This randomized controlled trial was performed on infertile women with poor ovarian response (POR) diagnosis who underwent IVF/ICSI cycles at Arash Women’s Hospital affiliated to Tehran University of Medical Sciences between September 2019 and May 2020. The trial protocol was approved by the Review Boards and Ethics Committee of Tehran University of Medical Sciences (ethics reference number: IR.TUMS.MEDICINE.REC.1398.544) and was registered in Iranian Registry of Clinical Trials Website (www.irct.ir, IRCT20110731007165N9).
The women with POR diagnosis undergoing IVF/ICSI cycles were determined on the basis of Bologna criteria (12) and existence of at least two of the following criteria: 1) a previous history of POR (retrieved oocytes ≤ 3) in a conventional stimulation protocol, 2) advanced maternal age (≥40 years) or any other risk factors for POR (e.g. a history of ovarian surgery) and 3) abnormal ovarian reserve test (i.e. antral follicle count (AFC) <5 follicles or anti-Mȕllerian hormone (AMH) < 1.1 ng/ml). The patients with age over 44 years, uterine factor and/or severe male factor infertility, hypothalamic amenorrhea, chronic diseases, preimplantation genetic detection diagnosis, history of recurrent miscarriage and repeated implantation failure were excluded from study. The eligible patients were randomly allocated into two groups: early antagonist protocol (intervention group) and flexible antagonist protocol (control group). The permuted block randomization was conducted by the statistician advisor with a computer-generated list. The type of treatment was placed in sealed envelopes and the assignment to intervention and control groups were performed by the out-of-study nurse. Each patient was participated in the study only once and if she had signed the written informed consent.
The pituitary down-regulation was achieved and maintained by the GnRH-ant protocol and controlled ovarian hyperstimulation (COH) was performed using the recombinant follicle stimulating hormone FSH (rFSH) (Gonal-F, Merck-Serono) and human menopausal gonadotropin (hMG; Menopur; Ferring) for all of patients. In both study groups serial two-dimensional follicle monitoring by transvaginal ultrasonography (Philips Affiniti 70 machine with a C10-3v Pure-Wave endovaginal probe) and hormonal assay (as needed) were performed.
The women in early antagonist group received rFSH (150-225 IU) and GnRH-ant (0.25 mg) (Cetrotide : Merck-Serono, or Orgalutran, MSD) simultaneously on the second day of the cycle. The patients in control group received daily injections of recombinant FSH (150-225 IU) from day 2 of the cycle and the GnRH-ant (Cetrotide: 0.25 mg daily) was initiated when the leading follicle diameter was ≥ 13 mm. In both groups, when at least two dominant follicles with 17 mm or greater in diameter was observed in ultrasound monitoring, the final oocyte maturation was triggered by human chorionic gonadotropin (hCG) (10000 IU, Choriomon, IBSA). The serum estradiol, progesterone and LH levels were measured at two points: baseline assessment (day 1 or 2 of menstrual cycle) and hCG administration day. The all-freeze strategy was applied for patients with progesterone level ≥ 1.2 ng/ml on hCG administration day.
The ovarian puncture was carried out 34-36 hours after hCG injection and IVF/ICSI process was then applied in accordance with our standards clinical procedures. The embryos quality were evaluated on the basis of Cummins et al.‘s (13) criteria with detecting the number of blastomeres, fragmentation, multinucleation and symmetry on the third day after oocyte retrieval. On the basis of the women’s age and embryos quality, up to three embryos were transferred at cleavage stage (day 3 after ovum pickup). Luteal phase was supported by using vaginal progesterone suppositories 400 mg BID (Cyclogest®, Actoverco, Iran) daily for 14 days until to pregnancy test day. In case of the positive pregnancy, vaginal progesterone was administrated until 10 weeks of gestation.
The primary outcome was the total number of retrieved and metaphase II (MII) oocytes. The implantation rate (the number of observed gestational sacs divided by the number of embryos transferred for each patient), clinical pregnancy (the presence of a gestational sac with fetal heart beat on vaginal ultrasound), early miscarriage (spontaneous loss of a clinical pregnancy ≤ 12 weeks of gestation) and ongoing pregnancy (pregnancies continued more than 12 weeks after ET) rates were considered as secondary outcomes.
Statistical Analysis
The study sample size (85 subjects in each group) was estimated using Epi-Info software with considering a difference of 0.5, significance level (alpha level) of 0.05 and 80% power. Statistical analysis was done using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) version 23.0. The comparisons of continuous variables between groups were provided by student’s t-test and presented as mean ± standard deviation (SD). The chi-square test was used for comparing the categorical variables between groups. The statistical significant level was considered at p value <0.05.