Study population
A retrospective cohort study was carried out in two reproductive centers in China, namely, the Affiliated Obstetrics and Gynaecology Hospital of Nanjing Medical University and the Affiliated Changzhou Maternity and Child Health Care Hospital of Nanjing Medical University. We included all frozen-thawed blastocyst transfer (FBT) cycles in women aged ≥ 35 years (N = 1813) between January 2017 and January 2021. All procedures and protocols performed in this study were approved by the ethics committee of the two hospitals and were conducted based on the principles of the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Eligible patients were 35–42 years old and underwent frozen-thawed autologous blastocyst transfer. The exclusion criteria were as follows: endometrium < 7 mm; evidence of a uterine malformation; cycles of preimplantation genetic testing (PGT); and ≥ 3 FET cycles. Finally, a sum of 1,284 FBT cycles were included to conduct the investigation (Fig. 1).
Ovarian Stimulation
Patients received either a mild stimulation regimen or a flexible gonadotrophin-releasing hormone (GnRH) antagonist regimen. For old patients who underwent the mild stimulation regimen, 5 mg of letrozole (Letrozole tablets, Hengrui, China) or 50 mg of clomiphene (Fertilan, Medovhrmie Ltd., Republic of Cyprus) was administrated from day-3 to day-7 combined with 75 or 150 IU of recombinant follicle-stimulating hormone (rFSH) once a day. In the flexible GnRH antagonist regimen, gonadotropin (recombinant follicle-stimulating hormone, Gonal-F, Merck Serono, Switzerland) at 150–300 IU/day was started on the third day of menstruation and the GnRH antagonist (Cetrorelix 0.25 mg, Pierre Fabre Medicament Production, Aquitaine Pharm International, France) was added when a leading follicle was ≥ 14 mm in mean diameter. Human chorionic gonadotrophin at 10000 IU (hCG, Lizhu Co., China) was injected to induce final oocyte maturation when at least 3 follicles were > 17 mm in mean diameter. After 35–36 h, the oocyte was retrieved. Oocytes were inseminated by regular in vitro fertilization (IVF) or intracytoplasmic sperm injection based on sperm quality. The embryos were cultured in accordance with our previously published protocol[8]. Two cleavage-stage embryos with good morphology were transferred or frozen and supernumerary embryos were cultured to blastocysts regardless of quality.
Blastocyst Grading
The blastocysts were graded based on Gardner's scoring system [9], which involves blastocyst expansion, inner cell mass (ICM), and trophectoderm (TE). Blastocysts were evaluated on the expansion of the blastocyst cavity on a scale of 1–6. When the blastocyst arrived at a cavity expansion level 3 or over, ICM and TE were evaluated based on the size and density of the cells (A/B/C), respectively. A blastocyst evaluated 3 and above with an A or B for either ICM or TE was considered as a good-quality embryo; if not, it was defined as a low-quality embryo (grades 3–6 AC/BC/CA/CB). Poor-quality blastocysts (grades 3–6 CC) were discarded due to their low developmental potential.
Endometrial Preparation For Fet Cycles And Luteal Phase Support
The endometrial preparation regimen for FET cycles was selected by the physicians at their own discretion. The natural cycle (NC) regimen was used for participants with regular menstrual cycles. In the NC regimen, ovulation was determined by using ultrasound to detect the leading follicle and measuring the progesterone levels. When the dominant follicle had reached at least 18 mm, endometrial thickness was 6 mm or thicker, and meanwhile, progesterone level was ≤ 1.5 ng/mL, 10000 IU of hCG was administered as the ovulatory trigger. After ultrasound confirmation of ovulation, luteal phase support (LPS) was started by administering 10 mg thrice a day of progesterone (dydrogesterone, Abbott Biologicals B.V., The Netherlands). In artificial cycles, an oral estrogen treatment was started since the second to fourth day of the menstruation for one week, at a dose of 4–6 mg/day (estradiol valerate tablets, Progynova, Bayer, France), which was adjusted to 6–8 mg depending on the serum estradiol (E2) levels and endometrial thickness. After approximately ten days later, when endometrial thickness ≥ 7 mm and serum E2 ≥ 200 pg/mL, the patients were given 90 mg of vaginal progesterone (Crinone, Merck Serono, UK) once a day and 10 mg of dydrogesterone thrice a day (P + 1). In all cases, LPS with progesterone was administered after embryo transfer. Once gestational sac and embryonic heartbeat were detected by transvaginal ultrasound, the LPS was maintained until 10–12 weeks of pregnancy.
Clinical Outcomes (Dup: Abstract ?)
The primary clinical outcome was the live birth rate (LBR) and the secondary clinical endpoints included clinical pregnancy rate (CPR), clinical miscarriage rate, and twin delivery rate (TDR). The delivery of a live infant after 28 weeks of pregnancy was considered a live birth. The presence of a gestational sac confirmed by transvaginal ultrasound 4 weeks after the transfer was considered a sign of clinical pregnancy. Miscarriage was defined as a loss of pregnancy after visualization of a gestational sac. TDR was calculated from the number of twin deliveries per FET cycle.
Statistical analysis
The SPSS 26.0 software (IBM Corp., Armonk, NY, USA) was used to perform all statistical analyses. Student’s t-test was used to compare continuous variables, whose data are presented as mean ± standard deviations. Pearson’s χ2 test or Fisher’s exact test was used to compare categorical variables, whose data are presented as proportions. A multivariate logistic regression analysis was used to assess the independent effect of the transferred blastocyst number on the LBR. The covariates included were: age, basal FSH, and the number of good-quality blastocysts transferred (1 or 2 vs. 0). The odds ratios (OR) or adjusted odds ratios (aOR) and 95% confidence intervals (CI) are used to display the results. Statistical significance was accepted at a P-value < 0.05.