperm injection-embryo transfer (ICSI-ET) cycle, despite transfer of two or more high-quality embryos per attempt, were enrolled. The study protocol was approved by the university’s Ethics Committee (IR.MUQ.REC.1404.020), and written informed consent was obtained from all participants. The trial was prospectively registered in the Iranian Registry of Clinical Trials (IRCT20220921056008N2), a WHO-recognized primary registry. Participants were excluded if they were aged 40 years, had a body mass index (BMI) >30 kg/m², endocrine or systemic disorders, antiphospholipid syndrome, severe endometriosis, recurrent miscarriage (≥3), chromosomal abnormalities, or uterine anomalies. The sample size was calculated based on the primary outcome (clinical pregnancy rate). According to previous studies ( 20 , 21 ), a clinical pregnancy rate of 23% was assumed for the control group and 56% for the treatment group. Using 80% power (β=20%) and a significance level of α=0.05, a sample size of 60 participants per group was calculated (total n=120, accounting for a 20% dropout rate, Fig .1 ). Eligible participants were randomly assigned in a 1:1 ratio to either the treatment group or the control group using a computer-generated random sequence (www.randomization.com). Block randomization with variable block sizes of 4 and 6 was employed to ensure balanced allocation throughout the study. Al- location concealment was maintained using sequentially numbered, opaque, sealed envelopes that were prepared by an independent statistician not involved in patient recruitment or outcome assessment. The random allocation sequence was generated before the start of the study, and the envelopes were opened only after the participant had completed all baseline assessments and was confirmed eligible for enrollment. Both groups followed identical endometrial preparation protocols. Physicians and participants were unblinded to treatment allocation. On cycle day 2, transvaginal ultrasound was performed to assess antral follicles, and baseline hormone levels, including follicle-stimulating hormone (FSH), prolactin (PRL), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and anti-Müllerian hormone (AMH), were measured. Ovarian stimulation was conducted using a GnRH antagonist protocol (Cinal-F 75–150 IU/ day, CinnaGen, Iran) ( 22 ) and triggering was performed with 10,000 IU HCG (IBSA, Switzerland) when ≥3 follicles reached >18 mm. Oocyte retrieval occurred 34-36 hours post-trigger, and ICSI was performed based on semen quality as assessed according to the World Health Organization (WHO) laboratory manual for the examination and processing of human semen ( 23 ). Semen parameters evaluated included semen volume (≥1.5 mL), sperm concentration (≥15 million/mL), progressive motility (≥32%), normal morphology (≥4%), and viability (≥58%). For samples meeting WHO reference values, the standard ICSI procedure was applied. Fertilized oo-cytes (with two pronuclei; 2PN) were cultured in HEPES -buffered medium (ORIGIO®) and embryos were graded (A-D) according to Hill et al.’s criteria ( 24 ), with only grades A-B selected for transfer. Embryos were vitrified using Cryotop (Kitazato, Japan) ( 25 ) and thawed prior to transfer, followed by ≥2 hours of post-warming culture. All patients received standard luteal-phase support. CONSORT flow diagram of participant enrollment, allocation, follow-up, and analysis. ICSI; Intracytoplasmic sperm injection and GCSF; Granulocyte colony stimulating factor. The primary outcome was clinical pregnancy, defined as the presence of a gestational sac with fetal heartbeat on transvaginal ultrasound 28-30 days after embryo transfer. Secondary outcomes included chemical pregnancy (serum β-hCG ≥5 mIU/mL 14 days post-transfer) and endometrial thickness measured on cycle days 12-13. Data were analyzed using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). The normality of continuous variables was assessed with the Shapiro-Wilk test. Normally distributed data are presented as mean ± standard deviation (SD) and compared using the independent samples t test, whereas non-normally distributed data are presented as median (interquartile range (IQR)) and compared using the Mann-Whitney U test. Categorical variables were analyzed using the Chi-square or Fisher’s exact test and are presented as n (%). A two-sided P<0.05 was considered statistically significant. The study initially enrolled 150 infertile women, of whom 30 were excluded during screening, leaving 120 participants for the final analysis ( Fig .1 ). Comparative analysis of baseline characteristics showed no significant differences between the treatment and control groups in age, duration of marriage, type of infertility, body mass index, or hormonal profiles, including FSH, LH, AMH, prolactin, and TSH ( Table 1 ). Demographic and clinical characteristics of study participants Data are presented as mean ± SD (Student’s t test). *; Type of infertility was analyzed using the Chi-square test. BMI; Body mass index, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, AMH; Anti-Müllerian hormone, PRL; Prolactin, and TSH; Thyroidstimulating hormone. Comparative analysis of ICSI cycle parameters demon-strated similar outcomes between two groups, with no statistically significant differences in previous ICSI attempts (P=0.099), number of retrieved oocytes (P=0.528), number of mature (metaphase II) oocytes (P=0.323), endometrial thickness (P=0.440), number of embryos transferred (P=0.648), or embryo quality scores (P=0.572, Table 2 ). Regarding pregnancy outcomes, the treatment group re -ceiving G-CSF plus HCG exhibited significantly higher rates of both chemical pregnancy (53.3 (32/60) vs. 30% (18/60), P=0.041) and clinical pregnancy (48.3 (24/60) vs. 28.3% (17/60), P=0.050) compared to the control group, supporting the therapeutic efficacy of this combined intervention ( Table 2 ). ICSI cycle parameters and pregnancy outcomes in the two study groups Data are presented as mean ± SD (Student’s t test) unless otherwise indicated or n (%). *; Categorical variables (embryo quality and pregnancy outcomes) were analyzed using the Chi-square test. Bold values indicate statistical significance. ICSI; Intracytoplasmic sperm injection, ET; Embryo transfer, and SD; Standard deviation. This randomized controlled trial evaluated the efficacy of combined G-CSF and HCG therapy in enhancing pregnancy outcomes among infertile women with a history of at least one unsuccessful ICSI cycle. To our knowledge, no previous studies have directly assessed this combination, making our investigation a valuable contribution toward identifying optimal strategies to improve reproductive success in this challenging patient population. Our results demonstrated that women receiving subcutaneous G-CSF together with intramuscular HCG achieved significantly higher chemical pregnancy rates (53.3% vs. 30%, P<0.05) and clinical pregnancy rates (48.3% vs. 28.3%, P<0.05) compared with controls. As the first study to examine systemic co-administration of G-CSF and HCG in this specific population, our findings provide strong evidence that this dual therapy may significantly enhance ICSI outcomes. The therapeutic potential of G-CSF in reproductive medicine was first highlighted by Scarpellini and Sbracia, who reported a live-birth rate of 82% in women with recurrent pregnancy loss treated with G-CSF, compared with 48% in the control group, with an excellent safety profile ( 26 ). Our findings are consistent with subsequent studies demonstrating improved IRs in IVF patients with repetitive failures ( 16 ). However, conflicting results reported by Eftekhar et al. ( 8 ), Barad et al. ( 9 ), and Li et al. ( 27 ) indicate that treatment efficacy may depend on factors such as the route of administration (systemic vs. intrauterine), patient characteristics (e.g., age, endometrial quality), and study design. Notably, positive outcomes were most consistently observed in studies utilizing subcutaneous administration in younger patients with normal endometrial development ( 28 ). The critical role of G-CSF in implantation is further supported by Salmassi et al. ( 29 ), who reported sustained elevation of G-CSF in successful pregnancies compared with declining levels in failed cycles. Additionally, Rahmati et al. ( 30 ) demonstrated that upregulation of the GCSF receptor may help overcome implantation failure, while Eftekhar et al. ( 8 ) observed improved pregnancy rates in patients with thin endometrium, even in the absence of significant endometrial thickening. Mechanistically, G-CSF appears to enhance fertility through multiple pathways: i. Promoting trophoblast function ( 31 , 32 ), ii. Regulating implantation-related genes ( 30 ), and iii. Inducing maternal immune tolerance via T helper 2 polarization and increased regulatory T cell activation ( 33 , 34 ). These complementary actions may explain the superior outcomes observed with systemic administration. Nevertheless, the literature on G-CSF efficacy is conflicting, with effectiveness influenced by both the route of administration and patient characteristics. While our study and others employing subcutaneous administration ( 20 , 30 ) reported positive outcomes, trials using intrauterine infusion, such as those by Eftekhar et al. ( 8 ) and Barad et al. ( 9 ), did not demonstrate significant improvements in pregnancy rates. Similarly, a meta-analysis by Li et al. ( 27 ) concluded that current evidence is insufficient to universally recommend GCSF for all patients with RIF. This discrepancy highlights that the method of administration (subcutaneous vs. intrauterine) and patient selection criteria (e.g., presence of thin endometrium) are critical determinants of treatment success. HCG functions as a critical signaling molecule that mediates embryo-endometrium communication, enhances endometrial receptivity, and activates gene expression pathways essential for successful implantation ( 35 , 36 ). In fertility treatments, including IVF, HCG -either in purified or recombinant form- is employed to promote oocyte maturation and facilitate follicular rupture during controlled ovarian stimulation ( 37 ). Despite its well-established roles, the effects of HCG on immune regulation during implantation remain in-completely understood. Evidence suggests that HCG promotes proangiogenic factors in the endometrium, modulates uterine NK cell activity, increases regulatory T cell populations, and enhances trophoblast invasion ( 38 - 40 ). Mansour et al. ( 14 ) were among the first to in -vestigate HCG administration in IVF patients, reporting a positive association with improved implantation and pregnancy outcomes. Additionally, Liu et al. ( 40 ) reported that HCG administration in women with RIF produced variable outcomes depending on the stage of embryo transfer. Notably, blastocyst transfers were associated with higher pregnancy rates compared with cleavage-stage transfers, a difference that may be partially attributable to the younger age of women in the blastocyst group. This study has several limitations. First, the non -blinded design and absence of a placebo control may introduce potential performance bias. Second, the single-center setting may limit the generalizability of our findings. Third, the study design did not include separate G-CSF or HCG monotherapy arms, which limits our ability to determine whether the observed effects are synergistic or primarily driven by one intervention. This issue warrants further investigation through larger trials with multiple active comparator arms. Fourth, although randomization achieved balanced baseline characteristics, other potential confounders such as subtle differences in embryo quality not captured by conventional grading or variations in patients’ underlying immunological profiles could have influenced the outcomes. A single subcutaneous dose of G-CSF (300 μg) combined with intramuscular HCG (2,500 IU) administered one hour before embryo transfer significantly improved ICSI outcomes in patients with a history of implantation failure, independent of age, endometrial parameters, or ovarian reserve markers. These preliminary findings should be interpreted with caution and require longer follow-up and systematic monitoring of potential adverse effects before any consideration of broader clinical adoption or standardized treatment protocols.