Study setting and allocation of patients
This prospective RCT was conducted at the reproductive centre of Shanghai East Hospital affiliated with Shanghai Tongji University School of Medicine between September 20, 2018, and December 31, 2019. This study was performed in accordance with the Declaration of Helsinki for medical research and Good Clinical Practice guidelines and was approved by the Ethics Committee of the Institutional Review Board (IRB) of the clinic (number: 2018-17). This trial was registered in the China Clinical Trial Registry on September 6, 2018 (number: ChiCTR1800018246). Written informed consent was obtained from all enrolled patients with infertility undergoing their first ovarian stimulation cycle.
The inclusion criteria were as follows: female age 22–40 years, spontaneous menstrual cycle (25–35 days), serum anti-Müllerian hormone (AMH) levels greater than 1.1 ng/ml, and first cycle of IVF or ICSI procedure. Exclusion criteria were endometriosis grade 3 or higher, basal oestradiol levels above 80 pg/ml, recurrent miscarriage, and any contraindications for COH.
A computer-generated list was used for randomisation. Patients who met the eligibility criteria were randomly allocated to each group via numbered sealed envelopes at a ratio of 1:1. A physician assigned the drugs according to the code, and an experienced nurse instructed the patients on how to use the drugs. Physicians, nurses, and embryologists were not blinded to allocation. A total of 348 women (174 in each group) were enrolled in this study.
On the second or third day of the menstrual period, transvaginal ultrasonography and basal serum hormone (follicle-stimulating hormone [FSH], LH, oestradiol [E2], and progesterone) levels were monitored to exclude any patients with cysts or elevated basal oestradiol levels. Tablets containing 10 mg oral medroxyprogesterone acetate (MPA, Shanghai Xinyi Pharmaceutical Co., China) and 150–225 (IU) of intramuscular human menopausal gonadotropin (hMG, Anhui Fengyuan Pharmaceutical Co., China) were administered daily starting from day 2 or 3 of the menstrual cycle (MC). The hMG dosage was adjusted according to serum E2 levels and follicle sizes. When three or more dominant follicles reached 18 mm in diameter, a double trigger was administered for final oocyte maturation. Subcutaneous (SC) GnRH agonist (0.1 mg triptorelin, Decapeptyl®; Ferring Pharmaceuticals, Germany) and intramuscular (IM) hCG (5,000 IU) injections were administered for final oocyte maturation. Oocyte retrieval was performed 36 h after administering the trigger.
GnRH antagonist (GnRH-ant) protocol
Participants receiving the flexible GnRH-ant protocol received daily injections of hMG (150–225 IU) from MC day 2–3 until the trigger day. The GnRH antagonist (Cetrotide, 0.25 mg, Merck-Serono) was administered when a rise in serum LH occurred or when leading follicular diameter >14 mm was observed. The dosage of hMG was adjusted according to the patient’s characteristics, serum LH level, and follicular response. Similarly, final oocyte maturation was performed using the double trigger, and oocyte retrieval was performed approximately 36 h later.
Oocyte collection and embryo culture
Oocyte retrieval was performed in accordance with the routine protocol, and all follicles > 10 mm in diameter were aspirated. Standard insemination or ICSI was performed within 6 h of retrieval. On the third day, embryos were examined for the number and regularity of blastomeres and the degree of embryonic fragmentation. If available, up to two top-quality embryos were transferred on the third day in the GnRH-ant group if the patient’s condition permitted fresh embryo transfer (low OHSS risk and optimal endometrium). The remaining top-quality embryos were frozen by vitrification, and the non-top-quality embryos were subjected to extended culturation. Only good morphological blastocysts were frozen on day 5 or day 6. In the PPOS group, all top-quality cleavage embryos were frozen on the third day, and the non-top-quality embryos were extensively cultured and cryopreserved using the Gardner and Schoolcraft system . The viable embryos included all top-quality cleavage embryos (including grade I and grade II, 8-cell blastomere embryos) and good morphological blastocysts [12, 13].
Endometrium preparation and frozen embryo transfer
All patients in the PPOS group underwent frozen embryo transfer (FET). In the GnRH-ant group, freeze-all procedures were performed only in patients with a high risk of OHSS, a serum P level on trigger day exceeding 1.5 ng/ml, or based on personal choice. FET was conducted during the second menstrual cycle after the oocyte pickup (OPU) cycle. The methods for endometrial preparation in FET cycles were natural cycle, mild stimulation, and hormone replacement treatment (HRT), and the choice was based on routine procedures used in our clinic and in previous articles . Briefly, the natural cycle was selected first for women with normal ovulation. HRT was preferred in patients with thin endometria. Oral and vaginal progesterone were simultaneously used for luteal support. When pregnancy was achieved, exogenous progesterone supplementation was continued until 10 weeks of gestation.
Outcome measures and definitions
The primary outcome measure was the CLBR. Secondary outcome measures included the number of oocytes retrieved, the incidence of premature LH surge, the duration and dosage of hMG treatment, numbers of mature oocytes and viable embryos, and rates of implantation and clinical pregnancy. Safety measurements included serious adverse events, such as moderate/severe OHSS, bleeding, and infection after oocyte retrieval. A completed OPU cycle was defined as one in which all embryos had been used or a live birth had been achieved. Low and high ovarian responses were defined as < 5 or > 15 retrieved oocytes, respectively. The implantation rate was defined as the number of gestational sacs visualised on ultrasound examination divided by the number of embryos transferred. Biochemical pregnancy was defined as serum β-hCG level ≥ 5 IU/l 2 weeks after embryo transfer. Clinical pregnancy was defined as at least one gestational sac on ultrasound 4 weeks after embryo transfer. The CLBR was calculated as the delivery of a living newborn after the 24th gestational week divided by the number of enrolled patients. A premature LH surge during COH was defined as a serum LH level > 15 IU/l accompanied by an increase in progesterone. The OHSS was classified into three grades (mild, moderate, and severe) according to the 2016 guidelines .
This was a prospective non-inferiority trial. In line with previous studies, the CLBR was 35% in the GnRH-ant protocol . This study was powered to detect a significant difference of 15% in the CLBR. A sample size of 167 patients in each group was estimated to achieve at least 80% power to establish superiority at the 5% level of significance (PASS, version 11, NCSS, WI, USA). Given the possibility of a 5% dropout rate, we designed the study to include a total of 174 patients in each group.
The normality of the data distribution was assessed using the Shapiro–Wilk test. Continuous data are presented as the mean ± standard deviation (SD), while categorical data are presented as numbers and percentages. The results were compared between the two groups using unpaired Student’s t-tests, Mann–Whitney nonparametric U-tests, Pearson’s chi-square tests, or Fisher’s exact tests. A two-sided P value of < 0.05 was considered statistically significant. All data were analysed using the Statistical Package for the Social Sciences for Windows (SPSS, version 25.0, Chicago, IL, USA).