This was a retrospective study in which we linked patient information from EMRs in the Chongqing Health Center for Women and Children database to evaluate women with non-poor ovarian response (> 3 oocytes retrieved) who started their first IVF treatment under the age of 38, and who had experienced three cycles of ovum-pick up (OPU) (including IVF and intracytoplasmic sperm injection [ICSI]) from January of 2016 to December of 2019. Oocyte quality and the likelihood of pregnancy and live birth in ART diminishes with increasing age in women, and drops markedly after 40 years of age [16–18]. The 139 patients included in this study underwent their IVF cycles with either a GnRH-a or GnRH-ant protocol. Exclusion criteria were as follows. Patients with PCOS, those whose oocytes were vitrified, whose oocyte retrieval was canceled, and those patients undergoing preimplantation genetic testing (PGT) cycles, or manifesting chromosomal abnormalities or uterine malformations were excluded. At the third OPU cycle, the patients treated with GH (n = 52) were compared with the GH-free group (n = 87). Importantly, all patients showed failure in their two previous IVF cycles (Fig. 1).
Patients in the GH group received three IU of recombinant human GH (Jintropin AQ, Gensci, Changchun, China) per day, from the initial day of downregulation using the long protocol or stimulation using the antagonist protocol until the day of the hCG trigger. The average duration of GH co-treatment was 30 days for patients receiving the long agonist protocol and 10 days for those receiving the antagonist protocol.
Stimulation Protocol
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 starting in the previous luteal phase. After administration of GnRH agonist for 14–21 days—and when the levels of estrogen were < 50 pg/mL, luteinizing hormone < 5 mIU/mL, and P < 1 ng/mL—a dose of recombinant follicle-stimulating hormone (rFSH) at a dose ranging from 75 to 300 IU was then administrated subcutaneously per day based on the woman’s age, anti-Müllerian hormone (AMH) level, and antral follicle count (AFC).
GnRH-antagonist protocol: Controlled ovarian stimulation (COS) was initiated on day 2 or 3 of the cycle at a dose of rFSH ranging from 75 to 300 IU. The GnRH antagonist (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) the presence of at least one follicle > 14 mm in diameter, (ii) a serum estrogen level of > 600 pg/mL, and (iii) a serum LH level of > 10 IU/L (11).
Trigger Day
When at least three follicles measured > 18 mm in diameter, human chorionic gonadotropin (hCG, Merck Serono, Italy) was administered, and some women undergoing GnRH-ant cycles received triptorelin acetate injection (GnRH-a, Ferring GmbH, Germany) as trigger. Transvaginal oocyte retrieval was performed 36 h later, and then embryo transfer (ET) on day 3 post-oocyte retrieval. Luteal-phase support was initiated immediately after oocyte retrieval by combined vaginal and oral progesterone. Surplus viable embryos or all-frozen embryos (used due to the potential for ovarian hyperstimulation syndrome [OHSS], or a thin endometrium, abnormal blood biochemical index, or other personal situations 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 were not suitable for cryopreservation on day 3 were cultured until day 5 or 6 and vitrified if they reached the blastocyst stage. Luteal-phase support with combined vaginal-oral progesterone was started 3 days before FET.
Vitrification And Storage
The Cryotop vitrification method was performed according to the manufacturer’s instructions and as reported by Kuwayama et al. [19], and the Kitazato 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 executed according to the manufacturer’s protocol. Embryos were then placed on a Cryotop sheet and excess vitrification solution was removed by aspiration using a pipette prior to the immersion of the carrier in LN2.
Warming Procedure
A Dewar flask of LN2 containing the carriers was placed close to the microscope. We used forceps to grasp the straw in the LN2 and place it in a dish containing 3 mL of 1.0 mol/L sucrose at 37°C for 1 min. All embryos were 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 culture. Post-warming survival of cryopreserved embryos was defined as the survival of more than one-half of the original cells that were intact.
Outcome Measures
The indices of embryonic quality were the day 2–4 cell (D2-4c) rate and day 3–8 cell (D3-8c) rate, defined as the proportion of embryos with 4/8 cells on day 2/3, respectively, of the total number of two-pronuclear (2PN)-stage embryos. The primary outcome was the number of available embryos and cumulative clinical pregnancy rate (CCPR) per OPU, the latter defined as the presence of a fetal sac during ultrasonographic examination at 6 weeks and which resulted from an ART cycle—including fresh and FETs that resulted from the associated ovarian stimulation. If a pregnancy occurred, then the patients were said to have obtained an outcome, regardless of subsequent cycles.
Statistics
Data are presented as means (SD) or number (%), as appropriate. We used the Wilcoxon rank-sum test to analyze continuous variables and a nonparametric paired test was used for self-paired variables. The CCPRs in the GH and GH-free groups were compared using Chi-squared test. The generalized estimating equation (GEE) models were used to evaluate the relative prognostic significance of treatment groups, women’s age, protocol, the ovarian sensitivity index (OSI=[number of retrieved oocytes/total gonadotropin dose] × 1000), insemination and primary cause of infertility in relation to CCPR, and the number of available embryos. We adjusted interactions between independent covariates. 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).