This is a retrospective real-world data study, which links the information of patients from the EMRs, Chongqing Health Center for Women and Children database to evaluate women who commenced their first COS cycle [including IVF and Intracytoplasmic Sperm Injection (ICSI)] including fresh and subsequent frozen-thawed cycles with no embryos left from January 2016 to December 2019. All patients included in this study were undergoing their first COS cycle with GnRH-a protocol or GnRH-ant protocol. Patients with preimplantation genetic testing (PGT) cycles, chromosome abnormalities and uterine malformation were excluded. Finally, a total of 18853 patients were analyzed, and 16827 patients underwent treatment with GnRH-a long protocol and 2026 patients underwent treatment with GnRH-ant protocol (Fig. 1).
The study involving human participants was reviewed and approved by the Ethics Committee Review Board of Chongqing Health Center for Women and Children (approval number: 2020-RGI-13) for retrospective analysis and clinical data reporting. Informed consent was waived by the committee because of the retrospective nature of the study.
GnRH-agonist long protocol
GnRH-a (Triptorelin 0.1 mg/d or 0.05 mg/d, sc. Decapeptyl Ferring, Germany) was used for pituitary downregulation from the previous luteal phase. After administration of GnRH agonist for 14-21 days, if the levels of estrogen <50 pg/mL, luteinizing hormone <5 mIU/mL and P <1 ng/mL, then a dose of recombinant follicle stimulating hormone (rFSH) ranging from 75 to 300 IU was administrated subcutaneously per day based on the woman’s age, antimüllerian hormone (AMH) level and antral follicle counts (AFC).
COS was initiated on day 2 or 3 of the cycle with a dose of rFSH ranging from 75 to 300 IU. GnRH antagonist of 0.25 mg (Orgalutran, Organon, The Netherlands or Cetrorelix, Merck serono, Switzerland) was given to patients daily if at least one of the following criteria was fulfilled: (i) with at least one follicle of >14 mm; (ii) serum estrogen level of >600 pg/mL; and (iii) serum LH level of >10 IU/L (11).
If there were at least three follicles were measured>18 mm in diameter, human chorionic gonadotropin (hCG, Merck Serono, Italy) was administered and part of GnRH-ant cycles used Triptorelin Acetate Injection (GnRH-a, Ferring GmbH, Germany) as trigger. After that, transvaginal oocyte retrieval was performed 36h, and then embryo transfer (ET) was performed on day 3 after oocyte retrieval. Luteal-phase support was started immediately after oocyte retrieval with vaginal combined oral progesterone. Most of the patients received double embryo transfer (DET) unless the patient had only one available embryo for single embryo transfer according to the national guidelines (12). Surplus available embryos or all-frozen embryos (due to OHSS, thin endometrium, abnormal blood biochemical index and personal causes of patients) were frozen for later transfer in subsequent frozen embryo transfer (FET) cycles. The vast majority of these embryos were frozen on day 3. Embryos that are not suitable for cryopreservation on day 3 were cultured till days 5 or 6 and vitrified if they reach the blastocyst stage. Luteal-phase support with vaginal combined oral progesterone was started three days before FET.
Vitrification and storage
The cryotop vitrification method was performed according to the manufacturer’s instructions as reported by Kuwayama et al (13). The Kitazato vitrification kit (Kita, Toyota, Japan) was used for vitrification. Embryos were equilibrated in VS1 solution for 5 min before exposure to vitrification solution (VS 2). The embryos were dipped in vitrification solution (VS 2) for 30 s, and the vitrification procedure was carried out according to the manufacturer’s protocol. The embryos were then placed on a Cryotop sheet and excess vitrification solution was removed by aspiration using a pipette before the immersion of the carrier in LN2.
A Dewar of LN2 containing the carriers was placed close to the microscope. Forceps were used to grasp the straw in the LN2 and placed it in a dish containing 3 mL of 1.0 mol/L sucrose at 37°C for 1 min. All the embryos were then transferred sequentially to 0.5 and 0.25 mol/L sucrose solutions at room temperature for 3 min each. The embryos were then washed several times in Quinn’s 1024 (Cooper Surgical, CT, USA) solution, and placed in G1 medium (Vitrolife, Kungsbacka, Sweden) for further culturing. Post-warming survival of cryopreserved embryos was defined as survival of more than one-half of the original cells that are intact.
If pregnancy is achieved, then luteal phase support was continued until 12 weeks' gestation in both the groups.
The indexes of embryo quality were the D2-4c (Day 2-4 cell) rate and D3-8c (Day 3-8 cell) rate defined as the proportion of embryos with 4/8 cells on Day 2/3 of the total number of two pronuclei (2PN) cleavege embryos. The primary outcome was the cumulative live birth per Ovum Pick-Up (OPU), which was defined as the first live born baby at ≥28 weeks’ gestation that results from a completed ART cycle, including all fresh and FETs that result from the associated ovarian stimulation. If a live birth occurs, then the patients can obtain an outcome regardless of subsequent cycles. According to this definition, multiple deliveries from the same pregnancy or multiple live births were considered as one live birth. CLBRs were calculated as the proportion of cycles that achieved the first live birth.
Data are presented as means (SD) or number (%) as appropriate. Wilcoxon rank-sum test was used for analyzing continuous variables. The cumulative pregnancy rates and CLBRs in the GnRH-a and GnRH-ant groups were compared by Chi-square test. The number of oocytes retrieved were categorized into five groups, namely 1-3 (poor), 4-9 (suboptimal), 10-15 (normal) and >16 (high) . The Cochran-Mantel-Haenszel (CMH) test by different levels of retrieved oocytes was used to compare the CLBR, normal fertilization rate, D2-4c rate and D3-8c rate between protocol groups. A multivariable logistic regression analysis were used to evaluate the relative prognostic significance of protocol groups, female age, body mass index (BMI), AMH, FSH, the number of retrieved oocytes, the number of available embryos, type of infertility in relation to CLBR. Interactions between independent covariates were adjusted. Utilized Propensity Score Matching (PSM) for sampling by up to 1:1 nearest neighbor matching with caliper (0.05) to balence the baseline between groups. P-values of <0.05 were considered to indicate statistical significance. All analyses were conducted using Stata (version 15.1, College Station, TX: StataCorp LLC).