Study population and grouping
This was a retrospective, single-center study of patients undergoing frozen embryo transfer (FET) from January 2016 to October 2018 at the Department of Reproductive Medicine Center in the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. Inclusion criteria included the following: (1) women 20 to 42 years of age, (2) basal follicle-stimulating hormone (FSH) <10 mIU/mL, (3) first IVF/ICSI (intracytoplasmic sperm injection) cycle, (4) first FET cycle after whole embryo freezing, (5) transfer of one or two day 5 blastocysts, and (6) endometrium ≥ 7 mm. The reasons for whole embryo freezing included the prevention of moderate and severe OHSS (ovarian hyperstimulation syndrome), increased progesterone level on human chorionic gonadotrophin (HCG) day, untreated hydrosalpinx, and personal reasons (acute internal, surgical and gynecological diseases, and non-medical factors). Exclusion criteria included the following: (1) oocyte donation and cycles with preimplantation genetic testing (PGT); (2) known uterine anomalies confirmed by three-dimensional sonography and/or hysteroscopy, including endometrial polyps, septal uterine cavity, submucosal fibroid, etc.; (4) presence of hydrosalpinx not corrected surgically prior to FET, (5) stage III–IV endometriosis or adenomyosis, and (6) uncontrolled endocrine and/or immune disorders or other systemic diseases, including hypertension, diabetes, thyroid disease, hyperprolactinemia, antiphospholipid syndrome, systemic lupus erythematosus, etc. Each patient signed an informed consent upon obtaining and analyzing their clinical data prior to the initiation of IVF/ICSI-ET treatment.
These women, aged 20–42 years old, were divided into five groups depending on the quantity and quality of the day 5 blastocyst: group A (n=1569) received one good-quality blastocyst, group B (n=1113) received two good-quality blastocysts, group C (n=313) received one good- and one average-quality blastocysts, group D (n=222) received two average-quality blastocysts, and group E (n=145) received one average-quality blastocyst.
Patients in this study were given a long protocol treatment of downregulation with a gonadotrophin-releasing hormone (GnRH) agonist (triptorelin, Diphereline®, Ipsen, France) or GnRH antagonist (cetrorelix, Cetrotide®, Merck, Germany) protocol for ovarian stimulation. Individually tailored doses of recombinant human follitropin (r-hFSH; GONAL-f ®, Merck Serono, Switzerland, or Puregon®, MSD, Netherlands) were administered, and then the dosages adjusted based on the follicular development indicated by ultrasound monitoring and serum estradiol levels. Urine human chorionic gonadotrophin (uHCG, Lizhu Group Co., China) or recombinant HCG (rHCG, Merck Serono) was administered to induce oocyte maturation when at least three follicles reached a mean diameter of 17 mm. Oocyte retrieval was performed 36–38 h after HCG injection, and oocytes were incubated for insemination by conventional IVF or ICSI as determined by sperm quality.
Embryo grading, vitrification, and warming
Blastocysts were graded and scored using the Garden criteria  according to blastocyst expansion, inner cell mass (ICM) development, and trophectoderm (TE) appearance. Patients in our study were only transferred day 5 blastocyst. Blastocysts graded 4 and over with ICM A or B and TE A or B were considered as good-quality embryo. Those blastocysts that presented as grade 4 and over with an ICM C or TE C and all blastocysts that graded 3 were regarded as average-quality embryos.
All available blastocysts were cryopreserved by vitrification method according to manufacturer’s instruction. Vitrified blastocysts were thawed by a rapid thawing method in the morning of embryo transfer. The number and stage of transferred embryos were determined by clinicians and the couples, giving priority to clinical factors including patients’ age and blastocyst qualities and quantities.
Frozen-thawed cycle and embryo transfer
Endometrial preparations for FET including the natural cycle (NC) program and hormone replacement therapy (HRT) program have been described previously . In short, the NC program was suitable for patients with a regular menstrual cycle and ovulation. One or two blastocysts were transferred on the sixth day after ovulation. HRT was applicable in patients with irregular menstrual cycles or poor endometrium development in NC. The endometrium preparation of HRT used daily oral estradiol valerate tablets (Progynova®, Bayer, Germany) from the second to the fourth day after menstruation, and embryo transfer was performed on the sixth day of progesterone (60 mg/day) injection. All patients received luteal support with progesterone after embryo transfer. If transvaginal ultrasound showed gestational sac and embryonic heartbeat 4–6 weeks after embryo transfer, luteal support was continued until 10 weeks of gestational age.
Statistical analysis was performed using the Statistical Package for Social Science (SPSS) version 22.0. The baseline characteristic was expressed as the mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA) or Student’s t-test. Categorical variables were described as frequencies and percentages and compared using chi-square test or Fisher’s exact test. P < 0.05 was considered statistically significant. Live birth was defined as the delivery of any viable neonate at 28 weeks of gestation or older. Clinical pregnancy was defined as the presence of gestational sac on ultrasound at 6–8 weeks of gestation, and low birth weight was defined as birth weight less than 2,500 g and very low birth weight less than 1,500 g.