Investigating dosage effects of ovulation inhibitors on oocyte maturation in assisted reproductive technology: A retrospective study among patients with normal ovarian reserve.

Globally, approximately 8–12% of reproductive-age couples grapple with infertility, particularly in developed countries, further intensifying the declining birth rates [ 1 ]. Infertility extends beyond mere health issues, inflicting substantial psychological and economic stress and exacerbating psychosocial distress in affected females [ 2 ]. Hence, the implications transcend individual health concerns, posing substantial challenges to societal demographics and family structural dynamics. With over three million cycles conducted in 2018, assisted reproductive technology (ART) has been progressively utilized worldwide [ 3 ]. However, the rate of successful live births remains comparatively modest [ 3 ]. This underscores the urgent need for continuous research and improvements in ART methodologies and practices. ART primarily comprises four processes: oocyte retrieval following ovarian stimulation, fertilization of oocytes with sperm, culturing of the resulting embryos, and embryo transfer [ 4 ]. The retrieval of a sufficient number of high-quality oocytes is a crucial aspect of ART. Therefore, selecting and understanding appropriate controlled ovarian stimulation (COS) methods are of paramount importance. COS methods largely comprise the administration of ovulation inhibitors to prevent luteinizing hormone (LH) surge and subsequent ovulation of developed oocytes before oocyte retrieval. First, an agonist method, initially using gonadotropin-releasing hormone (GnRH) agonists as ovulation inhibitors, was developed [ 5 ]. This was followed by the widespread adoption of the GnRH antagonist (GnRH-ant) protocol, involving the administration of GnRH antagonists as ovulation inhibitors [ 6 ] and, more recently, the progestin-primed ovarian stimulation (PPOS) protocol, which uses progestins [ 7 , 8 ]. Although ovulation inhibitors effectively suppress ovulation, they concurrently inhibit the secretion of LH and follicle-stimulating hormone (FSH), which are essential for oocyte development [ 9 ]. Consequently, the excessive use of these inhibitors may hinder oocyte maturation and development, ultimately impeding the retrieval of good-quality oocytes. Although previous studies have compared the impact of ovarian stimulation methods that use different daily ovulation inhibitor dosages on the number of oocytes and metaphase II (MII) oocytes. [ 7 , 10 , 11 ], they have not addressed the clinical question of which ovulation inhibitor doses within each method can be safely administered without compromising oocyte quality. Indeed, higher doses of ovulation inhibitors may negatively affect both the maturation and developmental potential of oocytes. To clarify this, the dose-dependent impact of total ovulation inhibitor use on oocyte quality must be assessed after adjusting for various confounding factors. Accordingly, in the current study, we employ Poisson regression analysis to assess the dose-dependent effects of ovulation inhibitors on oocyte maturation and fertilization rates using the PPOS and GnRH-ant protocols. Our findings demonstrate that within the applied dosages, these ovulation inhibitors did not significantly affect oocyte maturation or fertilization rates, providing valuable insights for optimizing infertility treatments and guiding the design of ART protocols.

The median age of the 299 patients in the PPOS group was 34.0 years ( Table 1 ). The rate of premature LH surge, defined as a serum LH level greater than 10 mIU/m, was 4.7%. The median total dosage of CMA was 22 mg (IQR 18.0–32.0), and 78.9% (236/299) of the patients had a fixed daily CMA dose ( Table 2 ). Univariable Poisson regression analysis revealed a statistically significant dose-dependent increase in the oocyte maturation rate, with an unadjusted RR of 1.004 per 1 mg of CMA (95% CI: 1.001–1.008; p = 0.017). The fertilization rate was 1.002 per 1 mg of CMA (95% CI: 0.987–1.017; p = 0.770; Table 3 , Fig 3 ). However, after adjusting for the 17 covariates in a multivariable Poisson regression analysis, the aRR of the maturation rate was 1.003 (95% CI: 0.999–1.007; p = 0.194; Table 3 ). After adjusting for the 18 covariates, the aRR of the fertilization rate was 1.002 (95% CI: 0.985–1.020; p = 0.783; Table 3 ). Multivariable Poisson regression analysis indicated no dose-dependent effects of CMA on oocyte maturation or fertilization rates during the PPOS protocol. This figure illustrates the impact of CMA dosage on oocyte maturation and fertilization rates, analyzed through univariable Poisson regression. CMA, chlormadinone acetate. This table demonstrates the specific dosage employed in each stimulation protocol. CMA, chlormadinone acetate; IQR, interquartile range; OS, ovarian stimulation; PPOS, progestin-primed ovarian stimulation This table outlines the influence of Chlormadinone Acetate (CMA) dosage on oocyte maturation and fertilization rates, examined through both univariable and multivariable Poisson regression analyses. CI, confidence interval; RR, relative risk. Bold text indicates significant p -values < 0.05. After excluding 3 patients who exhibited signs of ovulation prior oocyte retrieval, 605 patients who underwent GnRH-ant were included. The median age of the 605 participants in the GnRH-ant group was 35.0 years. The rate of premature LH surge was 19.0%. The median total cetrorelix dosage was 0.5 mg [IQR 0.5–0.5], and the median day on which cetrorelix injection was initiated was Day 8 [median, IQR: 8.0–9.0] of the menstrual cycle ( Table 2 ). The univariable Poisson regression analysis revealed no significant differences in the maturation rate, with an unadjusted RR of 1.028 per 0.25 mg of cetrorelix (95% CI: 0.987–1.069; p = 0.184), and in the fertilization rate, with an unadjusted RR of 1.038 per 0.25 mg of cetrorelix (95% CI: 0.882–1.221; p = 0.653; Table 4 , Fig 4 ). After adjusting for the 17 covariates, the aRR of the maturation rate was 1.009 (95% CI: 0.962–1.059; p = 0.717). After adjusting for the 18 covariates, the aRR of the fertilization rate was 1.022 (95% CI: 0.839–1.246; p = 0.829). Multivariable Poisson regression analysis revealed no dose-dependent effects of cetrorelix on oocyte maturation or fertilization rates during the GnRH-ant protocol ( Table 4 ). This figure illustrates the impact of cetrorelix dosage on oocyte maturation and fertilization rates, analyzed through univariable Poisson regression. This table outlines the influence of cetrorelix dosage on oocyte maturation and fertilization rates, examined through both univariable and multivariable Poisson regression analyses. CI, confidence interval; RR, relative risk. Bold text indicates significant p -values < 0.05.

Our study findings indicate that, within the employed dosage ranges, neither CMA nor cetrorelix significantly affects oocyte maturation or fertilization rates in patients with normal ovarian reserve. These findings underscore the suitability of increasing ovulation inhibitor dosages, if necessary. Our results provide valuable guidance to clinicians regarding the strategic utilization of ovulation inhibitors to enhance the effectiveness of infertility treatments.

This retrospective cohort study, conducted at a reproduction center between March 2018 and October 2020, included 1,298 Japanese patients with normal ovarian reserve undergoing their first in vitro fertilization (IVF) cycles. Participants were divided into two groups: PPOS with chlormadinone acetate (CMA) and GnRH-ant with cetrorelix. The inclusion criteria comprised individuals < 40 years of age, an anti-Müllerian hormone (AMH) level ≥ 1.1 ng/mL, and patients who had their own oocytes retrieved in their initial IVF cycle at our center. These criteria aligned with the lower limits established by the Bologna criteria for poor responders, characterized by a suboptimal response to COS, resulting in diminished oocyte retrieval and fewer embryos for transfer [ 12 ]. Patients with a history of ovarian surgery, oocyte cryopreservation, chronic diseases (such as cancer or diabetes), congenital uterine anomalies, polycystic ovary syndrome, or couples with severe forms of male infertility necessitating simple or microdissection testicular sperm extraction or chromosomal abnormalities, and those with incomplete or missing data were excluded. Additionally, patients who exhibited signs of ovulation before oocyte retrieval, which were defined as a serum progesterone level ≥ 5.0 ng/mL or ultrasonographical evidence of ovulation (disappearance of the primary follicle, corpus luteum formation, and appearance of ascites fluid), were excluded. Early ovulation before oocyte retrieval did not occur in the PPOS group, whereas it occurred in three patients in the GnRH-ant group, who were subsequently excluded from the analysis. After excluding 394 patients, 904 were included, with 299 undergoing PPOS with CMA and 605 undergoing GnRH-ant with cetrorelix ( Fig 1 ). A schematic representation illustrating the flow of participant selection and the subsequent Poisson regression analysis in a retrospective study conducted at a reproduction center. The study involved 1,298 Japanese patients with normal ovarian reserves undergoing their initial in vitro fertilization (IVF) cycles, categorized into two groups: progestin-primed ovarian stimulation (PPOS) with chlormadinone acetate (CMA) and GnRH-antagonist (GnRH-ant) with cetrorelix. AMH, anti-Müllerian hormone; BMI, body mass index; CMA, chlormadinone acetate; E 2 , estradiol; FSH, follicle-stimulating hormone; GnRH-ant, GnRH antagonist; IVF, in vitro fertilization; LH, luteinizing hormone; OS, ovarian stimulation; PCOS, polycystic ovary syndrome; PPOS, progestin-primed ovarian stimulation; SA, spontaneous abortion. Basal hormone levels refer to serum concentrations of FSH, LH, and E 2 . The baseline characteristics of patients undergoing COS procedures are detailed in Table 1 , presenting median and interquartile ranges (IQR). COS was initiated between Days 2 and 5 of the menstrual cycle. The initial dose of human menopausal gonadotropin (hMG; HMG Ferring; Ferring Pharmaceuticals, Tokyo, Japan; HMG Fuji; Fuji Pharma, Tokyo, Japan) or rFSH (Gonal-f; Merck, Tokyo, Japan) ranged from 150 to 450 IU, considering age, AMH levels, and body mass index (BMI). In the PPOS protocol, 2 mg of CMA (Lutral tablets; Fuji Pharma) was orally administered daily from Day 2 to Day 5 of the menstrual cycle until the trigger day. When the serum LH level exceeded 5 mIU/mL, the CMA dose was increased by 2 mg/day (up to a maximum of 6 mg). This table provides a detailed overview of the baseline characteristics of patients undergoing controlled ovarian stimulation (COS) procedures. AFC, antral follicle count; AMH, anti-Müllerian hormone; BMI, body mass index; E 2 , estradiol; FSH, follicle-stimulating hormone; GnRH-ant, GnRH-antagonist; hCG, human chorionic gonadotropin; hMG, human menopausal gonadotropin; ICSI, intra-cytoplasmic sperm injection; IQR, interquartile range; IVF, in vitro fertilization; LH, luteinizing hormone; OS, ovarian stimulation; PPOS, progestin-primed ovarian stimulation; rFSH, recombinant human follicle-stimulating hormone; SA, spontaneous abortion; basal hormone levels refer to serum concentrations of FSH, LH, and E 2. In the GnRH-ant protocol, patients received cetrorelix (0.25 mg/mL; Merck, Tokyo, Japan) every other day, commencing between Day 8 and Day 10 of the cycle or when leading follicles reached ≥ 14 mm in diameter. The COS protocol is shown in Fig 2 . Patients at high risk of ovarian hyperstimulation syndrome, identified by a serum AMH level ≥ 5.0 ng/mL or antral follicle count (AFC) ≥ 15 on transvaginal ultrasonography, were administered 2.5 mg/day of an aromatase inhibitor (Femara; Novartis, Tokyo, Japan) for 2–5 days. Those with a serum AMH level < 2.0 ng/mL received 50–100 mg of clomifene citrate (Clomid; Fuji Pharma) daily throughout stimulation. Ovulation was induced with human chorionic gonadotropin (hCG; HCG Mochida; Mochida Pharmaceutical Co., Tokyo, Japan), a GnRH agonist (Buserecur; Fuji Pharma), or a dual trigger when leading follicles exceeded > 18 mm in diameter, followed by oocyte retrieval 36 ± 2 h later. Oocytes were fertilized via conventional IVF, intra-cytoplasmic sperm injection (ICSI), or split-ICSI, based on semen parameters and the number of retrieved oocytes. This figure outlines the controlled ovarian stimulation (COS) protocols, specifically detailing the methods and schedules of ovulation inhibitor usage for both progestin-primed ovarian stimulation (PPOS) and GnRH-antagonist (GnRH-ant). CMA, chlormadinone acetate; hMG, human menopausal gonadotropin; rFSH, recombinant human follicle-stimulating hormone. The primary outcome was the oocyte maturation rate. For both ICSI and cIVF methods, the oocyte maturation rate was calculated as the number of mature oocytes divided by the total number of oocytes retrieved. In ICSI, mature oocytes were specifically defined as those in the MII stage. In cIVF method, mature oocytes were identified by the presence of a visible polar body, observed the day after insemination, with germinal vesicle and metaphase I stage oocytes excluded. The secondary outcome was the fertilization rate, defined as the ratio of two pronuclear embryos to the number of retrieved oocytes with cIVF or mature oocytes with ICSI. The primary outcome was analyzed using univariable and multivariable Poisson regression analyses, incorporating 17 variables: age, BMI, cause of infertility, duration of infertility, infertility type, serum AMH level, basal hormone level (FSH, LH, and estradiol [E 2 ]), antral follicle count, history of spontaneous abortion, previous IVF attempts, duration of ovarian stimulation, total gonadotropin dose, type of oral medication, type of gonadotropin, and type of trigger. These covariates were selected a priori based on clinical plausibility and previous studies [ 13 , 14 ]. The secondary outcome was subjected to underwent both univariable and multivariable Poisson regression analyses with adjustments for 18 variables, including the previously mentioned 17 factors and fertility methods. The unadjusted relative risk (RR) and adjusted RR (aRR) were calculated, and the 95% confidence interval (CI) per 1 mg of CMA or 0.25 mg of cetrorelix increased in a dose-dependent manner. All tests were two-tailed, with p -values < 0.05 considered statistically significant for comparisons between the two groups. R software version 4.2.2 R was used for all statistical analyses of clinical data. This study adhered to the Ethical Guidelines for Medical Research Involving Human Subjects of the Ministry of Health, Labor, and Welfare, Japan. The retrospective design and associated procedures were approved by the Ethical Review Board of Osaka University Hospital (Suita, Osaka, Japan) (No. 19197 and No. 21113–2). In this retrospective study, we consulted the electronic medical records of patients from April 1, 2021 to December 31, 2023. Authors had access to information that could identify individual participants during or after data collection. Samples were collected between November 1, 2021 and February 1, 2022 from patients who provided written informed consent before inclusion and participated in COS cycles at the Reproduction Clinic Osaka, Osaka, Japan.