Study design and participants
The data was collected from Center for Reproductive Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. In vitro fertilization patients who had vitrified cryopreserved embryos and following the first frozen embryo transfer cycles from January 2004 to August 2019.A total of 31143 patients met the inclusion criteria and were grouped according to the storage time (20926 patients in Group 1 with storage time <3 months, 6472 patients in Group 2 with storage time between 3 and 6 months, 2237 patients in Group 3 with storage time between 6 and 12 months and 746 in Group 4 with storage time between 12 and 24 months, 762 patients in Group 5 with storage time >24 months).The study had been approved by the ethics committee of the Third Affiliated Hospital of Guangzhou Medical University. And written informed consent was obtained from each participant.
Women were monitored and managed according to the hospital’s clinical protocols. Various controlled ovarian stimulation (COS) protocols were used, with 150-450 IU/day of recombinant FSH or human menopausal gonadotropin in a gonadotropin-releasing hormone antagonist protocol, a long agonist protocol, or a short agonist protocol. The protocols were determined according to each patient's characteristics (age, body mass index (BMI), AFC and AMH). Transvaginal oocyte retrieval was scheduled 35-36 h after HCG injection. ART was performed per standard operating procedure of the hospital. Fertilization was assessed by the appearance of two pronuclei. The luteal phase was supported by vaginal administration of micronized progesterone (400 mg/d) started on the day of ovarian puncture.
Embryo Cryopreservation Techniques and Thawing Protocols
The embryos were frozen and thawed in accordance with the protocols of the Vitrification Kit (Kato Corporation, Shizuoka, Japan). Initially, the embryos were exposed to an equilibration solution (ES) for 5 min at room temperature(cleavage embryo) or 2min at 37°C (blastocyst). After that, the embryos were transferred to a vitrification solution (VS) for 45s. Finally, embryos were set on a cryotop strip (Kato Company, Japan) in a small volume and were plunged into liquid nitrogen as soon as possible. The embryologists have been well trained to perform vitrification technically. The embryos were stored at a constant temperature of -196°C in the liquid phase of liquid nitrogen tank (Taylor Wharton HC35, Theodore, AL, USA). The level of liquid nitrogen is kept under constant surveillance manually by highly skilled embryologists to prevent suboptimal storage conditions, and the liquid nitrogen tank is refilled manually twice a week.
Vitrified embryos were thawed by a rapid thawing method on the morning of embryo transfer. For the thawing process of vitrified embryos, the embryos unloaded from the carriers were immediately submerged into the thawing solution for 1 min at 37°C. Then, the embryos were transferred into the diluent thawing solution for 3 min at room temperature. At the final step, the embryos were moved to the wash solution twice for 3 min at room temperature. After that, the embryos were cultured in culture medium at 37°C under the gas phase of 5% CO2 and 5% O2 in an incubator until transfer.
Endometrial preparation for FET cycle, and embryo transfer
Endometrial preparation for FET cycle in this study was achieved by natural cycle (NC) or hormone replace treatment (HRT) programs. The ovulation in NC program was determined by monitoring follicular development with transvaginal ultrasonography and hormone levels. The patients in HRT-FET cycles were treated with daily oral estradiol valerate tablets (Progynova, Bayer, Germany) since the second to fourth day of menstruation. When the endometrial thickness reached 7 mm or thicker, 40 mg/day progesterone was intramuscularly administered daily. One or two thawed embryos were transfer on the fourth (cleavage-stage embryo) or sixth day (blastocyst) after ovulation or progesterone injection using a soft-tipped Wallace (PortexLed., Hythe, United Kingdom) catheter under ultrasound guidance. 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.
The reproductive outcome of this study included embryo viability, implantation competence, pregnancy outcome. Neonatal outcomes included sex ratio, birth weight, birth height and congenital anomalies. Live birth was defined as the delivery of any viable neonate who was 28 weeks of gestation or older. Clinical pregnancy was defined as the present of gestational sac on ultrasound at 6–8 weeks of gestation; low birth weight was defined as the birth weight less than 2500 g and high birth weight more than 4000 g.
The statistical analysis was performed using the Statistical Package for Social Science (SPSS) version 22.0. Descriptive data are presented as mean with 1 SD. The differences between groups were tested using the ANOVA test for continuous variables and the Pearson’s chisquare test for categorical variables. Multivariable logistic regression was performed to explore the effect of storage time on pregnancy outcome or neonatal outcome after controlling for potential confounders, including maternal age and embryo quality. P < 0.05 was considered statistically significant.