Efficacy of Stop GnRH-Agonist/Antagonist versus GnRH-Antagonist Protocols in In Vitro Fertilization Cycles of Patients with Polycystic Ovary Syndrome: A Pilot Randomized Controlled Trial.

Polycystic ovary syndrome (PCOS) is a common endocrine condition that affects reproductive health. This syndrome is marked by elevated androgen levels, persistent anovulation, irregular menstruation, along polycystic ovarian morphology evident on ultrasound ( 1 , 2 ). Approximately 70% of anovulatory infertile individuals struggle with this syndrome, which is a substantial contributor to infertility ( 3 , 4 ). In vitro fertilization (IVF) is a viable treatment option for patients with PCOS who have not responded successfully to other ovulation stimulation methods ( 5 ). A fundamental component of the IVF procedure is controlled ovarian stimulation (COS), which gathers several fertilizable oocytes and generates numerous embryos, ultimately improving the chances of conception ( 6 ). Gonadotropins are typically administered in combination with gonadotropin-releasing hormone (GnRH) analogs as part of COS regimens. The COS protocols that are widely used are the long GnRH-agonist (GnRHag) and the multiple-dose GnRH-antagonist (GnRH-ant) regimens ( 7 ). However, individuals with PCOS encounter peculiar challenges during the COS process due to their increased sensitivity to gonadotropins and greater baseline levels of luteinizing hormone (LH) ( 8 ). Their susceptibility to ovarian hyperstimulation syndrome (OHSS), a serious complication that complicates COS treatment, is greatly heightened by these features. As a result, creating special COS protocols to reduce these risks is essential to enhancing PCOS patients' treatment success ( 9 ). The Stop GnRH-agonist/GnRH-antagonist protocol is a newly developed protocol for utilizing GnRH agonist to first downregulate the hypothalamic-pituitary-gonadal axis and use GnRH antagonist during the stimulation phase and which aims to combine the advantages of both traditional GnRH-agonist and GnRH-antagonist protocols while mitigating their respective drawbacks. This approach seeks to lower the risk of OHSS while preserving adaptable control over the stimulation procedure ( 10 ). While the Stop GnRH-agonist/GnRH-antagonist regimen has theoretical advantages, its direct comparison with the conventional GnRH antagonist protocol in PCOS patients remains unexplored. This study aims to address this gap by assessing the efficacy and safety of the Stop protocol in comparison to the conventional antagonist protocol, providing new insights into its potential role in COS for PCOS patients.

Forty participants were enrolled in our study ( Fig .1 ). Among participants, around 30% (12/40) reported a history of previous pregnancy. Additionally, 15% (6/40) of the study population reported a history of prior abortions. Almost all participants were born and currently reside in urban areas (97.5%). One participant (2.5%) reported a history of hypertension; no participants reported diabetes, epileptic disorders, or skeletal diseases. Flow chart of the study process. GnRH; Gonadotropin hormone-releasing hormone. All demographic characteristics were similar between the two groups, with no significant differences observed in age, BMI, laboratory results, and duration of infertility (P>0.05). Detailed characteristic data is shown in Table 1. In the conventional protocol group, the number of follicles measuring between 16-18 mm was found to be significantly greater compared to the stop protocol group (P<0.027). However, there were no significant differences observed between the two groups for other sizes of follicles. No significant differences were observed in the total number of oocytes retrieved (P=0.702), the number of oocytes at the metaphase 1 (P=0.285) and 2 (P=0.431) stages, or the number of germinated (P=0.338) and degenerated (P=0.484) oocytes. However, it is worth mentioning that the conventional method group underwent stimulation less (mean ± SD, 10.74 ± 1.8) than the other group (12.0 ± 1.1, P=0.003). The occurrence of mild or moderate OHSS was not significantly different between the two groups (P=0.500). Additionally, the total dose of gonadotropins administered did not differ significantly between the groups (P=0.496, Table 2 ). Basic characteristics of PCOS patients undergoing stop GnRH-agonist/ GnRH-antagonist or conventional GnRH antagonist protocols Data are presented as n (%) for categorical variables, median (interquartile range) for non-normally distributed continuous variables, and mean ± standard deviation for normally distributed continuous variables. *; P<0.05 are considered statistically significant, BMI; Body mass index, AMH; Anti-müllerian hormone, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, a ; Mann-Whitney U test, b ; Student’s independent t test, and c ; Chi-square test. Cycle characteristics and pregnancy outcomes PCOS patients undergoing stop GnRH-agonist/GnRH-antagonist or conventional GnRH Antagonist protocols Data are presented as n (%) for categorical variables, median (interquartile range) for non-normally distributed continuous variables, and mean ± standard deviation for normally distributed continuous variables. *; P<0.05 is considered significant, OHSS; Ovarian hyperstimulation syndrome, a ; Mann-Whitney U, b ; Student’s independent t test, c ; Chi-square. Variables with statistically significant differences (P<0.05) are highlighted in bold.

To the best of our knowledge, this study is the first to directly compare the Stop GnRH-Agonist/GnRH-Antagonist protocol with the conventional GnRH Antagonist protocol in PCOS patients undergoing IVF. Our findings provide important insights into the efficacy and safety of these protocols in this population. The results indicate no significant difference in the total number of oocytes retrieved between the two protocols, suggesting that the Stop protocol does not compromise oocyte yield. Similarly, there were no significant differences in the number of oocytes at the metaphase I and II stages, or in the number of germinated and degenerated oocytes. These findings imply that the two COS methods produce comparable outcomes in oocyte quality and quantity. These findings align with prior research, such as the study by Khezri et al. ( 12 ), which examined a similar protocol in poor ovarian responders. They reported that while the number of mature oocytes did not differ significantly between protocols, the GnRH agonist stopantagonist group exhibited a significantly higher number of dominant follicles and embryos, as well as a greater percentage of high-quality embryos. One notable finding was the significantly shorter stimulation duration in the conventional protocol group compared to the stop protocol group. This could suggest that the stop protocol may require longer stimulation, possibly due to the washout period following GnRH agonist cessation. However, this did not translate into a significantly increased gonadotropin dose, as both groups received comparable total gonadotropin doses. Additionally, the incidence of OHSS did not differ significantly between the groups, indicating that both protocols offer similar safety profiles in ovarian hyperstimulation risk. Interestingly, the conventional protocol group had a significantly higher number of follicles measuring 16-18 mm compared to the stop protocol group. This suggests that the follicular growth pattern may differ between the two protocols, potentially influencing synchronization and maturation rates. The clinical significance of this finding remains uncertain and warrants further investigation in larger trials assessing pregnancy and live birth rates. Previous research on GnRH agonist and antagonist protocols in PCOS patients has highlighted the need for individualized COS strategies to balance efficacy and safety ( 13 - 16 ). Better cycle control and follicular development synchronization may be achieved by the long GnRH-agonist regimen, which is frequently employed. It entails initial downregulation followed by stimulation. But this method is linked to an increased risk of OHSS, particularly in those with PCOS ( 17 ). In contrast, the GnRH antagonist approach offers a shorter treatment period and a lower risk of OHSS since it avoids early downregulation, which sets it apart from the long GnRH-agonist procedure. Furthermore, this technique enables more customized and adaptable stimulation. In poor responders, a GnRH antagonist regimen may be more cost-effective and shorter in treatment duration than a long GnRH regimen ( 17 ). Additionally, OHSS incidence is significantly lower among normal responders, whereas pregnancy and live birth rates are comparable in GnRH antagonists compared to long-term typical GnRH agonist regimens ( 7 ). Nonetheless, certain research indicates that it could lead to less consistent follicular growth and perhaps reduced rates of conception in specific groups ( 18 ). Compared to the use of GnRH agonist or antagonist alone, the stop GnRH-agonist/GnRH-antagonist program has a number of advantages to consider. These benefits become clearer in specific instances, such as poor responders or patients who have had recurrent IVF failures. With respect to this approach, there are noticeably more top-quality embryos produced, and their percentage compared to the number of MII oocytes extracted is greater. This is a vital benefit for people whose prior embryo quality was subpar. Clinical pregnancy rates were shown to be respectable and occasionally higher with this combined protocol ( 19 ). The combined protocol provides flexibility in final follicular maturation triggers, allowing the use of GnRH agonists instead of hCG, thereby reducing the risk of OHSS in high responders. This feature allows clinicians to fine-tune the stimulation process up to the very end of the cycle, optimizing outcomes ( 20 ). The sequential use of a GnRH agonist followed by a GnRH antagonist may stabilize the hormonal milieu more effectively than either approach alone, enhancing endometrial receptivity and embryo implantation rates by minimizing hormone fluctuations. GnRH agonist pretreatment improves ovarian synchronization, while its discontinuation during stimulation may reduce gonadotropin requirements. Additionally, prolonged LH suppression by the GnRH agonist, combined with the immediate LH suppression of the GnRH antagonist, mitigates premature LH surges and may enhance embryo quality. Initiating stimulation with recombinant FSH optimizes mature oocyte yield, and the dual trigger (GnRH agonist plus hCG) for final oocyte maturation may further improve IVF outcomes ( 10 ). Despite the encouraging results, our research has several limitations. As this was only a pilot study, the generalizability of the findings is limited due to the small sample size. Additionally, the single-site methodology may introduce site-specific biases. The focus was primarily on immediate outcomes, such as follicle count and oocyte yield, without considering long-term results like pregnancy and live birth rates. Furthermore, the study only included participants aged 20-35 with an AMH level greater than 3.5 ng/mL, which restricts the applicability of our results. Therefore, caution is warranted when extrapolating our findings, as they may not accurately represent the broader IVF population.

The Stop GnRH-Agonist/GnRH-Antagonist protocol appears to be a viable alternative for PCOS patients undergoing IVF, providing comparable oocyte yields and safety profiles to the conventional GnRH antagonist protocol. While this approach may require a slightly longer stimulation duration, it does not increase gonadotropin requirements or OHSS risk. Further large-scale studies are needed to validate these findings and assess long-term reproductive outcomes, including pregnancy and live birth rates.

This pilot randomized, controlled clinical trial was carried out at the academic infertility clinic of Vali-EAsr Hospital, Imam Complex in Tehran, Iran from March 2023 to December 2023 and included participants who were candidates for IVF treatment. Eligible patients were women between 20 and 35 years of age who had been diagnosed with PCOS according to the Rotterdam criteria established by the European Society of Human Reproduction and Embryology (ESHRE) ( 11 ). We only included patients with anti-Müllerian hormone (AMH) levels exceeding 3.5 ng/ml. Additionally, male partners of participants were either required to exhibit normal semen analysis results or meet the minimum criteria for intracytoplasmic sperm injection (ICSI). Patients were excluded from the study if they had a history of autoimmune diseases, coagulation disorders, hypo- or hyperthyroidism, uterine anomalies, chromosomal or genetic disorders, chronic renal or metabolic diseases, any type of malignancy, endometriosis stage 3 or 4, recurrent pregnancy loss, or repeated implantation failure (RIF). Additionally, patients who have previously experienced adverse reactions to ovulation stimulation drugs were excluded. Lastly, individuals who do not exhibit adequate follicle growth suitable for puncture and patients who refuse to complete the entire treatment course were also excluded from the study. All patients treated following the ethical principles of the Helsinki Declaration. The study is permitted by the ethics board of the Tehran University of Medical Sciences (IR.TUMS.IKHC.REC.1402.525). Informed consent was obtained from all patients before participation. This study was registered in the Iranian Registry of Clinical Trials (IRCT20180409039247N10). We estimated the sample size based on practical considerations and previous studies in the field. Specifically, our sample size estimation was guided by the study conducted by Khezri et al. ( 12 ), which compared similar protocols in poor ovarian responders. The calculation was based on the number of mature oocytes retrieved (metaphase 2 oocytes) and was determined using the following formula for comparing means between two independent groups: n = [2 × (Z(1-α/2) + Z(1-β))² × σ²] / Δ² Where: Z(1-α/2) = 1.96 (for a 95% confidence level) Z(1-β) = 0.84 (for 80% power) σ = 2.54 (pooled standard deviation of mature oocytes from observed data) Δ = 0.8 (expected difference in the number of mature oocytes between groups) Substituting these values: n = [2 × (1.96 + 0.84)² × (2.54)²] / (0.8)² n ≈ 19.7 (rounded to 20 per group) Post hoc power analysis, based on the observed means and standard deviations of mature oocytes retrieved in each group, indicated a power of approximately 68% at a significance level of 0.05. While this power is slightly below the conventional 80% threshold, it is acceptable for a pilot study intended to explore feasibility and inform future research. A computer-generated randomization list was created by an independent statistician blinded to the trial. To ensure balanced allocation, the block randomization method was used with an allocation ratio of 1:1. A block size of 4 was employed to accommodate the total sample size of 40 participants. Participants remained unaware of their group allocation throughout the study. On day 2 or 3 of the cycle, a baseline transvaginal ultrasound was performed for patients in both protocols to check for ovarian cysts and assess antral follicle count (AFC). Blood samples were taken from all participants to measure baseline levels of follicle-stimulating hormone (FSH), LH, and AMH. Initiation phase: The stop protocol began with daily subcutaneous administration of a GnRH agonist in the mid-luteal phase (approximately day 21 of the menstrual cycle) until the onset of menses. Downregulation confirmation: Downregulation was verified using a vaginal ultrasound before ceasing GnRH agonist administration. Gonadotropin stimulation: After a two-day washout period (cycle days 2-3), gonadotropin stimulation commenced, with doses individualized based on patient characteristics (age, ovarian reserve, and previous response). Daily doses ranged from 150 IU to 225 IU of HMG. GnRH antagonist introduction: A GnRH antagonist (ganirelix) was introduced on stimulation day 5 and continued for two days. Follicular monitoring: Ultrasound was performed on day 7, and the GnRH antagonist was resumed when the leading follicle reached 13-14 mm. Antagonist administration continued until at least 2-3 follicles larger than 18 mm were observed. Final oocyte maturation: Oocyte maturation was triggered using either 250 μg recombinant hCG or a dual trigger (0.2 mg triptorelin + 1,000-1,500 IU hCG) when 2-3 follicles exceeded 18 mm. Ovum pick-up and embryo evaluation: Oocyte retrieval occurred 34-40 hours post-trigger, followed by embryo tracking 48 hours later. Embryos were frozen, and their grades were documented. The GnRH antagonist protocol involved administering gonadotropins starting from day 2 or 3 of the menstrual cycle. The initial dose of gonadotropins was tailored similarly to the Stop protocol, with adjustments based on ovarian response monitored via ultrasound. Patients received gonadotropins for at least 5 days. From the 6th day onwards, vaginal ultrasounds were performed every 2 or 3 days. When the leading follicle reached a size of 13-14 mm, the GnRH antagonist (ganirelix) was subcutaneously introduced. Once at least 2-3 follicles exceeded 18 mm, the final trigger was administered using hCG or a GnRH agonist. Ovum pick-up was performed 34-40 hours later. The primary outcome was the number of mature oocytes retrieved. Secondary outcomes encompassed the number of follicles >12 mm on the day of hCG administration, and incidence of OHSS. Transvaginal ultrasounds are conducted every 2-3 days to monitor follicle growth. Data were collected using a self-designed questionnaire administered to all participants at the enrollment visit. The questionnaire gathered information on demographics and medical history, including age, height, weight, smoking status, birthplace, reproductive history (gravidity, parity, abortion history), infertility type (primary or secondary), and previous medical history. The assessment of oocyte and embryo quality was performed by an expert embryologist. Eventually, a gynecology and obstetrics specialist gathered data on the hormonal profile (serum AMH, FSH, and LH levels), treatment protocol details, and follicle characteristics. Data was analyzed using SPSS version 21.0 (IBM, USA). Continuous variables were summarized using the mean and standard deviation for normally distributed data and the median with interquartile range for skewed distributions. Categorical variables were presented as frequencies and percentages. For group comparisons, the independent t test was used for normally distributed continuous variables, while the Mann-Whitney U test was applied to non-normally distributed data. Categorical variables were compared using the Chi-square test or Fisher’s exact test when appropriate. A significance level of P<0.05 was considered statistically significant.