Impact of GnRH antagonist pretreatment on oocyte yield after ovarian stimulation: A retrospective analysis

The use of gonadotropin-releasing hormone (GnRH) antagonists has been progressively increased in Assisted Reproductive Technique (ART) clinics worldwide, GnRH antagonists act by suppressing immediately and irreversibly the gonadotropin secretion, which results in a shorter duration of treatment with less patient distress [ 1 – 3 ]. Moreover, the use of GnRH antagonist protocol is associated with lower risk of hospital admission due to ovarian hyperstimulation syndrome (OHSS) [ 4 ]. On the other hand, several ART centers use the GnRH agonist protocol as first line option due to several reasons. First, the GnRH antagonist protocol has been associated with asynchrony antral follicle growth under certain condition [ 5 ]; second, the start of ovarian stimulation in a GnRH antagonist protocol relies on the occurrence of spontaneous menses [ 6 – 8 ] whereas the GnRH agonist protocol is more flexible allowing a more controlled scheduling of oocyte retrievals which means also the reduction or even the avoidance of oocyte retrievals during the weekend. In the clinical practice, pretreatment with Oral Contraceptive Pill (OCP) is used in antagonist protocols to obtain a more flexible scheduling of the start of ovarian stimulation. However, this practice is associated a decrease of ongoing pregnancy rate (OPR) [ 9 , 10 ] as well as fresh and cumulative live birth rates (LBRs) [ 11 ]. Furthermore, an increase of duration of ovarian stimulation with higher gonadotropin consumption has been reported [ 12 ]. In addition, higher serum gonadotropin concentrations as well as higher E2 concentration are found at the onset of ovarium stimulation in GnRH antagonist protocol when compared with a pituitary down regulation protocol. As a result, the unsuppressed FSH level at the start of a GnRH antagonist cycles allows the initial growth of a few leading follicles before the addition of exogenous recombinant FSH (rFSH) [ 1 , 13 , 14 ]. Menstrual administration of an antagonist before starting ovarian stimulation might reduce size and improve homogeneity of antral follicles [ 5 ]. It has already been shown that elevated progesterone at the onset of ART cycles, and reduced fertility outcome, can be solved by the administration of GnRH antagonists for 3 consecutive days before the start of Ovarian Stimulation (OS) [ 15 ]. Furthermore, a pilot study conducted in women under 36 years old, found that GnRH antagonist pretreatment during 3 consecutive days before the initiation of ovarian stimulation had a trend towards a higher number of retrieved cumulus-oocyte complexes (COCs) with improved pregnancy outcome [ 16 ]. Using a similar protocol, improved maturation and fertilization rates of retrieved oocytes was showed [ 17 ]. The current study aims to investigate whether a 3-day pretreatment course with a GnRH antagonist in the early follicular phase may increase the number of oocytes retrieved in a GnRH antagonist stimulation protocol using a large data set.

In total, 430 patients undergoing 860 cycles were included. The average female age was 34.4 ± 4.8 years. Indications for fertility treatment included unexplained infertility (34.3%), male-factor infertility (33.3%), age (16.9%), PCOS (8.2%), Tubal-factor infertility (4.7%) and endometriosis (2.6%). All cycles were divided into two groups: group 1 (standard, 430 cycles) and group 2 (pretreatment, 430 cycles). The average cohort AMH value was 2.61 ± 2.52. Basal progesterone (assessed on day 2 or 3 of the menstrual cycle) was significantly higher in group 2 (0.66 ± 0.72 vs 0.51 ± 0.3, p <0.005) ( Table 1 ). Prior-triggering hormonal assessment revealed that E2, P, LH and FSH levels were significantly higher in Group 2 than in Group 1 (2289.7 ± 1355.6 vs 1628.4 ± 971.3, p<0.001; 1.02 ± 0.65 vs 0.88 ± 0.53, p<0.001; 3.9 ± 4.65 vs 2.5 ± 3.17, p<0.001; 18.08 ± 7.2 vs 15.8 ± 6.9, p<0.001, respectively) ( Table 2 ).The mean duration of stimulation was similar in both groups (10.3 ± 1.6 vs 10.3 ± 2.2; p = 0.28) ( Table 3 ). The starting dose of gonadotropin and the total amount of gonadotropins used were significantly higher in group 2 than in group 1 (234 ± 60.9 vs 196.7 ± 54.4 p<0.001; 2419 ± 758.4 vs 2020 ± 674.9, p<0.001) ( Table 3 ). In both groups, rFSH, was more used than hMG [389/531(73.3) vs 142/531(26.7); 284/531 (53.5) vs 247/53 (46.5), p<0.001] ( Table 3 ). The total number of obtained COCs and the number of mature oocytes were significantly higher in group 2 than in group 1 (10 ± 6.6 vs 7.8 ± 5.5, p<0.001; 8 ± 5 vs 5.8 ± 4, p<0.001, respectively; difference in means 2 . 2 and 95% CI : from 1 . 6 to 2 . 9) . Fertilization rate, number of cryopreserved D3 Embryos, embryo utilization rate and the incidence and severity of OHSS were similar between the two groups [68 ± 27 vs 70 ± 25, p = 0.27; 0.3 ± 0.8 vs 0.47 ± 1.1, p = 0.08; 52 ± 36 vs 51 ± 33, p = 0.32; No OHSS: 531/531 (100) vs 529/531(99.6), Mild OHSS: 0/531 (0) vs1/531 (2), Moderate OHSS: 0/531 (0) vs 1/531 (2), p = 0.36]. The number of cryopreserved blastocysts was significantly higher in group 2 than in group 1 (1.09 ± 2.2 vs 0.28 ± 0.7, p <0.001) ( Table 4 ). a calculated as number of oocytes fertilized divided by number of COC, multiplied by 100 b calculated as number the number of embryos utilized (transferred or cryopreserved) per number of 2PN zygotes The generalized estimating equation (GEE) analysis showed that the pretreatment strategy had a significant positive effect on the number of COCs (coefficient 2.4, 95% C.I. 3.15 to 1.76, p <0.001), after adjusting for the confounders (age, indication of infertility, stimulation dose, type and duration of stimulation). On the other hand, the older age had a significant negative effect on the number of COCs (coefficient -.28, 95% C.I. -.38 to -.18, p<0.001) ( Table 5 ). Outcome: number of COCs, predictors: Maternal age, indication, type and dose of gonadotropins, duration of stimulation. Note. C.I. (confidence interval)

A 3-day pretreatment course with a GnRH antagonist administered in the early follicular phase seems to increase the number of oocytes retrieved in a GnRH antagonist stimulation protocol. Furthermore, as the initiation of ovarian stimulation in a GnRH antagonist protocol relies on the unpredictable occurrence of spontaneous menses, addition of three days of GnRH antagonist pretreatment may enhance scheduling flexibility without reducing efficacy. Larger cohort studies are needed to validate these findings.

This was a retrospective, single-centre cohort study (crossover, match–control design) at a tertiary referral university hospital including all consecutive women undergoing ovarian stimulation for In Vitro Fertilisation/ Intracytoplasmic Sperm Injection (IVF/ICSI) at Brussels IVF, the University Hospital of Brussels in Belgium from January 2011 to December 2020. The study was approved by the institutional Review Board of Universitair Ziekenhuis Brussel (approval B.U.N. 143201838385). Eligible patients were those who did not get pregnant after one standard GnRH antagonist stimulation cycle (“standard cycle”) and proceeded with one GnRH antagonist stimulation cycle preceded by early administration of GnRH antagonist for 3 days (“pretreatment cycle”) with fresh embryo transfer or frozen embryo transfer. All women may have used the same or a lower initial dose of gonadotropins in their first IVF cycle (standard cycle), both cycles needed to be performed in a time interval of <12 months. The age of included patients ranged from 20 to 44 years. Patients were excluded from the study if they had planned to undergo ovarian stimulation for preimplantation genetic diagnosis or screening, oocyte donation, social or medical egg freezing and in vitro maturation (IVM) of oocytes. All women that had basal progesterone levels >1.5ng/ml, were deemed non-eligible. All cycles were divided into two groups: group 1 (standard cycles) and group 2 (pretreatment cycles). In standard cycles ovarian stimulation was started on day 2 or 3 of the menstrual cycle with daily injections of gonadotrophins, followed by a daily dose of 0.25 mg of GnRH antagonist in a fixed protocol, starting 6 days later. In pretreatment cycles patients started antagonist pretreatment on day 2 or 3 of the menstrual cycle for 3 days onwards. The day after finishing the pretreatment (day 5 or 6 of menstrual cycle) they started with daily injections of gonadotrophins, followed by a daily dose of 0.25 mg of GnRH antagonist in a fixed protocol, starting on the sixth day of stimulation. Gonadotropins used were rFSH Gonal-F®, Merck Pharmaceuticals, Darmstadt, Germany; Ovaleap®, Theramex, Ireland Limited; Puregon®, Organon, Whitehouse Station, NJ, USA; or highly purified HMG (hpHMG) Menopur®, Ferring Pharmaceuticals, St. Prex, Switzerland. Cycle monitoring involved serum assessments of E2, P, FSH, LH, and serial transvaginal ultrasound examinations [ 18 ]. Ovulation was induced by administering hCG upon the observation of three follicles with a diameter of 17 mm [ 19 ]. Oocyte retrieval took place 36 hours thereafter. Collected oocytes were inseminated either via conventional IVF, ICSI or via IVF/ICSI. Embryos were cultured up to Day 3 or Day 5 following oocyte retrieval and the embryo transfer (ET) was performed under ultrasound guidance. Luteal phase support consisted in vaginal progesterone tablets of 200 mg three times daily, administered from the day after oocyte retrieval onwards until 7 weeks of pregnancy [ 20 , 21 ]. In case of frozen ET of embryos obtained from the same cycle, hormonal replacement therapy (HRT), natural cycle (NC) and NC with triggered ovulation protocols were used to prepare the endometrium. The primary outcome parameter was the total number of retrieved COCs after ovarian stimulation. The secondary outcomes were consumption (IU) of gonadotrophins and duration (days) of ovarian stimulation. Continuous data are presented as mean ± standard deviation (SD) and median with interquartile range (IQR). Categorical data are described by number of cases, including the numerator and denominator, and percentages. Differences in continuous variables (including the primary endpoint: total number of retrieved COCs after ovarian stimulation) between patients’ 2nd IVF cycle (with GnRH antagonist pretreatment) and their preceding cycle were calculated via dependent-sample t-tests or Wilcoxon signed-rank tests, as appropriate. Categorical variables were analyzed via Mc Nemar test, as appropriate. Continuous variables were analyzed by regression models with estimation by generalized estimating equations (GEE) to assess the effect of antagonist pretreatment in the number of oocytes and embryo utilization rate, after accounting for several confounders such as dose of gonadotropin used, type of gonadotropin used, age, cause of infertility and duration of ovarian stimulation. GEE was used to account for the within subject correlation in outcomes for repeated treatments. Results are presented with adjusted odds ratios (ORs) and 95% confidence intervals (CIs). All statistical tests used a two-tailed α of 0.05. Analyses were performed using STATA 13.0. A p-value <0.05 was considered as statistically significant.

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