Study design and participants
Since April 1, 2020, the abovementioned protocols (all-37°C and 37°C-RT) for thawing embryos have been used at our center, but their relative clinical outcomes are unknown. Accordingly, we conducted a retrospective case-control matched study that included 1,734 FET cycles performed from January 1, 2020, to January 30, 2022, at the Department of Reproductive Genetics, the First Affiliated Hospital of Kunming Medical University. In this study, we evaluated the effects of the two thawing protocols on the recovery rate of embryos frozen by vitrification and the clinical outcomes after FET. Only women aged 38 or younger who had undergone conventional IVF or intracytoplasmic sperm microinjection (ICSI) insemination and were on their first FET cycle were included. Women were excluded if they had been diagnosed with recurrent miscarriage, uterine anomalies, or adenomyosis. We also excluded cycles with a thin endometrium (< 7 mm) and thawed oocytes, as well as no high-quality embryos or blastocysts (i.e., Grade III or IV cleavage stage embryos and blastocysts inferior to grade 3BB).
The study was conducted according to the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University, and no informed consent was required because the study was a retrospective study.
Sample size calculation
To enhance the reliability of this study, the required sample was determined based on the CPR for FET cycles performed from January 1, 2020, to January 1, 2021. The CPR was 56.2% per FET cycle during this period, from which it was calculated that 334 cycles would be needed in each group to achieve a power of 85% and detect a 20% between-group difference. Case and control groups were matched at a ratio of 1:1, reducing the interferences of common confounders.
Insemination methods and embryo culture
The insemination methods of all FET cycles in our center were conventional IVF or ICSI. Oocytes after injection or zygotes 16–18 h after insemination were transferred separately to 30-µL of G-1™ PLUS and covered with mineral oil (Vitrolife Sweden AB, V.Frӧlunda, Sweden). Fertilization was observed under an inverted phase contrast microscope (Nikon, Japan) at a magnification of 200–400 ×. Embryos showing normal fertilization, defined as the presence of two clear pronuclei (2PN). Then, the dishes were placed in a 37 ℃ Multi-Gas Incubator (Astec, Japan, 7.0% CO2; 5.0% O2; 88% N2) to culture embryos. On Day 3 (D3), cleavage embryos were composed of 6–12 symmetric blastomeres, with < 20% fragmentation, normal zona pellucida, and no multinucleation was considered good quality [32, 33]. One or two cleavage embryos of good quality with the highest scores were used for embryo transfer or vitrification. The remaining usable cleavage embryos were transferred separately to 30-µL of G-2™ PLUS and covered with mineral oil (Vitrolife Sweden AB, V.Frӧlunda, Sweden) for blastocyst culture. On Day 5 or Day 6 (D5 or D6), the morphology of blastocysts was evaluated according to the Gardner&Schoolcraft scoring system. Expanded blastocysts were evaluated as stages 3–6 based on the degree of expansion of the blastocyst cavity; the appearance of inner cell mass and trophectoderm was scored as A, B, or C [34]. One or two blastocysts with a grade of 3BB or higher were selected for embryo transfer or vitrification.
Vitrification and thawing protocol
Kitazato Vitrification Kit VT101 (Nakajima, Fuji, Shizuoka, Japan) was used for vitrification, and all steps in the vitrification procedure were performed at RT according to the kit instructions. First, the equilibrium solution (ES; 7.5% v/v ethylene glycol; 7.5% v/v dimethyl sulfoxide; 20% v/v replace and supplement serum; 0.005% v/v gentamicin; 83.995% v/v 199HEPES buffer solution) and the vitrification solution (VS; 17% v/v sucrose; 15% v/v ethylene glycol; 15% v/v dimethyl sulfoxide; 20% v/v replace and supplement serum; 0.005% v/v gentamicin; 51.995% v/v 199HEPES buffer solution) were stood at RT (20–25°C) for at least 30 min. The selected embryos were placed in ES for 8 min and then washed by transfer via pipette to three successive microdroplets of VS by pipetting. After the final wash, the embryos were immediately placed in a Cryotop (Kitazato Corporation), then the Cryotop was quickly immersed in liquid N2 (-196°C). The period from the transfer of the embryo into the first VS microdroplet to the immersion of the embryo into liquid N2 was equal to 45–60 s. Before freezing, blastocysts were manually shrunk with a laser to allow fluid from the blastocyst cavity to flow out [35].
The two protocols employed for thawing cryopreserved embryos with the Kitazato Thawing Kit VT102 (Yanagishima, Fuji, Shizuoka, Japan) were as follows. In the 37°C-RT protocol, the TS (34% v/v sucrose; 20% v/v replace and supplement serum; 0.005% v/v gentamicin; 64.995% v/v 199HEPES buffer solution) droplets were pre-warmed in a 37°C Multi-Gas Incubator without gas (Astec, Japan, 7.0% CO2; 5.0% O2; 88% N2) for at least 30 min. Then the DS (17% v/v sucrose; 20% v/v replace and supplement serum; 0.005% v/v gentamicin; 81.995% v/v 199HEPES buffer solution) and WS1 and WS2 (20% v/v replace and supplement serum; 0.005% v/v gentamicin; 98.995% v/v 199HEPES buffer solution) were aspirated and covered with mineral oil at 37°C to rewarm before being exposed to RT, which could reduce the osmotic pressure change of solutions. The vitrification-preserved embryos were placed in the TS droplet at 37°C for 1 min, then placed in a DS droplet for 3 min, then transferred to a WS1 droplet for 5 min, and finally transferred to a WS2 droplet for 5 min. All steps of embryo movement into DS to WS2 were performed on a workstation at RT. In the all-37°C protocol, the TS, DS, WS1, and WS2 were pre-warmed for at least 30 min at 37°C under the same conditions. All subsequent thawing steps were performed on a 37°C workstation. The embryos were kept in the TS droplet for 1 min, then transferred to the DS droplet for 3 min, then to the WS1 solution for 2.5 min, and finally to the WS2 droplet for 2.5 min. The blastocyst-thawing procedure was the same as the embryo thawing procedure. Finally, the embryos were promptly transferred to BM dishes and observed under an inverted phase contrast microscope (Nikon, Japan) at a magnification of 200–400 × to evaluate their development and morphology after thawing. Embryos that survived thawing were transferred to double-well plates and incubated for 2–4 h at 37°C in a Multi-Gas Incubator (Astec, Japan, 7.0% CO2; 5.0% O2; 88% N2).
Endothelial preparation protocol and embryo transfer
Depending on the physiology of the patients, their endometria for FET were prepared following a natural cycle protocol, an ovulation-stimulation protocol, a hormone-replacement protocol, or a pituitary downregulation hormone-replacement protocol. When the thickness of the endometrium was greater than or equal to 8 mm, embryos were then transferred to the prepared endometrium. Blood was collected 12–14 days after the endometrial implantation to track serum hCG concentrations in real-time. Clinical pregnancy was defined as a positive β-hCG test (β-hCG > 10 mIU/mL), followed by ultrasound detection of a fetal heart or gestational sac 2–3 weeks later. An early miscarriage was defined as the ultrasound-detected disappearance of the gestational sac before the twelfth week of pregnancy.
Observational indicators and outcomes
The demographic characteristics and clinical indicators associated with FET cycles for both male and female partners were compared between the all-37°C (case) and 37°C-RT (control) groups. Characteristics and indicators compared were the age and body mass index (BMI) of the male partners and the age, BMI, type of infertility, serum anti-Müllerian hormone (AMH) concentration, endometrial preparation protocol, endometrial thickness, insemination method, number of oocytes aspirated, number of embryos thawed, and embryo transfer stage (D3/D5/D6) of the female partners.
The primary outcome was CPR. Secondary outcomes were the survival rate, early miscarriage rate (eMR), and IR. The CPR was defined as the total number of cycles with at least one gestational sac with positive cardiac activity at the seventh week of amenorrhea divided by the total number of FET cycles. The survival rate was calculated as the number of embryos with at least 50% of their cells surviving after thawing divided by the total number of thawed embryos. The eMR was defined as the total number of pregnancy losses before 12 weeks of gestation divided by the total number of transferred embryos. The IR was defined as the number of gestational sacs monitored by ultrasound divided by the number of transferred embryos.
Statistical analysis
The case and control groups were matched 1:1 for potential confounding factors, namely, the woman’s age, the embryo transfer stage (D3/D5/D6), the number of transferred embryos, and the endometrial preparation method.
The baseline characteristics of the two groups were compared. Continuous data were analyzed for normality using the Shapiro–Wilk test and normal distribution plots. As no continuous variables had a normal distribution at baseline, their values are presented as medians with interquartile ranges. Categorical variables are presented as numbers with percentages. Continuous variables were compared using the Mann–Whitney U test. The distributions of categorical variables were compared using a chi-square test or Fisher’s exact test. All tests were two-tailed, with P < 0.05 considered to be statistically significant. SPSS version 26 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.