In the present retrospective cohort study, a review of medical records from July 1, 2014, through September 30, 2020, was performed for all IVF/ICSI-fresh ET cycles that underwent the GnRH antagonist protocol at the Reproductive and Genetic Center of Integrative Medicine, The Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The study was approved by the Ethics Committee of the Reproductive Medicine Ethics Committee of the Affiliated Hospital of the Shandong University of Traditional Chinese Medicine. Informed consent was waived owing to the retrospective study design.
Inclusion and exclusion criteria
The inclusion criteria were women: (i) aged<50 years; (ii) with a body mass index (BMI) of 18–35 kg/m2; and (iii) who received GnRH antagonist and underwent fresh ET during the IVF/ICSI cycles. Our exclusion criteria were women: (i) with other underlying diseases that could not tolerate childbearing; (ii) suffering from an endocrine disorder (diabetes mellitus, hyperprolactinemia, thyroid dysfunction, congenital adrenal hyperplasia, Cushing syndrome); (iii) with a history of recurrent spontaneous abortion; and (iv) with uterine anomaly, endometriosis, or chromosomal abnormalities that cause infertility. In cases of missing data, telephone surveys were conducted.
After applying the inclusion and exclusion criteria, a total of 520 IVF/ICSI with fresh ET cycles were included and then divided into two groups: dual-trigger and hCG trigger. Applying either hCG alone or the dual-trigger for final oocyte maturation depended on the attending physician.
All patients were treated with the GnRH antagonist protocol in the flexible mode for COH. On the second day of the menstrual cycle, baseline hormone levels, such as follicle-stimulating hormone (FSH), LH and estrogen (E2), were measured and transvaginal ultrasound for antral follicles was conducted to evaluate the baseline ovary status. If the conditions permitted, 150–225 IU/d of recombinant human follicle-stimulating hormone (rFSH, GONAL-F, Merck Serono, Darmstadt, Germany) with or without application of human menopausal gonadotropin (HMG, Lizhu Pharma, Shaoguan, China) was injected from the third day of the menstrual cycle, according to the age, BMI, antral follicle count, and previous response to COH. Besides, the addition of human recombinant luteinizing hormone (rLH, Luveris, Merck Serono) and recombinant human growth hormone (rhGH, Genheal, United Cell Biotechnology, Shanghai, China) during COH was at the physician’s discretion. The dosage of Gn was adjusted lying on the follicular growth and serum level of E2. When the dominant follicle reaches 12–14 mm in diameter, the GnRH antagonist 0.25 mg/d (Cetrotide, Merck Serono) was administered until the day of final oocyte maturation. The trigger was administered when at least two follicles reached a diameter of 18 mm or three follicles reached a diameter of 17 mm. The hCG trigger group (n=458 patients) received 250 mg of recombinant hCG (Ovidrel, Merck Serono) alone, and the dual-trigger group (n=62 patients) received 0.2 mg triptorelin (Decapeptyl, Ferring Pharmaceuticals, Saint-Prex, Switzerland) plus 250 μg of recombinant hCG. The oocyte was retrieved by transvaginal ultrasonography after 36 h. Whether fertilization was conducted by IVF or ICSI depended on the results of semen analysis or prior fertilization condition.
Normal fertilization was confirmed when two pronuclei and two polar bodies were observed after 16–18 h of insemination. Amphicytula were cultured in a cleavage medium (Cook Medical, Bloomington, IN, USA), and the embryo development was assessed daily (i.e., number, shape, evenness, and fragment rate of embryos). Fresh ET was performed 3 days after oocyte retrieval. The number of transferred embryos was 1–2 depending on the embryo quality and patient age. A high-quality embryo was defined as one that met the following three criteria established by the Istanbul consensus workshop (20): (i) ≥6 cells 3 days after fertilization, (ii) <10% fragmentation, and (iii) symmetric blastomeres. Then, the remaining embryos that did not satisfy the criteria were transferred and cultured to the blastocyst stage in the blastocyst medium (Cook Medical). Blastocysts were graded according to the Gardner classification (21); blastocysts were considered to be of excellent quality if the following criteria were met: (i) complete expansion of the blastocyst, and the cavity completely occupies the total volume of the embryo (grade 3); (ii) the inner cell mass was composed of several loosely grouped cells (grade B); and (iii) the trophectoderm contained few cells that formed loose epithelium (grade B). Embryos that were not transferred were cryopreserved by vitrification.
All patients received 40 mg/d of progestin injection (Xianju, Taizhou, China) for LPS from the day of oocyte retrieval until the pregnancy test was performed 2 weeks after fresh ET. In case of a positive pregnancy test, the LPS strategy was continued until 10 weeks of gestation.
Laboratory and clinical data were collected as follows: the number of oocytes retrieved, rates of normal fertilization, number of embryos, number of high-quality embryos, implantation rate (IR), clinical pregnancy rate (CPR), ectopic pregnancy rate (EPR), miscarriage rate (MR), and LBR. Normal fertilization rate was defined as the number of normal fertilized oocytes divided by the total number of retrieved oocytes. The implantation rate was calculated as the number of gestational sacs divided by the total number of transferred embryos. Clinical pregnancy was defined as the presence of gestational sacs on ultrasonogram at 5–6 weeks of pregnancy. A diagnosis of ectopic pregnancy was made either by laparoscopy or sonographic visualization of an extrauterine gestational sac. The miscarriage rate was calculated as a pregnancy that was terminated before 28 gestational weeks divided by the total clinical pregnancy events. The LBR was calculated by dividing the total deliveries of viable infants over 28 gestational weeks by the total number of fresh ET cycles.
Data analyses were carried out using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). We compared laboratory and clinical outcomes for the two groups in a propensity score matching (PSM) cohort to minimize potential deviation (Fig. 1). The propensity scores were calculated using binary logistic regression analyses based on the following patient variables at baseline: patient age; infertility duration; infertility type (primary or secondary); gravidity; parity; number of previous IVF/ICSI attempts; BMI; level of anti-Mullerian hormone (AMH); basic hormone levels of FSH, LH, and E2; stimulation duration and dosage of Gn; insemination treatment; and the number and quality of embryos transferred. A 1:1 pair matching was performed using the caliper-matching method, and a 0.02 propensity score tolerance was imposed on the maximum propensity score distance. Shapiro–Wilk test was used to assess the normality of data distribution. Continuous variables were expressed as mean±standard deviations (SD) and then compared using Student’s t-tests or Mann–Whitney U-tests. Categorical variables were presented as frequencies and percentages, and the between-group differences were analyzed using the chi-square test. P<0.05 was considered statistically significant. We also used binary logistic regression analysis to assess the association between trigger protocols or other potential factors and pregnancy outcomes. We calculated the adjusted odds ratio (OR) with a 95% confidence interval (CI).