This retrospective study was approved by the Medical Ethics Committee of Nanjing Drum Tower Hospital affiliated to Nanjing University Medical School on 6 May 2021 (reference number 2021–163-01).
All medical files including frozen-thawed blastocyst cycles from January 2013 to December 2019 at the Reproductive Medicine Centre of Nanjing Drum Tower Hospital were retrospectively reviewed. Inclusion criteria: (1) patients undergoing single blastocyst transfer with live singleton births and known perinatal outcomes; (2) patients have experienced at least one frozen-thawed cycle and did not have a live birth in the preceding cycle. Patients with endometriosis, abnormal uterine pathology or undergoing preimplantation genetic testing or those with history of recurrent pregnancy loss were excluded. In total, 647 live born singletons were included in this study. They were divided into two groups according to the transferred embryo cryopreservation time: vitrified-warmed blastocysts (once-vitrified group, n = 592) and vitrified-warmed blastocysts derived from thawed cleaved embryos (re-vitrified group, n = 55).
IVF/ICSI procedure and embryo culture
Depending on the patient’s age, ovarian reserve, and ovarian responses in the previous ovulation cycle, gonadotrophin-releasing hormone (GnRH) agonist protocol or antagonist protocol was used .
GnRH-agonist protocol: In mid luteal phase of the preceding cycle, a short-acting GnRH-a (Triptorelin, Ferring AG, Germany) was administered daily for 14 days and follicular ultrasonography, serum luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2) and progesterone were measured. 150–300 IU recombinant FSH (Gonal-F, Merck Serono, Switzerland) was administered daily when the serum FSH and LH levels were < 5 mIU/mL and E2 was < 50 pg/mL. GnRH-a was continued until the trigger day. The dosage of recombinant FSH was adjusted according to the ovarian response, and human menopausal gonadotropin (HMG, LIVZON, China) or recombinant LH (Luveris, Merck Serono, Switzerland) was added as needed.
GnRH-antagonist protocol: 150–300 IU recombinant FSH was initiated on Day 2 or 3 of the menstrual cycle until trigger day. The dosage of recombinant FSH was adjusted, and HMG or recombinant LH was added according to the ovarian response evaluated by transvaginal ultrasonography and serum hormone levels. 0.25 mg cetrorelix (Cetrotide, Merck Serono, France) was used daily when the leading follicles reached a mean diameter of 14 mm until trigger day.
For both protocols, if two to three dominant follicles reached a diameter of 18 mm, 250 μg recombinant human chorionic gonadotropin (hCG) (Ovitrelle, Merck Serono, France) was injected. The oocytes were retrieved under transvaginal ultrasound guidance 36–38 hours after recombinant hCG administration. Retrieved oocytes were then fertilized in conventional IVF or intracytoplasmic sperm injection (ICSI). Two pronuclei or two polar bodies can be observed in normal fertilization 16–18 h post insemination. Embryos were cultured in G1/G2 sequential media (Vitrolife, Goteborg, Sweden) at 37 °C in 6% CO2, 5% O2 and 89% N2 high-humidity incubator. Blastocyst morphological evaluation was based on the Gardner scoring system .
Vitrification cryopreservation and thawing procedures
The vitrification cryopreservation and thawing procedures were carried out using a vitrification or warming kit from Kitazato (Tokyo, Japan). Before vitrification, the blastocyst was artificially shrunken by laser drilling. Cleavage-stage embryos or shrunken blastocysts were preequilibrated in equilibration solution for 5–8 min at room temperature and then placed into vitrification solution. One minute later, the embryos were loaded onto the surface of Cryotop (Kitazato, Tokyo, Japan) and then submerged into liquid nitrogen immediately, using open vitrification devices.
The warming procedure was referred to our center’s previous research . The embryo was immediately transferred to preequilibrated thawing solution for 1 min and dilution solution for 3 min and then washed twice in washing solutions 1 and 2 for 5 min each; the embryo was then cultured in a 100 μl droplet of culture medium G2 (Vitrolife, Goteborg, Sweden) overlaid with light paraffin oil (Vitrolife, Goteborg, Sweden) in the incubator at 37 °C in a humidified atmosphere with 5% O2, 6% CO2 and 89% N2 for 2–4 h and then assessed. Laser-assisted hatching was applied to vitrified–warmed blastocysts except for those blastocysts with an expansion degree of V or VI. Generally, blastocysts with re-expanded blastocoel cavities with clear morphology and a bright lustre were considered to have survived.
Overall baseline clinical characteristics for each patient included age, body mass index (BMI), basal FSH, duration of infertility, parity, type of infertility and cause of infertility, insemination methods (IVF or ICSI), endometrium preparation protocol for FET, and endometrial thickness.
Any singleton birth ≥24 weeks of gestation was considered a live singleton birth. The birth weight, gestational age at delivery and sex were recorded for all live singletons. The neonatal outcomes evaluated were preterm birth (PTB, delivery between 24 and 37 weeks), low birthweight (LBW, birthweight < 2500 g), macrosomia (birthweight > 4000 g), average birthweight, sex ratio, small for gestational age (SGA), and large for gestational age (LGA). SGA was defined as a weight below the 10th percentile for gestational age, and LGA was defined as having a birthweight greater than the 90th percentile for gestational age at birth .
Continuous variables were described by using the mean ± SD or median (interquartile range, IQR), while categorical variables were described as absolute numbers and percentages. Student’s test was used to compare the differences of normally distribution parameters, while for non-normal distribution parameters, Wilcoxon rank sum test were used. Pearson’s chi-squared test or Fisher’s precision probability test was used to compare the differences of categorical variables. To help account for the nonrandomized administration of re-vitrified embryos, we used propensity-score methods to reduce the effects of confounding. Matching was performed with the use of a 1:1 matching protocol without replacement, with a caliper width equal to 0.01 of the standard deviation of the logit of the propensity score. P values < 0.05 were considered statistically significant. The variables included maternal age, parity, basal FSH, BMI, duration of infertility, type of infertility, parity, and cause of infertility. To analyse the associations between the cryopreserved time and perinatal outcomes, logistic regression models were conducted for each outcome indicator using the before and after matching data, and odds ratios (OR) and their 95% CI before and after adjusting for confounders were calculated. Statistical analyses were performed using SPSS statistical package version 26.0.