This was a retrospective cohort study initiated at the Center for Reproductive Medicine in the Peking University Shenzhen Hospital, China between January 2017 and July 2018. A total of 705 FET patients in which patients aged < 40 were included and 1486 embryos were formed, of which 1366 embryos were eventually transferred. All patients underwent only one FET cycle. In our research, the final transplanted slow- growing embryos were divided into early thawing group and routine thawing group according to the physician’s decision, all of which were cultured overnight and were transferred one day later. This study has been approved by the Ethics Review Committee of Shenzhen Hospital of Peking University.
Ovarian stimulation, IVF or ICSI treatment, Embryo freezing and thawing
Embryos were gained from controlled ovarian stimulation with conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). All embryos were assessed according to the routine evaluation system  and were cryopreserved on Day 3 of embryo culture. All the cryopreservation was performed using vitrification protocols. For Day 3 vitrification, embryos with at least 4 blastomeres and ≤ 25% fragmentation were selected for cryopreservation. Only the slowing growing embryos which were composed of 4 to 6 blastomeres were included in this study.
Embryos were thawed at the following two different time points and classified into two different groups (Fig. 1). As for the natural cycle and controlled ovarian stimulation cycle, embryos were thawed after 2 days (early thawing group) or 3 days (routine thawing group) of ovulation. As for the hormone replacement treatment (HRT) cycle, embryos were thawed after 3 days (early thawing group) or 4 days (routine thawing group) of progesterone administration.
Embryos were then warmed 1 day before transferring. The surviving embryos were cultured overnight and transferred at the 2nd day. Morphological survival was confirmed by counting the intact cells on the number of cells present at cryopreservation. Embryos with at least 50% intact cells were considered surviving and were further cultured. Further cleavage was evaluated the next morning and characterized into top quality, good quality and poor quality according to the previous standard . Embryos with further cleavage and showing signs of compaction or blastulation were considered as top quality. Good quality embryos had at least eight blastomeres and further cleavage of at least two cells, while poor quality embryos had less than eight blastomeres and/or no further cleavage and/or limited further cleavage of only one cell. The further cleavage rate was defined as the percentage of embryos with at least division of two cells after one-day culture on the total number of transferred embryos.
Preparation of the endometrium
As for natural cycle and natural cycle with ovulation stimulation cycle: Ultrasound examination was applied to monitor the follicular growth from day 8–10 of the menstrual cycle and consecutively examined every 2–4 days. After the luteinizing hormone (LH) surge, dominant follicle collapse was confirmed as the ovulation day (day 0) . Besides, ovulation day was double confirmed according to serum progesterone (P) level. If dominant follicle disappeared, P serum level was checked. In the natural cycle with hCG, after ultrasound evidence of follicles ≥ 18 mm and endometrium thickness ≥ 7 mm, 6,000 IU urinary hCG (Choriomon, IBSA, Lugano, Switzerland) was administered to trigger ovulation.
As for HRT cycle and GnRH-a pretreatment plus HRT cycle: before the start of the cycle, the basal hormone value of endocrine evaluation was confirmed by the measurement of estradiol (E2), P, luteinizing hormone (LH) and follicle stimulating hormone (FSH). Then a dose of 2 mg estradiol valerate (Progynovaw, Bayer-Schering Pharma AG, Berlin, Germany confirm) twice a day was given to the patients for 7 days, followed by 6 days of estradiol valerate at a dose of 2 mg three times per day. On Day 13 of estradiol valerate treatment, endometrial thickness was measured by ultrasound and hormonal analysis was performed by serum E2 and P examination. In FET cycle, the same embryonic time convention is usually followed, and the date of ovulatory in the natural cycle is equivalent to the 1st day of P administration in the artificial cycle . If the serum P level was higher than.1.5 ng/ml, the cycle was cancelled. If endometrial thickness was ≥ 7 mm, P supplementation was started. The choice for using GnRH-a was depended on the physician’s decision.
Assessment of pregnancy outcome
Conception was defined as arbitrary serum beta hCG is higher than 50 IU/L at 14 days following embryo transfer. Clinical pregnancy was confirmed by ultrasound scan with at least one fetus with heart beat by 7 weeks pregnancy. The implantation rate was that the number of gestational sacs observed divided by the number of embryos transferred and the live birth was defined as at least one baby born.
The pregnancy outcome of FET cycle was studied, including conception, clinical pregnancy, implantation, biochemical miscarriage, first trimester pregnancy loss, second trimester pregnancy loss, ongoing pregnancy and livebirth.
The p-value, risk ratio and confidence interval were analyzed by Social Science 19 (SPSS, Inc., Chicago, IL, USA). The categorical data are expressed as percentage and analyzed by using the chi-square test or Fisher's accurate test according to the sample size. The odds ratio (OR) and 95% confidence interval (CI) were compared to evaluate the difference. According to the normality of the results, the continuous variables were analyzed by independent t test or Mann-Whitney u test, and all the data were two-tailed test. Logistic regression analysis was used to adjust for confounders, including regimen of endometrial preparation and the timing of thawing embryos. The significance level was set at p < 0.05.