An all-37 °C thawing method improves the clinical outcomes of vitrified frozen-thawed embryo transfer: a retrospective study using a case–control matching analysis

The purpose of this study is to assess the impact of different temperatures and incubation times on the clinical outcomes of FET cycles during the thawing procedure and to select a better thawing method to improve clinical outcomes. This retrospective study included 1734 FET cycles from January 1, 2020, to January 30, 2022. Embryos vitrified using a KITAZATO Vitrification Kit were thawed at 37 °C in all steps (the case group, denoted the “all-37 °C” group) or at 37 °C and then at room temperature (RT; the control group, denoted the “37 °C-RT” group), according to the kit instructions. The groups were matched 1:1 to avoid confounding. After case–control matching, 366 all-37 °C cycles and 366 37 °C-RT cycles were included. The baseline characteristics were similar (all P > 0.05) between the two groups after matching. FET of the all-37 °C group yielded a higher clinical pregnancy rate (CPR; P = 0.009) and implantation rate (IR; P = 0.019) than FET of the 37 °C-RT group. For blastocyst transfers, the CPR (P = 0.019) and IR (P = 0.025) were significantly higher in the all-37 °C group than in the 37 °C-RT group. For D3-embryo transfers, the CPR and IR were non-significantly higher in the all-37 °C group than in the 37 °C-RT group (P > 0.05). Thawing vitrified embryos at 37 °C in all steps with shortening wash time can enhance CPR and IR in FET cycles. Well-designed prospective studies are warranted to further evaluate the efficacy and safety of the all-37 °C thawing method.


Introduction
The freezing and thawing of oocytes/embryos/blastocysts is an important step in assisted human reproduction procedures, the widespread use of which has increased the chances of pregnancy for women undergoing in vitro fertilization (IVF)-embryo transfer [1]. In particular, the use of frozen-thawed embryo transfer (FET) has increased dramatically in recent years, as it offers a high live-birth rate (LBR) and a low risk of adverse pregnancy outcomes such as preterm birth, low birth weight, small for gestational age, multiple pregnancies, and ovarian hyperstimulation syndrome, thereby enhancing the safety and efficacy of the treatment [2][3][4][5][6].
Cells contain a high volume of water, the freezing of which must be regulated during cryopreservation, as ice crystals formed inside cells during cooling or warming may cause serious damage [7,8]. Cryoprotectants are used to Gaofeng Yan and Youlin Yao contributed equally to this work. reduce physical damage during chilling, but they contain a variety of chemicals that in high concentrations can have toxic effects on and cause osmotic damage to cells [9,10]. The two methods commonly used for preserving embryos/ blastocysts/oocytes are slow freezing and vitrification [11]. Slow freezing uses a low concentration of cryoprotectant and a sufficiently low entire cooling rate to effectively dehydrate cells and reduce ice-crystal formation [12]. In contrast, vitrification uses a high concentration of cryoprotectant and rapid cooling to vitrify the inside and outside of cells without producing ice crystals, and the cryoprotectant concentration inside the cell during vitrification is lower than that during slow freezing [13,14], which reduces damage to cells during freezing. Compared with slow freezing, the use of vitrification for freezing embryos has less of an effect on cell metabolism and the genome and increases the survival and developmental potential of embryos [15,16]. Vitrification has also been shown to improve clinical pregnancy outcomes compared with slow freezing; specifically, vitrification results in a higher LBR and does not increase unfavorable neonatal outcomes. Therefore, most fertility clinics are opting for vitrification over slow freezing [17][18][19].
Despite significant advances in the vitrification technique for assisted reproduction, a variety of parameters-such as cryoprotectant composition and concentration, equilibration duration, chilling, and warming temperatures and ratesdamage cells and affect embryo survival and development [20][21][22]. The majority of studies have concentrated on the effects of freezing on embryo injury, survival, and development, but some have shown that the rate of warming has a greater effect on embryos [8,[23][24][25][26]. That is, even though few or small ice crystals may have been formed during the freezing process, small ice crystals can recrystallize into larger ice crystals during the thawing process and cause embryo cell damage. For example, Jin et al. found that embryos did not exhibit visible ice-crystal formation during freezing but were more likely to show visible ice crystals after slow thawing at moderate temperatures. Consequently, the survival rate of vitrified frozen embryos thawed at a moderate rate (25 °C in the air for 120 s) was much lower than that of embryos that were thawed rapidly [7]. A rapid warming rate prevents the recrystallization of small intracellular ice crystals and decreases cell damage, thereby increasing the survival and developmental potential of oocytes and embryos [27][28][29][30].
The KITAZATO Vitrification Freezing and Thawing Kit is one of the most commonly used vitrification kits in assisted human reproduction procedures. The freezing solution in this kit contains high concentrations of sucrose, ethylene glycol, and dimethyl sulfoxide. The thawing procedure suggested by the manufacturer involves two temperature steps: warming at 37 °C followed by incubating at room temperature (RT) (i.e., incubating embryos in thawing solution [TS] at 37 °C for 1 min, in dilution solution [DS] at RT for 3 min, in washing solution 1 [WS1] at RT for 5 min, and WS2 at RT for 5 min). This method thaws oocytes or embryos mainly at RT to decrease the chemical toxicity of cryoprotectants. Truong et al. investigated the effect of antioxidants on frozen-thawed blastocysts using the Rapid-Warm Blast Kit (Vitrolife). All thawing steps were performed at 37 °C and excellent results were obtained [31]. However, it remains unknown which embryo thawing procedure is optimal for FET: thawing at 37 °C in all steps (all-37 °C) or at 37 °C followed by RT (37 °C-RT). To this end, we compared the FET outcomes of all-37 °C embryos with those of 37 °C-RT embryos. Our findings suggested that thawing at 37 °C in all steps improves the clinical pregnancy rate (CPR) and implantation rate (IR).

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 survival rate of embryos frozen by vitrification and the clinical outcomes after transfer. 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 lowquality 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 (REF 10128, Vitrolife Sweden AB, Gothenburg, Sweden) and covered with OVOIL ™ (REF 10029, Vitrolife Sweden AB, Gothenburg, Sweden). Fertilization was observed under an inverted phase contrast microscope (Ti2-U, Nikon, Japan) at a magnification of 200-400× . Embryos showing normal fertilization, defined as the presence of two clear pronuclei (2PN). Then, the dishes (REF 353001, BD Falcon, United States) were placed in a 37 ℃ Multi-Gas Incubator (APM-50D, Astec, Japan, 7.0% CO 2 ; 5.0% O 2 ; 88% N 2 ) 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]. 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 (REF 10132, Vitrolife Sweden AB, Gothenburg, Sweden) and covered with OVOIL ™ 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. 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 [33]. 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 (REF 91011, Kitazato Corporation, 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 rewarmed to RT on the IVF workstation (L-126Dual, K-systems, Denmark). The selected embryos were placed in 300 μL of ES for 8 min and then washed by transfer via pipette to three successive microdroplets 100 μL of VS. After the final wash, the embryos were immediately placed in a Cryotop (REF 81111-81115, Kitazato Corporation, Japan), then the Cryotop was quickly immersed in liquid nitrogen (− 196 °C). The period from the transfer of the embryos into the first VS microdroplet to the immersion of the embryos into liquid nitrogen was equal to 45-60 s. Before freezing, blastocysts were manually shrunk with a laser (Saturn Active, Research Instruments, United Kingdom) to allow fluid from the blastocysts cavity to flow out [34].
The two protocols employed for thawing vitrified embryos with the KITAZATO Thawing Kit VT102 (REF 91021, Kitazato Corporation, Japan) were as follows. In the 37 °C-RT protocol, the 500 μL of TS (34% v/v sucrose; 20% v/v replace and supplement serum; 0.005% v/v gentamicin; 64.995% v/v 199HEPES buffer solution) were prewarmed in a 37 °C Multi-Gas Incubator without gas for at least 30 min. Then 200-μL each of 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 covered with OVOIL ™ at 37 °C to rewarm before being exposed to RT, which can prevent the rise in solution osmotic pressure by reducing the amount of water that evaporates during the rewarming and thawing process. The vitrified 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, according to the instruction manual. All steps of embryo movement into DS to WS2 were performed on the IVF 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 IVF 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. Finally, the embryos were promptly transferred to culture dishes (REF 353037, BD Falcon, United States) and observed under an inverted phase contrast microscope at a magnification of 200-400× to evaluate their development and morphology after thawing. Embryos that survived thawing were transferred to G-2 ™ PLUS and incubated for 2-4 h at 37 °C in a Multi-Gas Incubator.

Endometrial 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 hormonereplacement 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 clinical pregnancies. 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. Figure 1 represents the patient selection process. 1734 FET cycles met our inclusion criteria, 305 of which were excluded for the following reasons: female partner diagnosed with recurrent miscarriage, uterine abnormality, or adenomyosis (n = 91); low-quality embryos or blastocysts (n = 169); endometrial thickness less than 7 mm (n = 32); and cycles with thawed oocytes (n = 13). Of the remaining 1429 FET cycles, embryos in 369 cycles were thawed following the all-37 °C protocol, and embryos in 1060 cycles were thawed following the 37 °C-RT protocol. After case-control matching, 366 cycles from each group were included in our analysis. Table 1 summarizes the baseline characteristics of the two patient groups. There were no significant differences (P > 0.05) between the two groups in terms of the age or BMI of either partner. The female partners in each group were not significantly different (P > 0.05) in terms of the type of infertility, serum AMH concentrations, number of oocytes obtained, or insemination method. The female partners' endometrial preparation protocols during the FET cycle were the same for both groups (P = 1), and there was no significant difference in the number of frozen embryos thawed (P = 0.930) or the embryo transfer stage (P = 0.332) between the groups. Table 2   Cycles were excluded due to -women having been diagnosed with recurrent pregnancy loss, uterine malformations, and/or adenomyosis (n = 91) -low-quality D3 embryo or blastocyst (n = 169) -an endometrial thickness < 7 mm (n = 32) -having cycles with thawed oocytes (n = 13) Cycles with embryos thawed using the 37℃-RT protocol (n = 1,060) or the all-37℃ protocol (n = 369) Case-control matching (1:1) for the age of the women, embryo transfer stage (D3 embryo or blastocyst), number of transferred embryos, and endometrial preparation protocol 37℃-RT group (n = 366)

Results
All-37℃ group (n = 366) Cycles that met the inclusion criteria (n = 1,734) frozen D3-embryos and blastocysts were not significantly different between the two groups (P > 0.05).

Discussion
As the protocol recommended by the vast majority of currently available thawing kits involves the thawing of vitrified oocytes/embryos/blastocysts at 37 °C and RT, which has resulted in successful outcomes [35][36][37], few studies have compared the advantages and disadvantages of this protocol with those of other temperature protocols. To the best of our knowledge, this is the first study to assess the effects of two thawing procedures (all-37 °C versus 37 °C-RT) using the same thawing kit on the clinical outcomes of FET cycles. The findings of this large retrospective case-control matched study suggest that thawing at all-37 °C significantly improved the clinical outcome of FET cycles compared with thawing at 37 °C-RT. Several studies have examined various temperatures of TS and durations in attempts to increase the survival and developmental potential of frozen-thawed embryos. There are mainly two basic aspects in which temperature would have an impact on vitrified embryos. On one hand, high temperature is supposed to increase the toxicity of cryoprotectants. For instance, exposure to dimethyl sulfoxide (DMSO) at 37 °C will depolymerize actin microfilaments and impact the cytoskeleton of oocytes [38]. High warming temperature, on the other hand, results in rapid warming rates, which would benefit embryos by preventing damage from recrystallization of intracellular water during the thawing step [7,28]. In a study using mouse embryos by Seki et al., warming at 37 °C significantly increased the survival rate of vitrified embryos and the subsequent blastocyst formation rate as compared to warming at 23 °C. The authors assumed that the improved outcomes were due to a faster warming rate at 37 °C compared with that at 23 °C [30]. However, the effect of temperature itself on the embryo during the recovery process cannot be ignored. 37 °C is the standard temperature for in vitro embryo culture. Higher or lower temperatures will affect embryonic development potential [39,40]. As a result, almost all commercial thawing kits set the TS temperature in their manuals at 37 °C to achieve a rapid warming speed. However, temperature at the following steps branch into RT and 37 °C in different kits due to the dilemma of balancing damages between the toxic solution and low temperature. For example, the diluting (in DS) and washing (in WS) steps of the Kitazato and Irvine kits are suggested to be performed at RT, while 37 °C are advised at both steps when the Vitrolife kit is used [36,41]. In the current study, we examined the impact on survival rate and subsequent clinical outcomes of DS, WS1, and WS2 thawing procedures at 37 °C and RT. Our results showed that there were similar survival rates between the all-37 °C and 37 °C-RT groups, which may be attributable to the high-similarity of the recovery rates of the two thawing procedures (98.8 vs 98.3%). The all-37 °C thawing protocol significantly increased CPR and IR as compared to the 37 °C-RT procedure. Therefore, a long-term RT exposure of 13 min and the total duration time in DS, WS1, and WS2, may impair embryonic development potential.
An alternative strategy to reduce toxic damage from cryoprotectants is to decrease the duration of embryos in these solutions. The stability of microfilaments in oocytes exposed to ethylene glycol, a widely used penetrating cryoprotectant, for a short period at RT were not affected, while a prolonged exposure resulted in actin filament breaking and impaired cell function [42]. Based on this consideration, we reduced the duration time in WS to offset the potential damage caused by the elevated temperature, as suggested by previous researches. Hong et al. compared the outcomes of different duration times with an all-37 °C method and found that a 2.5 min duration time for each step gained better embryo quality than a 5 min interval [43]. Another study to evaluate the effects of different Cryotops by Momozawa et al. [44] adopted a thawing strategy similarly to our all-37 °C method, that is 1 min in TS, 3 min in DS, and totally 5 min in WS. Thus, a total of 5 min in WS (WS1 and WS2) is supposed to be enough for the cryoprotectants displacement as the exchange rate rises between inside and outside cells along with the temperature because the permeability of the cell membrane to water and cryoprotectants is positively related to temperature [45]. Embryos at different stages have distinct responses to the vitrification and/or thawing procedures. The tolerance of embryos to vitrification increases as the embryo develops, which might contribute to the faster rate at which cryoprotectants such as glycerol and ethylene glycol are transmitted in blastocysts than in cleavage-stage embryos, as the expression of aquaporin 3 (AQP3) increases with embryo development [46][47][48]. We performed subgroup analysis on various embryo stages and showed that, the CPR and IR of blastocyst transfer are significantly increased in the all-37 °C group compared with the 37 °C-RT group, while the differences in both CPR and IR were not significant for D3-embryo transfer between the two groups, which may be due to the different sample sizes. The CPR and IR of D3-embryo transfer increased by 22.7% and 16.1%, respectively, in all-37 °C group when compared with 37 °C-RT group. The increased percentage of CPR and IR are even higher in D3-embryo transfer than in blastocyst transfer (14.1% for CPR and 14% for IR).
Our study has several strengths. First, we adopted strict inclusion and exclusion criteria to exclude women with potential confounding characteristics that might have influenced the FET outcomes, excluded low-quality embryos/ blastocysts, and adopted a large sample size, all of which strengthen the reliability of our results. Second, all embryos' quality evaluation, freezing, and thawing operations were performed according to standard protocols by two experienced embryologists, which reduced operational and observational bias. Third, a 1:1 case-control matching approach was adopted, which allowed us to control for the effects of potential confounding factors.
However, our study has some limitations. First, this was a retrospective study, which may have introduced unavoidable selection and confounding bias. Second, our study spanned a relatively long period, during which the operations of different steps may have been inconsistent and thus affected our results. Finally, our results were based on data from only a single reproductive center and a limited sample size. Therefore, larger sample sizes from multiple centers should be adopted in future studies to support our findings.

Conclusions
Our findings suggested that compared with the traditional 37 °C-RT thawing protocol for vitrified embryos, the all-37 °C thawing protocol for vitrified embryos improves the clinical outcomes of FET cycles. This may be due to the avoidance of damage to development potential from RT exposure and shortened duration of cryoprotectant persistence in embryos. A multicenter prospective randomized controlled trial is required to confirm the advantages of the all-37 °C thawing protocol.
Author contributions SZ, GY, and YY designed the study. WY, LL, LW, and DZ collected the data. SZ and GY performed the data analyses and wrote the manuscript. All authors approved the final manuscript.
Funding This study was supported by Kunming Medical University (No. CXTD202105).

Data availability
The authors confirm that the data supporting the finding of this study are available within the article.

Conflict of interest The authors declare no competing interests.
Ethical approval This 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.