Study population and grouping
A retrospective study that included 2,038 FET cycles with poor-quality blastocysts was conducted at the Reproductive Medicine Center of the Third Affiliated Hospital of Guangzhou Medical University from January 2014 to December 2019. The inclusion criteria were as follows: (1) poor-quality blastocyst transfer; (2) age ≤40 years; and (3) endometrial thickness ≥ 7 mm. The exclusion criteria were as follows: (1) donated oocytes or embryos; (2) cycles with preimplantation genetic testing (PGT); (3) transferred blastocysts frozen on day 7; (4) stage III to IV endometriosis or adenomyosis; (5) known uterine anomalies including intrauterine adhesion, septal uterine cavity, endometrial polyps, and submucosal fibroid; (6) untreated hydrosalpinx; (7) uncontrolled endocrine and/or immune disorders or other systemic diseases, including hypertension, diabetes, thyroid disease, hyperprolactinemia, antiphospholipid syndrome, and systemic lupus erythematosus.
Patients were categorized into four groups according to blastocyst development speed and the number of embryos transferred, namely the D5-SBT group (n=476), D5-DBT group (n=365), D6-SBT group (n=730), and D6-DBT group (n=467). Patients in the SBT group were again divided into four subgroups based on the blastocyst morphology: D5-AC/BC (n=407), D5-CA/CB (n=69), D6-AC/BC (n=580), and D6-CA /CB (n=150). The study was approved by the local Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University.
Blastocyst quality assessment and vitrification
Blastocysts were scored according to the Gardner grading system  and recorded as high-quality blastocysts if they reached at least an expansion stage 3 with A or B for ICM and TE. The embryos included in this study were all poor-quality blastocysts, which are defined as at least an expansion stage 3 with ICM “C” or TE “C.” In all FET cycles, no more than two blastocysts were transferred.
The precise vitrification and thawing protocol of the blastocysts was carried out according to the manufacturer’s instructions. Briefly, blastocyst vitrification was carried out using a Cyrotop carrier system (Kitazato Biophama Co. Ltd.. Shizuoka. Japan), in conjunction with DMSO-EG-S as a cryoprotectant. For thawing, embryos were transferred into dilution solution in a sequential manner (1 M-0.5 M-0.25 M-0 M sucrose).
Endometrial preparation for the FET cycle embryo transfer
Endometrial preparation for the FET cycle in this study was achieved using natural cycle (NC) or hormone replacement treatment (HRT) programs. In short, NC was applied for patients with regular menstrual cycles and ovulation. The ovulation in the NC protocol was determined by monitoring the follicular development with transvaginal ultrasonography and hormone levels. HRT was applicable for patients with irregular menstrual cycles or poor endometrium development in NC. The patients were treated with daily oral estradiol valerate tablets (Progynova, Bayer, Germany) from the second to the fourth day of menstruation. When endometrial thickness reached 7 mm or thicker, exogenous progesterone was administered daily.
One or two thawed blastocysts were transferred on the sixth day after ovulation or progesterone exposure using a soft-tipped Wallace (Portex Led., Hythe, United Kingdom) catheter under ultrasound guidance. All patients received luteal support with progesterone after embryo transfer and continued to 10 weeks of gestational age if a pregnancy occurred.
The primary outcome of this study was the live birth rate (LBR). Secondary endpoints included rates of implantation, clinical pregnancy, multiple pregnancy, spontaneous miscarriage, and neonatal outcomes. Neonatal outcomes included preterm birth, birth weight, height, and low birth weight.
Live birth was defined as the delivery of any viable infant who was 28 weeks of gestation or older, and twins delivered by one mother were calculated as one live birth. Clinical pregnancy was defined as the presence of gestational sac transvaginal ultrasound at 6–8 weeks of gestation. Early miscarriage was defined as a spontaneous pregnancy demise at less than 12 weeks of gestation. Preterm birth was defined as a delivery before completing 37 weeks of gestation, and low birth weight was defined as a birth weight less than 2,500 g.
The statistical analysis was performed with the use of the Statistical Package for Social Science (SPSS) version 22.0. The baseline characteristics were expressed as the mean ± standard deviation (SD), and differences in variables were compared using Student’s t-test or one-way analysis of variance (ANOVA). Categorical variables were described as frequencies and percentages and compared using the chi-square test and Fisher’s exact test when the number of events was less than 5. A P-value < 0.05 was considered statistically significant.