Simplified warming protocol for vitrified human embryos: a comprehensive analysis across various developmental stages and morphological qualities

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Methods : This is a retrospective cohort study conducted at a single IVF center. A total of 1501 single vitrified-warmed embryo transfer cycles, involving 1351 blastocyst- and 150 cleavage-stage embryos, performed from January to December 2024, were analyzed. Post-warming survival rate and embryo transfer outcomes were compared between a conventional three-step warming protocol (thawing solution [TS] for 1 minute, dilution solution [DS] for 2 minutes, and washing solution [WS] for 3 minutes, n = 741) and simplified warming protocols, including a two-step (TS for 1 minute, followed by DS for 2 minutes, n = 461) and a one-step (TS for 1 minute, n = 298). The analysis primarily focused on blastocyst-stage embryos, for which detailed subgroup analyses were performed based on developmental stage, morphological quality, and day of blastulation. In addition, a separate analysis was conducted for cleavage-stage embryos. Results For blastocyst-stage embryos, post-warming survival rates were excellent and comparable among the three-step, two-step, and one-step protocols (100%, 99.3%, 99.6%; p = 0.1112). There were also no statistically significant differences in the fetal heartbeat confirmation rates (47.7%, 46.2%, 43.9%; p = 0.5765). Furthermore, this equivalence was consistently observed across all subgroups, including those based on developmental stage (full-blastocyst: 43.2%, 39.6%, 33.7%, p = 0.3111; expanded-blastocyst: 49.6%, 49.5%, 49.1%, p = 0.9950), morphological quality (poor-grade: 27.5%, 23.5%, 23.4%, p = 0.7636; good-grade: 52.1%, 51.6%, 48.4%, p = 0.6390), and day of blastulation (day 5: 52.7%, 52.9%, 50.3%, p = 0.8234; day 6: 34.4%, 28.5%, 27.4%, p = 0.4088). For cleavage-stage embryos, all protocols resulted in 100% post-warming survival with comparable fetal heartbeat confirmation rates (11.1%, 12.5%, 10.8%; p = 0.9717). Conclusions A simplified one- or two-step warming protocol is a safe and effective protocol for warming of vitrified human embryos. This approach is applicable to a variety of embryos, regardless of their characteristics. blastocyst cleavage cryopreservation embryo warming embryo vitrification one-step warming two-step warming osmotic stress rehydration dilution 1. Introduction The transition of oocyte and embryo cryopreservation methods from slow freezing to vitrification has enabled their viability to be maintained at an extremely high level, and the “freeze all” strategy allows the avoidance of ovarian hyperstimulation syndrome and the preparation of an appropriate implantation environment before embryo transfer [1] . Furthermore, this has expanded the possibilities of assisted reproductive technology in its application to preimplantation genetic testing and fertility preservation [2, 3]. In recent years, as vitrification and subsequent warming have become established as standard IVF laboratory practices, research focus has shifted to optimizing the reduction of laboratory workload [4, 5]. Although vitrification protocols typically recommend immersing embryos in equilibration solution for up to 15 minutes, recent research has shown that the duration of equilibration does not impact embryo viability [6–8]. Notably, the fast vitrification procedure described by Liebermann et al. demonstrates good clinical outcomes with only 4 minutes of equilibration time [9]. The optimization focus of the procedure is also on the warming process subsequent to vitrification. Traditional warming protocols utilize multiple solutions to create stepwise osmotic pressures, based on the principle of cryobiology, which emphasizes the importance of safely rehydrating cryopreserved cells while avoiding hypotonic stress that could lead to irreversible cellular damage [10–14]. Although this careful approach has proven effective, it is skill-dependent and time-consuming, posing challenges in laboratory workflows that involve a high volume of IVF cycles. As a result, there is a growing interest in simplifying the warming protocol to enhance efficiency and reduce the time embryos spend outside the incubator [15] . Recent studies indicate that a simplified warming protocol does not negatively impact the clinical outcomes of vitrified blastocysts and may even improve these outcomes [16–19]. These findings challenge the long-held belief in cryobiology that stepwise rehydration is essential for maintaining embryonic viability. However, two critical questions remain largely unanswered in the existing literature. First, it is unclear whether this simplified protocol is effective for various embryo cohorts, including different developmental stages, morphological qualities , and day of blastulation. Second, the majority of studies focus only on blastocysts, and the effects of sudden osmotic stress with a simplified warming procedure on cleavage-stage embryos, which have fundamentally different cellular structures and physiological functions, are not well understood. A previous in vitro study using discarded embryos demonstrated that a simplified warming protocol did not impair the survival and subsequent developmental potential of cleavage-stage embryos. However, the effects on pregnancy and miscarriage were not evaluated [20]. This study aims to address these critical knowledge gaps by investigating whether the omission of a stepwise osmotic dilution step during warming affects the clinical outcomes of human embryos with various characteristics. We conducted a large-scale retrospective comparative analysis and comprehensive safety evaluation of three warming protocols: a conventional three-step, an intermediate two-step, and a simplified one-step protocol. 2. Materials and Methods 2.1 Study design This retrospective cohort study was conducted with the approval of the Institutional Review Board at the study institution. In accordance with ethical guidelines for medical research involving human subjects, patient consent for data use was obtained through an opt-out approach, with notifications posted on the clinic's official website and internal bulletin boards. During the period from January to December 2024, this study included single vitrified-warmed embryo transfer cycles using three different warming protocols. The conventional three-step protocol was performed from January to September 2024, and the one-step protocol was introduced from April to September 2024. For the overlapping period between April and September, protocol assignment was randomized. The two-step protocol was then exclusively used from October to December 2024. The phased implementation of these protocols was to identify the optimal warming procedure. Cycles were excluded from the study if they met any of the following criteria: (1) use of embryos that underwent preimplantation genetic testing for aneuploidy ; (2) embryos not confirmed as two-pronuclear at the time of fertilization assessment; (3) embryos derived from cryopreserved sperm; (4) embryos transported from other IVF centers; (5) double-embryo transfer cycles; or (6) embryos cultured with complete zona pellucida removal. After applying these exclusion criteria, a total of 1501 cycles were included in the final analysis, comprising 1351 blastocyst- and 150 cleavage-stage transfers. 2.2 Obtaining vitrified blastocyst-/cleavage-stage embryos All patients underwent controlled ovarian stimulation (COS) tailored to their individual AMH levels, maternal age, and clinical backgrounds. The COS was performed using established protocols at the institution, including mild stimulation with clomiphene citrate, GnRH antagonist, and GnRH agonist, progestin-promoted ovarian stimulation protocols. In some cycles, oocyte retrieval was performed under natural unstimulated cycles. Final oocyte maturation was triggered with either human chorionic gonadotropin, a GnRH agonist, or a combination of both. Transvaginal ultrasound-guided oocyte retrieval was performed 35–38 hours post-trigger. The retrieved cumulus-oocyte complexes (COCs) were washed in HEPES-buffered medium (P+ HEPES medium®; Naka Medical, Tokyo, Japan) and pre-cultured in insemination medium (P+ insemination medium®; Naka Medical) under standard culture conditions (37.0°C, 6.0% CO 2 , 5.0% O 2 ). Retrieved COCs were inseminated via either conventional in vitro fertilization (C-IVF) or intracytoplasmic sperm injection (ICSI) after removal of cumulus cells, based on semen parameters and clinical background. The embryos were cultured in droplets of ONESTEP medium® (Naka Medical) covered with OVOIL® (Vitrolife, Gothenburg, Sweden) under conditions of 37.0°C, 6.0% CO₂, and 5.0% O₂. Embryos that reached the cleavage stage (day 2 or 3) or blastocyst stage (day 5 or 6) with good morphology satisfying our criteria (cleavage-stage: grade ≥3 according to Veeck criteria; blastocyst-stage: grade 3 ≥CC according to Gardner criteria) were cryopreserved using the vitrification protocol [22, 23]. Vitrification was performed using the Cryotop® methods (Kitazato, Shizuoka, Japan) according to the manufacturer's protocol. Briefly, embryos were first rinsed in an equilibration solution for up to 15 minutes, until they re-expanded to at least 90% of their initial volume. Subsequently, they were transferred to a vitrification solution for 1–1.5 minutes before being loaded onto the Cryotop® device and plunged into liquid nitrogen for storage. 2. 3 Vitrified embryo warming and embryo transfer protocol Vitrified cleavage- and blastocyst-stage embryos were selected for transfer based on the Veeck or Gardner criteria and were warmed using a commercially available warming solution [22, 23]. Three different protocols were utilized for warming the vitrified embryos. The conventional three-step protocol was a 6-minute procedure involving the sequential rinsing of embryos to thawing solution (TS) for 1 minute, followed by dilution solution (DS) for 2 minutes, and finally washing solution (WS) for 3 minutes. This approach ensured stepwise removal of the cryoprotectant. The simplified protocols included a modified two-step protocol, which involved rinsing of embryos with TS for 1 minute and then with DS for 2 minutes, and a one-step protocol, where embryos were rinsed only with TS for 1 minute prior to recovery culture. The commercial kits used for the three-step protocol were Cryotech warming solution (REPROLIFE, Tokyo, Japan), whereas the kits for the two-step and one-step protocols were the Vit Kit-Warm NX (Irvine Scientific, Santa Ana, CA, USA). After warming, the embryos were cultured for ≥2 hours under conditions of 37°C, 6% CO₂, and 5% O₂ until embryo transfer. For blastocyst-stage embryos, after rinsing with TS, laser-assisted hatching (LAH) was performed on embryos in areas with sufficient perivitelline space. Embryo survival was defined as blastocysts without dark-collared fragmented cells and with a clearly observable blastocoel. For cleavage-stage embryos, survival was defined as having at least 50% of blastomeres remaining intact after warming. All embryos were transferred using an embryo transfer catheter (Kitazato) under abdominal ultrasound guidance. Gestational sacs and fetal heartbeats were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed. 2.4 Statistical Analysis Statistical analysis was performed using JMP Pro 18.00 (SAS Institute, Cary, NC, USA). The cycle and embryo backgrounds were compared using either one-way ANOVA test or Chi-square test. To analyze post-warming survival rates and embryo transfer outcomes across the three protocols (three-step, two-step, and one-step), a Chi-square test was employed, with a significance level set at p < 0.05. The outcomes at the blastocyst-stage were analyzed using a multivariate logistic regression model to adjust for confounding factors such as patient age, AMH level, BMI, LAH performed during the warming process, developmental stage, morphological quality, and day of blastulation. 3. Results Background of the analyzed patients and blastocysts The background of patients who underwent blastocyst-stage embryo warming and the corresponding embryo characteristics are shown in Table 1. There was a significant difference in the mean maternal age among the groups, with the three-step group having the highest average age (35.95 ± 0.18 years) and the two-step group having the lowest (35.28 ± 0.29 years) (p=0.0062). No significant differences were found in the mean AMH levels (p=0.2338) and BMI (p=0.7945). Regarding blastocyst characteristics, there were no significant differences in the distribution of blastocyst developmental stage (full-blastocyst vs. expanded-blastocyst; p=0.4574), morphological quality (poor grade [<BB] vs. good grade [≥BB]; p=0.9223), and the day of blastulation (Day 5 vs. Day 6; p=0.9782) among the three protocols. A significant difference was noted in the rate of LAH performed at the time of warming: LAH was most frequently performed in the three-step protocol (96.52%), followed by the two-step protocol (94.13%), and then the one-step protocol (84.62%; p<0.0001). Post-warming survival rate and clinical outcomes after embryo transfer in blastocyst-stage embryos The post-warming survival rates of the blastocysts were excellent and comparable across all three protocols: 100%, 99.30%, and 99.62% for the three-, two-, and one-step protocols, respectively (p=0.1112) (Table 1). After embryo transfer, there were no significant differences in the rates of gestational sac confirmation (p=0.6032), fetal heartbeat confirmation (p=0.5765), or pregnancy loss (p=0.6187). Multivariate logistic regression analysis indicated that maternal age at blastocyst vitrification, development stage, morphological quality, and day of blastulation were factors associated with the outcomes of embryo transfer (Table 2). Notably, simplified warming protocols did not impact these outcomes. Subgroup analysis based on developmental stage, morphological quality, and day of blastulation We performed subgroup analyses to determine whether a simplified warming protocol for blastocyst-stage embryos is effective across embryos with various characteristics. The equivalence in fetal heartbeat confirmation rates between the three-step, two-step, and one-step protocols was consistently observed across all analyzed subgroups, including those based on developmental stage (full-blastocyst: 43.2%, 39.6%, and 33.7%, p=0.3111; expanded-blastocyst: 49.6%, 49.5%, and 49.1%, p=0.9950), morphological quality (poor-grade: 27.5%, 23.5%, and 23.4%, p=0.7636; good-grade: 52.1%, 51.6%, and 48.4%, p=0.6390), and day of blastulation (Day 5: 52.7%, 52.9%, and 50.3%, p=0.8234; Day 6: 34.4%, 28.5%, and 27.4%, p=0.4088) (Table 3–5). Furthermore, a subgroup analysis based on patient age at blastocyst vitrification revealed that the simplified warming protocol had no adverse effects, even on embryos derived from advanced maternal age patients (Supplemental table 1) . Survival rate and clinical outcomes of cleavage-stage embryos In the analysis of 150 cleavage-stage embryos after warming, there were no differences in patient backgrounds among the groups (Table 6). The blastomere number, embryo grade, and day of vitrification were similar across all protocols. All warming protocols showed 100% survival rate after warming, with no observed loss of blastomeres. A comparable proportion of embryos in each protocol progressed with cell division during the recovery culture period of at least 2 hours before transfer: 21.0%, 15.6%, and 24.3% for the three-, two-, and one-step protocols, respectively (p = 0.6693). The primary clinical outcomes were similar regardless of the warming protocol used. The rates of fetal heartbeat confirmation were 11.11%, 12.50%, and 10.81% for the three-, two-, and one-step protocols, respectively, with no significant differences (p = 0.9717). However, there was a significantly higher proportion of pregnancy losses in the two simplified protocol groups (p = 0.0284), though the low event rate suggests that this finding should be interpreted with caution. Table 1. Background of blastocyst-stage embryos used for embryo transfer and their survival and clinical outcomes Variables Three-step Two-step One-step p value No. of warmed embryos 661 429 261 - Age (years, mean ± SD) 35.95 ± 0.18 35.08 ± 0.22 35.28 ± 0.29 0.0062 AMH (ng/mL, mean ± SD) 3.47 ± 0.12 3.74 ± 0.15 3.39 ± 0.19 0.2338 BMI (kg/m 2 , mean ± SD) 21.88 ± 0.14 21.78 ± 0.18 21.97 ± 0.23 0.7945 LAH performed at the time of warming, n(%) 638 (96.52) 401 (94.13) 220 (84.62) 0.0001 Embryo backgrounds, n(%) Full-blastocyst 199 (30.11) 139 (32.40) 89 (34.10) 0.4574 Expanded-blastocyst 462 (69.89) 290 (67.60) 172 (65.90) Poor grade (Gardner criteria <BB) 120 (18.15) 82 (19.11) 48 (18.39) 0.9223 Good grade (Gardner criteria ≥BB) 541 (81.85) 347 (80.89) 213 (81.61) Day 5 blastocyst 478 (72.31) 310 (72.26) 187 (71.65) 0.9782 Day 6 blastocyst 183 (27.69) 119 (27.74) 74 (28.35) Survival rate, n(%) 661 (100) 426 (99.30) 260 (99.62) 0.1112 No. of transferred blastocysts 661 426 260 Gestational sac, n(%) 351 (53.10) 226 (53.05) 129 (49.62) 0.6032 Fetal heartbeat, n(%) 315 (47.66) 197 (46.24) 114 (43.85) 0.5765 Pregnancy loss, n(%) 75 (21.37) 51 (22.57) 33 (25.58) 0.6187 Results are shown as mean ± standard deviation or percentages when appropriate. Age, AMH level, and BMI are shown for female patients. Poor-grade indicated by grades CC, BC, CB, AC, and CA blastocysts; good-grade indicated by grades BB, AB, BA, and AA blastocysts. Categorical data were analyzed by Chi-square test, and other backgrounds were analyzed by one-way ANOVA test. Gestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed. P values in bold typeface indicate statistical significance. AMH, anti-Müllerian hormone; BMI, body mass index; LAH, laser assisted hatching Table 2. Logistic regression analysis of variables affecting clinical outcome in vitrified warm embryo transfer cycles Variables Gestational sac Fetal heartbeat Pregnancy loss aOR 95%CI p value aOR 95%CI p value aOR 95%CI p value Age (years, mean ± SD) 0.906 0.881–0.932 0.0001 0.908 0.882–0.933 0.0001 1.095 1.046–1.148 0.0001 AMH (ng/mL, mean ± SD) 1.005 0.965–1.048 0.8100 1.021 0.981–1.063 0.3132 0.969 0.903–1.035 0.3646 BMI (kg/m 2 , mean ± SD) 0.992 0.960–1.024 0.6015 0.991 0.959–1.023 0.5622 1.02 0.970–1.071 0.4374 LAH performed at the time of warming, n(%) 0.833 0.515–1.346 0.4553 0.738 0.460–1.184 0.2080 2.319 0.990–5.430 0.0528 Developmental stage Full-blastocyst (reference) - - - - - - - - - Expanded-blastocyst 1.785 1.379–2.310 0.0001 1.494 1.156–1.932 0.0022 1.094 0.714–1.678 0.6787 Morphological quality Poor grade (reference) - - - - - - - - - Good grade 2.474 1.772–3.455 0.0001 2.218 1.573–3.126 0.0001 0.594 0.334–1.058 0.0768 Day of blastulation Day 6 (reference) - - - - - - - - - Day 5 1.74 1.302–2.327 0.0002 1.635 1.219–2.193 0.001 0.83 0.516–1.336 0.4426 Warming protocols Three-step (reference) - - - - - - - - - Two-step 0.916 0.702–1.196 0.5197 0.865 0.664–1.128 0.2845 1.241 0.811–1.897 0.3195 One-step 0.824 0.600–1.132 0.2326 0.796 0.580–1.092 0.1575 1.553 0.944–2.554 0.0831 Adjusted odd ratios for gestational sac, fetal heartbeat, and pregnancy loss were calculated using a logistic regression model containing terms for patient age, AMH level, BMI, LAH performed during the warming process, developmental stage, morphological quality, day of blastulation, and warming protocols. P values in bold typeface indicate statistical significance. aOR, adjusted odd ratio; 95%CI, 95% confidence interval Table 3. Blastocyst survival and clinical outcomes classified by developmental stage Variables Three-step Two-step One-step p value Full-blastocyst No. of warmed blastocysts 199 139 89 - Survival rate, n(%) 199 (100) 139 (100) 89 (100) - No. of transferred blastocysts 199 139 89 - Gestational sac, n(%) 90 (45.23) 63 (45.32) 34 (38.20) 0.4896 Fetal heartbeat, n(%) 86 (43.22) 55 (39.57) 30 (33.71) 0.3111 Pregnancy loss, n(%) 17 (18.89) 15 (23.81) 9 (26.47) 0.5988 Expanded-blastocyst No. of warmed blastocysts 462 290 172 - Survival rate, n(%) 462 (100) 287 (98.97) 171 (99.42) 0.1037 No. of transferred blastocysts 462 287 171 - Gestational sac, n(%) 261 (56.49) 163 (56.79) 95 (55.56) 0.9659 Fetal heartbeat, n(%) 229 (49.57) 142 (49.48) 84 (49.12) 0.9950 Pregnancy loss, n(%) 58 (22.22) 36 (22.09) 24 (25.26) 0.8090 Gestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed. Table 4. Blastocyst survival and clinical outcomes classified by morphological quality Variables Three-step Two-step One-step p value Poor-grade (Gardner criteria <BB) blastocyst No. of warmed blastocysts 120 82 48 - Survival rate, n(%) 120 (100) 81 (98.78) 47 (97.92) 0.3420 No. of transferred blastocysts 120 81 47 - Gestational sac, n(%) 37 (30.83) 23 (28.40) 12 (25.53) 0.7849 Fetal heartbeat, n(%) 33 (27.50) 19 (23.46) 11 (23.40) 0.7636 Pregnancy loss, n(%) 13 (35.14) 10 (43.48) 3 (25.00) 0.5492 Good-grade (Gardner criteria ≥BB) blastocyst No. of warmed blastocysts 541 347 213 - Survival rate, n(%) 541 (100) 343 (99.42) 213 (100) 0.1134 No. of transferred blastocysts 541 345 213 - Gestational sac, n(%) 314 (58.04) 203 (58.84) 117 (54.93) 0.6443 Fetal heartbeat, n(%) 282 (52.13) 178 (51.59) 103 (48.36) 0.6390 Pregnancy loss, n(%) 62 (19.75) 41 (20.20) 30 (25.64) 0.3873 Poor-grade indicated by grades CC, BC, CB, AC, and CA blastocysts; good-grade indicated by grades BB, AB, BA, and AA blastocysts according to Gardner criteria. Gestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed. Table 5. Blastocyst survival and clinical outcome classified by day of blastulation Variables Three-step Two-step One-step p value Day 5 blastocyst No. of warmed blastocysts 478 310 187 - Survival rate, n(%) 478 (100) 310 (100) 187 (100) - No. of transferred blastocysts 478 310 187 - Gestational sac, n(%) 279 (58.37) 186 (60.00) 106 (56.68) 0.7622 Fetal heartbeat, n(%) 252 (52.72) 164 (52.90) 94 (50.27) 0.8234 Pregnancy loss, n(%) 53 (19.00) 38 (20.43) 27 (25.47) 0.3727 Day 6 blastocyst No. of warmed blastocysts 183 119 74 - Survival rate, n(%) 183 (100) 116 (97.48) 73 (98.65) 0.4438 No. of transferred blastocysts 183 116 73 - Gestational sac, n(%) 72 (39.34) 40 (34.48) 23 (31.51) 0.4438 Fetal heartbeat, n(%) 63 (34.43) 33 (28.45) 20 (27.40) 0.4088 Pregnancy loss, n(%) 22 (30.56) 13 (32.50) 6 (26.09) 0.8665 Gestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed. Table 6. Background of cleavage-stage embryos used for embryo transfer and their survival and clinical outcomes Variables Three-step Two-step One-step p value No. of warmed embryos 81 32 37 - Age (years, mean ± SD) 39.64 ± 0.59 38.78 ± 0.89 39.89 ± 0.83 0.6259 AMH (ng/mL, mean ± SD) 1.80 ± 0.25 1.82 ± 0.41 2.23 ± 0.37 0.6121 BMI (kg/m 2 , mean ± SD) 22.31 ± 0.38 21.67 ± 0.62 22.41 ± 0.57 0.6229 Embryo backgrounds, n(%) 2–4-cell 53 (65.43) 23 (71.88) 29 (78.38) 0.5682 5–6-cell 20 (24.69) 6 (18.75) 7 (18.92) >6-cell 8 (9.88) 3 (9.38) 1 (2.70) Poor grade (Veeck criteria 3) 57 (70.37) 19 (59.38) 17 (45.95) 0.1559 Fair grade (Veeck criteria 2) 16 (19.75) 8 (25.00) 13 (35.14) Good grade (Veeck criteria 1) 8 (9.88) 5 (15.63) 7 (18.92) Day 2 embryo 72 (88.89) 30 (93.75) 36 (97.30) 0.2713 Day 3 embryo 9 (11.11) 2 (6.25) 1 (2.70) Survival rate, n(%) 81 (100) 32 (100) 37 (100) - No. of transferred blastocysts 81 32 37 Gestational sac, n(%) 9 (11.11) 5 (15.63) 5 (13.51) 0.7968 Fetal heartbeat, n(%) 9 (11.11) 4 (12.50) 4 (10.81) 0.9717 Pregnancy loss, n(%) 0 (0) 1 (20.00) 3 (60.00) 0.0284 Results are shown as mean ± standard deviation or percentages when appropriate. Age, basal AMH level, and BMI are shown for female patients. Categorical data were analyzed by Chi-square test, and other backgrounds were analyzed by one-way ANOVA test. Gestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed. P values in bold typeface indicate statistical significance. Discussion This study provides comprehensive evidence supporting that simplified warming protocols do not compromise outcomes for either blastocyst- or cleavage-stage embryos. Our primary finding is that the post-warming survival rate and subsequent embryo transfer outcomes were comparable across three different protocols: a conventional three-step, an intermediate two-step, and a highly simplified one-step protocol. Notably, this equivalence was consistently observed even in various embryo cohorts, including different embryo morphological and developmental stages. These results suggest that vitrified human embryos can tolerate sudden osmotic changes resulting from a simplified warming procedure. In cryobiology, it has been theoretically established that stepwise rehydration is essential for cryopreserved cells to avoid cryoinjury [ 11 ]. However, recent clinical evidence has questioned this theory in vitrified human blastocysts. Liebermann et al. demonstrated that a one-step rehydration protocol not only achieves equivalent post-warming survival rates but also improves ongoing pregnancy rates and reduces miscarriage rates compared to conventional multi-step rehydration protocols [ 16 ]. Similarly, retrospective analysis by Jiang et al. and Licata et al. confirmed that a simplified warming protocol achieves equivalent live birth rates and clinical outcomes compared to a conventional protocol [ 18 , 19 ]. In our study, we analyzed 1351 blastocyst transfers and found that the simplified protocol achieved equivalent post-warming survival rate and embryo transfer outcomes as the conventional protocol. Shioya et al. previously demonstrated that a simplified warming protocol does not impair key indicators of blastocyst viability, including re-expansion potential, cell survival rate assessed by Hoechst33258/Propidium iodide staining, and trophoblast outgrowth using discarded embryos [ 20 ]. The consistency between this previous in-vitro finding and the clinical results of the present study supports the safety of omitting the stepwise dilution process. The sudden osmotic pressure changes from a hypertonic warming solution (TS; 1656 mOsm/kg) to an isotonic culture medium (265 mOsm/kg), as in simplified protocols, can cause significant hypotonic stress. In fact, Ezoe et al. reported that the osmotic shift from TS to culture medium causes cell membrane collapse due to over-rehydration [ 24 ]. Theoretically, this could result in a rapid influx of water into the blastocoel, inner cell mass, and trophectoderm, potentially leading to cell membrane rupture and impaired embryo viability [ 12 , 13 ]. Contrary to these theoretical concerns, Chan et al. concluded that a direct rehydration protocol for vitrified embryos without warming solution not only resulted in 100% survival, but also allowed repeated vitrification, warming, and biopsy, so that stepwise rehydration with careful attention to osmotic pressure was not necessary [ 25 ]. A key finding of this study is the consistent efficacy of the simplified warming protocol across all embryonic qualities and developmental stages. Previous literature has highlighted that blastocysts with certain pre-vitrification features are less tolerant to traditional cryopreservation protocols; Desai et al. showed a 5% decrease in post-warming survival rate in a standard warming protocol for day 6 blastocysts compared to day 5 blastocysts, indicating that the number of days required for blastulation influences embryo survival [ 26 ]. Furthermore, Cimadomo et al. reported that, in addition to the developmental speed to the blastocyst stage, the morphological quality before vitrification is an important factor affecting post-warming survival [ 27 ]. In delayed-development or poor quality blastocysts, the energy required for responding to physiological stresses and blastocoel re-expansion through the vitrification-warming process may be depleted, which may be the cause of reduced cryosurvival. Additionally, Mukaida et al. suggested that highly expanded-blastocysts are at greater risk due to their large fluid volume, which increases the chance of ice crystal formation [ 28 ]. Considering the variety of embryos subjected to warming, traditional warming protocols are designed to carefully rehydrate embryos that are particularly vulnerable to sudden osmotic stress. Rapid influx of water may damage poor-quality cellular structures and potentially lead to reduced survival rates. Thus, it was necessary to determine whether these characteristics of vitrified blastocysts affect viability with simplified warming. However, there were no significant differences in embryo survival and ET outcomes among the three protocols, either in poor quality, delayed development, or advanced expansion stage embryos, and the effectiveness of simplified warming was not affected by embryo characteristics. These unexpected results suggest that the tolerance of embryos to hypotonic stress and the resumption of metabolic activity from the vitrified state may not depend on embryo quality in simplified warming. On the other hand, the simplified warming protocol reported by Liebermann et al. showed better clinical outcomes [ 16 ]. We speculate that the adverse effects of prolonged in vitro handling in a multi-step protocol may outweigh the risks associated with rapid osmotic changes. For embryos with limited energy reserves, it may be important to minimize the time spent outside the incubator. Therefore, our findings indicate that clinicians and embryologists do not need to limit the use of warming protocols based on age or embryo characteristics, enabling the universal application of a safe and efficient warming protocol. This study provides clinical data on the application of a simplified warming protocol for cleavage-stage embryos. While blastocyst-stage embryo transfer is becoming a common strategy, cleavage-stage transfer remains a necessary clinical option in some countries. The results of the present study showed that the survival rate of cleavage-stage embryos was 100% in all protocols, and the fetal heartbeat confirmation rate was also equivalent. However, it is important to address the statistically significant differences in pregnancy loss rates between these groups. Due to the small number of pregnancies, these results should be interpreted with caution. Further investigation with a larger sample size is necessary to assess these specific outcomes more clearly. This study has several limitations. First, as this is a retrospective, single-center study, there is an inherent risk of selection bias. Second, the warming kits were obtained from two different manufacturers. Although the main components of the solutions are similar, slight differences in their composition may be confounding factors. However, commercialized vitrification/warming kits are available from various manufacturers, and their composition does not appear to affect embryo survival and pregnancy rates [ 29 , 30 ]. Kato et al. used three commercially available vitrification/warming kits for simplified warming, and their different compositions made no difference in blastocyst re-expansion rates and clinical outcomes [31]. Therefore, the use of different warming solutions across the protocols does not detract from the significance of our findings. Conclusion In conclusion, a simplified warming protocol is a safe and effective method for warming vitrified human embryos. This approach is applicable to both blastocyst- and cleavage-stage embryos, regardless of embryo characteristics. The broader implication of this research supports the adoption of more efficient, evidence-based standard operating procedures in IVF laboratories. Implementing simplified protocols can significantly reduce procedure times, minimize embryo handling, and lower operational time, thereby improving the overall quality and efficiency of assisted reproductive technology without compromising the chances of a successful pregnancy. Declarations Ethical Approval: This retrospective cohort study was conducted with the approval of the Institutional Review Board at the study institution. Patient consent for data use was obtained through an opt-out approach. Conflict of Interest: The authors declare that they have no conflicts of interest. Trial registration number: N/A. Funding: Not applicable. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by M.S., M.O.-K., K.K., M.F., and K.T. The first draft of the manuscript was written by M.S. All authors read and approved the final manuscript. Acknowledgement We thank the staff of Takahashi Women’s Clinic. References Bourdon M, Maignien C, Pocate-Cheriet K, Plu Bureau G, Marcellin L, Patrat C, Chapron C, Santulli P. The freeze-all strategy after IVF: which indications? Reprod Biomed Online 2021;42:529-545. Bosch E, De Vos M, Humaidan P. The Future of Cryopreservation in Assisted Reproductive Technologies. Front Endocrinol 2020;11:67. Casciani V, Monseur B, Cimadomo D, Alvero R, Rienzi L. Oocyte and embryo cryopreservation in assisted reproductive technology: past achievements and current challenges. Fertil Steril 2023;120:506-520. Gallardo M, Saenz J, Risco R. Human oocytes and zygotes are ready for ultra-fast vitrification after 2 minutes of exposure to standard CPA solutions. Sci Rep 2019;9:15986. Sciorio R, Tramontano L, Campos G, Greco PF, Mondrone G, Surbone A, Greco E, Talevi R, Pluchino N, Fleming S. Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review. Front Cell Dev Biol 2024;12:1398049. Schiewe MC, Reichelderfer R, Wozniak K, De Romana C, Nordbak M, Baek K, Chung K. Ultra-fast vitrification and rapid elution of human oocytes: part I. germinal vesicle model validation. Reprod Biomed Online 2024;49:104691. Wozniak K, Reichelderfer R, Ghaemi S, Hupp D, Fuzesi P, Ringler G, Marrs RP, Schiewe MC. Ultra-fast vitrification and rapid elution of human oocytes: Part II - verification of blastocyst development from mature oocytes. Reprod Biomed Online 2024;49:104690 Costa-Borges N, Matia-Algué Q, Coello A, Mestres E, Acacio M, Flores-Saiffe Farias A, Castello C, Gallardo M, Chavez-Badiola A, Marco-Jiménez F et al. Preclinical validation of fast oocyte vitrification and warming protocols with comparable efficiencies to a standard method. Hum Reprod 2025;40:1066-1076. Liebermann J, Brohammer R, Wagner Y, Smith R, Even K, Hirshfeld-Cytron J, Uhler ML. Fast and furious: successful survival and resumption of meiosis in immature human oocytes vitrified and warmed using a short protocol. Reprod Biomed Online 2024;49:103976. Rall WF and Fahy GM. Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature 1985;313:573-575. Mazur P, Schneider U. Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications. Cell Biophys 1986;8:259–285. Pedro PB, Zhu SE, Makino N, Sakurai T, Edashige K, Kasai M. Effects of hypotonic stress on the survival of mouse oocytes and embryos at various stages. Cryobiology 1997;35:150–158. Kasai M. Advances in the cryopreservation of mammalian oocytes and embryos: development of ultrarapid vitrification. Reprod Med Biol 2002;1:1–9. Vanderzwalmen P, Ectors F, Panagiotidis Y, Schuff M, Murtinger M, Wirleitner B. The Evolution of the Cryopreservation Techniques in Reproductive Medicine—Exploring the Character of the Vitrified State Intra- and Extracellularly to Better Understand Cell Survival after Cryopreservation. Reproductive Medicine 2020;1:142-157. Manns JN, Katz S, Whelan J III, Patrick JL, Holt T, Merline AM, Taylor TH. Ultra-fast blastocyst, warming technique reduces warming times to 1 min and yields similar survival and re-expansion compared to blastocysts warmed using a standard method. Fertil Steril 2021;116:e165. Liebermann J, Hrvojevic K, Hirshfeld-Cytron J, Brohammer R, Wagner Y, Susralski A, Jasulaitis S, Chan S, Takhsh E, Uhler M. Fast and furious: pregnancy outcome with one-step rehydration in the warming protocol for human blastocysts. Reprod Biomed Online 2024;48:103731. Bartolacci A and Albertini DF. The new ice age: the promise and challenges of rapid oocyte warming protocols. J Assist Reprod Genet 2024;41:2969-2971. Jiang VS, Cherouveim P, Naert MN, Dimitriadis I, Souter I, Bormann CL. Live birth outcomes following single-step blastocyst warming technique - optimizing efficiency without impacting live birth rates. J Assist Reprod Genet 2024;41:1193-1202. Licata E, VerMilyea M, Ducote B, Ferguson T, Bianco C, Gallo M, Paciotti G, Passerini R, Meneghini C, Fabiani C et al. A diluted one-step warming protocol: survival of vitrified blastocyst. J Assist Reprod Genet 2025;42:1101-1107. Shioya M, Hashizume R, Okabe-Kinoshita M, Kojima K, Nishi S, Nakano S, Koga K, Fujita M, Takahashi K. One-step warming of vitrified human cleavage and blastocyst stage embryos does not adversely impact embryo survivability and subsequent developmental potential. Hum Reprod 2025;40:261-269. Veeck LL. An atlas of human gametes and conceptuses. An illustrated reference for assisted reproductive technology; 1999. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73:1155-1158. Ezoe K, Miki T, Fujiwara N, Kato K. Influence of the shortened warming protocol on human blastocyst viability: an in-vitro experimental study. Reprod Biomed Online 2025;50:104454. Chan DYL, Xu M, Wu W, Fung KK,Zhang L, Wan HM, Chan CPS, Chung JPW. Live birth resulting from the direct warming and direct rehydration of vitrified human blastocyst in embryo culture medium: a pilot study. Fertil Reprod 2025;7:73–79. Desai N, Ploskonka S, Goodman L, Attaran M, Goldberg JM, Austin C, Falcone T. Delayed blastulation, multinucleation, and expansion grade are independently associated with live-birth rates in frozen blastocyst transfer cycles. Fertil Steril 2016;106:1370-1378. Cimadomo D, Capalbo A, Levi-Setti PE, Soscia D, Orlando G, Albani E, Parini V, Stoppa M, Dovere L, Tacconi L et al. Associations of blastocyst features, trophectoderm biopsy and other laboratory practice with post-warming behavior and implantation. Hum Reprod 2018;33:1992-2001. Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M, Takahashi K. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003;18:384-391. Lopes AS, Frederickx V, Van Kerkhoven G, Campo R, Puttemans P, Gordts S. Survival, re-expansion and cell survival of human blastocysts following vitrification and warming using two vitrification systems. J Assist Reprod Genet 2015;32:83-90. Gunst J, Vynck M, Hostens K, Standaert V, Roggeman S, van de Vijver A. Comparative Assessment of Survival and Clinical Outcome Between Two Commercial Vitrification Kits with Different Warming Protocols After Blastocyst Culture: Potential Perspectives Toward Simplified Warming Procedures. Reprod Sci 2023;30:3212-3221. Kato R, Itoi F, Tajima A, Oono N, Mori K, Watanabe S, Tanaka R, Akita M, Touyama H, Ibata M et al. One-step warming technique for vitrified human blastocysts using commercially available thawing solutions. Reprod Biomed Online 2025 (in press). Additional Declarations No competing interests reported. Supplementary Files SupplementalTable1.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7611070","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":524468224,"identity":"e1e04b21-db10-4846-9d25-a88e4d9cf1cd","order_by":0,"name":"Masashi Shioya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYBACCQkGhoMfKmzs+NkbgFwDC6K0MD6WOJOWLNlzAKRFgigtzAa8bYcYN9xIAPMJa5Gc3X5NQoLtALPkzOdXN/wokGDgb+9OwKtFWuZMmUQBzx0+fumcsps9QIdJnDm7Aa8WOYmcNAkJiWfMkrNz0m7wALUYSOQSoYXH4DDjhptn0m7+IUaLtET6YQOeBKCWG+zHbhNli+SMHGAgHwAFcg7bbRkDCR6CfpG4kf7g4Md/oKg8/uzmmz82cvztvfi1MDDwGKAweAgoBwH2B+iMUTAKRsEoGAWoAAAUt0pPOJv8zgAAAABJRU5ErkJggg==","orcid":"","institution":"Takahashi Women’s Clinic","correspondingAuthor":true,"prefix":"","firstName":"Masashi","middleName":"","lastName":"Shioya","suffix":""},{"id":524468225,"identity":"81b98a5c-50a1-4e2e-90aa-af3de75d1cee","order_by":1,"name":"Miki Okabe-kinoshita","email":"","orcid":"","institution":"Takahashi Women’s 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transfer\u003c/span\u003e\u003cspan lang=\"\"\u003e\u0026nbsp;[1]\u003c/span\u003e\u003cspan lang=\"\"\u003e. Furthermore, this has expanded the possibilities of assisted reproductive technology in its application to preimplantation genetic testing and fertility preservation [2, 3]. In recent years, as vitrification and subsequent warming have become established as standard IVF laboratory practices, research focus has shifted to optimizing the reduction of laboratory workload [4, 5]. Although vitrification protocols typically recommend immersing embryos in equilibration solution for up to 15 minutes, recent research has shown that the duration of equilibration does not impact embryo viability [6\u0026ndash;8]. Notably, the fast vitrification procedure described by\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"\"\u003eLiebermann et al. demonstrates good clinical outcomes with only 4 minutes of equilibration time [9].\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan lang=\"\"\u003eThe optimization focus of the procedure is also on the warming process subsequent to vitrification. Traditional warming protocols utilize multiple solutions to create stepwise osmotic pressures, based on the principle of cryobiology, which emphasizes the importance of safely rehydrating cryopreserved cells while avoiding hypotonic stress that could lead to irreversible cellular damage [10\u0026ndash;14]. Although this careful approach has proven effective, it is skill-dependent and time-consuming, posing challenges in laboratory workflows that involve a high volume of IVF cycles. As a result, there is a growing interest in simplifying the warming protocol to enhance efficiency and reduce the time embryos spend outside the incubator\u003c/span\u003e\u003cspan lang=\"\"\u003e\u0026nbsp;[15]\u003c/span\u003e\u003cspan lang=\"\"\u003e. Recent studies indicate that a\u0026nbsp;\u003c/span\u003esimplified\u0026nbsp;\u003cspan lang=\"\"\u003ewarming protocol does not negatively impact the clinical outcomes of vitrified blastocysts and may even improve these outcomes [16\u0026ndash;19]. These findings challenge the long-held belief in cryobiology that stepwise rehydration is essential for maintaining embryonic viability.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan lang=\"\"\u003eHowever, two critical questions remain largely unanswered in the existing literature. First, it is unclear whether this\u0026nbsp;\u003c/span\u003esimplified\u0026nbsp;\u003cspan lang=\"\"\u003eprotocol is effective for various embryo cohorts, including different developmental stages, morphological\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"\"\u003equalities\u003c/span\u003e\u003cspan lang=\"\"\u003e, and day of blastulation. Second, the majority of studies focus only on blastocysts, and the effects of sudden osmotic stress with a\u0026nbsp;\u003c/span\u003esimplified\u003cspan lang=\"\"\u003e\u0026nbsp;warming procedure on cleavage-stage embryos, which have fundamentally different cellular structures and physiological functions, are not well understood.\u003c/span\u003e\u003cspan lang=\"\"\u003e\u0026nbsp;A previous in vitro study using discarded embryos demonstrated that a simplified warming protocol did not impair the survival and subsequent developmental potential of cleavage-stage embryos. However, the effects on pregnancy and miscarriage were not evaluated\u0026nbsp;\u003c/span\u003e\u003cspan lang=\"\"\u003e[20].\u003c/span\u003e\u003cspan lang=\"\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThis study aims to address these critical knowledge gaps by investigating whether the omission of a stepwise osmotic dilution step during warming affects the clinical outcomes of human embryos with various characteristics. We conducted a large-scale retrospective comparative analysis and comprehensive safety evaluation of three warming protocols: a conventional three-step, an intermediate two-step, and a simplified one-step protocol.\u003c/p\u003e"},{"header":"2.\tMaterials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003e\u003cem\u003eStudy design\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective cohort study was conducted with the approval of the Institutional Review Board at the study institution. In accordance with ethical guidelines for medical research involving human subjects, patient consent for data use was obtained through an opt-out approach, with notifications posted on the clinic's official website and internal bulletin boards.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring the period from January to December 2024, this study included single vitrified-warmed embryo transfer cycles using three different warming protocols. The conventional three-step protocol was performed from January to September 2024, and the one-step protocol was introduced from April to September 2024. For the overlapping period between April and September, protocol assignment was randomized. The two-step protocol was then exclusively used from October to December 2024. The phased implementation of these protocols was to identify the optimal warming procedure.\u003c/p\u003e\n\u003cp\u003eCycles were excluded from the study if they met any of the following criteria: (1) use of embryos that underwent preimplantation genetic testing for aneuploidy ; (2) embryos not confirmed as two-pronuclear \u0026nbsp;at the time of fertilization assessment; (3) embryos derived from cryopreserved sperm; (4) embryos transported from other IVF centers; (5) double-embryo transfer cycles; or (6) embryos cultured with complete zona pellucida removal. After applying these exclusion criteria, a total of 1501 cycles were included in the final analysis, comprising 1351 blastocyst- and 150 cleavage-stage transfers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.2 Obtaining vitrified blastocyst-/cleavage-stage embryos\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients underwent controlled ovarian stimulation (COS) tailored to their individual AMH levels, maternal age, and clinical backgrounds. The COS was performed using established protocols at the institution, including mild stimulation with clomiphene citrate, GnRH antagonist, and GnRH agonist, progestin-promoted ovarian stimulation \u0026nbsp;protocols. In some cycles, oocyte retrieval was performed under natural unstimulated cycles.\u003c/p\u003e\n\u003cp\u003eFinal oocyte maturation was triggered with either human chorionic gonadotropin, a GnRH agonist, or a combination of both. Transvaginal ultrasound-guided oocyte retrieval was performed 35–38 hours post-trigger. The retrieved cumulus-oocyte complexes (COCs) were washed in HEPES-buffered medium (P+ HEPES medium®; Naka Medical, Tokyo, Japan) and pre-cultured in insemination medium (P+ insemination medium®; Naka Medical) under standard culture conditions (37.0°C, 6.0% CO\u003csub\u003e2\u003c/sub\u003e, 5.0% O\u003csub\u003e2\u003c/sub\u003e). Retrieved COCs were inseminated via either conventional in vitro fertilization (C-IVF) or intracytoplasmic sperm injection (ICSI) after removal of cumulus cells, based on semen parameters and clinical background.\u003c/p\u003e\n\u003cp\u003eThe embryos were cultured in droplets of ONESTEP medium® (Naka Medical) covered with OVOIL® (Vitrolife,\u0026nbsp;Gothenburg, Sweden) under conditions of 37.0°C, 6.0% CO₂, and 5.0% O₂.\u0026nbsp;Embryos that reached the cleavage stage (day 2 or 3) or blastocyst stage (day 5 or 6) with good morphology satisfying our criteria (cleavage-stage: grade ≥3 according to Veeck criteria; blastocyst-stage: grade 3 ≥CC according to Gardner criteria) were cryopreserved using the vitrification protocol [22, 23]. Vitrification was performed using the Cryotop® methods (Kitazato, Shizuoka, Japan) according to the manufacturer's protocol. Briefly, embryos were first rinsed in an equilibration solution for up to 15 minutes, until they re-expanded to at least 90% of their initial volume. Subsequently, they were transferred to a vitrification solution for 1–1.5 minutes before being loaded onto the Cryotop® device and plunged into liquid nitrogen for storage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e3\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Vitrified embryo warming and embryo transfer protocol\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVitrified cleavage- and blastocyst-stage embryos were selected for transfer based on the Veeck or Gardner criteria and were warmed using a commercially available warming solution [22, 23]. Three different protocols were utilized for warming the vitrified embryos. The conventional three-step protocol was a 6-minute procedure involving the sequential rinsing of embryos to thawing solution (TS) for 1 minute, followed by dilution solution (DS) for 2 minutes, and finally washing solution (WS) for 3 minutes. This approach ensured stepwise removal of the cryoprotectant. The\u0026nbsp;simplified\u0026nbsp;protocols included a modified two-step protocol, which involved rinsing of embryos with TS for 1 minute and then with DS for 2 minutes, and a one-step protocol, where embryos were rinsed only with TS for 1 minute prior to recovery culture. The commercial kits used for the three-step protocol were Cryotech warming solution (REPROLIFE, Tokyo, Japan), whereas the kits for the two-step and one-step protocols were the Vit Kit-Warm NX (Irvine Scientific, Santa Ana, CA, USA).\u0026nbsp;After warming, the embryos were cultured for ≥2 hours under conditions of 37°C, 6% CO₂, and 5% O₂ until embryo transfer.\u0026nbsp;For blastocyst-stage embryos, after rinsing with TS, laser-assisted hatching (LAH) was performed on embryos in areas with sufficient perivitelline space. Embryo survival was defined as blastocysts without dark-collared fragmented cells and with a clearly observable blastocoel.\u0026nbsp;For cleavage-stage embryos, survival was defined as having at least 50% of blastomeres remaining intact after warming. All embryos were transferred using an embryo transfer catheter (Kitazato) under abdominal ultrasound guidance. Gestational sacs and fetal heartbeats were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.4 Statistical\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eAnalysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using JMP Pro 18.00 (SAS Institute, Cary, NC, USA). The cycle and embryo backgrounds were compared using either one-way ANOVA test or Chi-square test. To analyze post-warming survival rates and embryo transfer outcomes across the three protocols (three-step, two-step, and one-step), a Chi-square test was employed, with a significance level set at p \u0026lt; 0.05. The outcomes at the blastocyst-stage were analyzed using a multivariate logistic regression model to adjust for confounding factors such as patient age, AMH level, BMI, LAH performed during the warming process, developmental stage, morphological quality, and day of blastulation.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBackground of the analyzed patients and blastocysts\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe background of patients who underwent blastocyst-stage embryo warming and the corresponding embryo characteristics are shown in Table 1. There was a significant difference in the mean maternal age among the groups, with the three-step group having the highest average age (35.95 \u0026plusmn; 0.18 years) and the two-step group having the lowest (35.28 \u0026plusmn; 0.29 years) (p=0.0062). No significant differences were found in the mean AMH levels (p=0.2338) and BMI (p=0.7945). Regarding blastocyst characteristics, there were no significant differences in the distribution of blastocyst developmental stage (full-blastocyst vs. expanded-blastocyst; p=0.4574), morphological quality (poor grade [\u0026lt;BB] vs. good grade [\u0026ge;BB]; p=0.9223), and the day of blastulation (Day 5 vs. Day 6; p=0.9782) among the three protocols. A significant difference was noted in the rate of LAH performed at the time of warming: LAH was most frequently performed in the three-step protocol (96.52%), followed by the two-step protocol (94.13%), and then the one-step protocol (84.62%; p\u0026lt;0.0001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePost-warming survival rate and clinical outcomes after embryo transfer in blastocyst-stage embryos\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe post-warming survival rates of the blastocysts were excellent and comparable across all three protocols: 100%, 99.30%, and 99.62% for the three-, two-, and one-step protocols, respectively (p=0.1112) (Table 1). After embryo transfer, there were no significant differences in the rates of gestational sac confirmation (p=0.6032), fetal heartbeat confirmation (p=0.5765), or pregnancy loss (p=0.6187). Multivariate logistic regression analysis indicated that maternal age at blastocyst vitrification, development stage, morphological\u0026nbsp;quality, and day of blastulation were factors associated with the outcomes of embryo transfer (Table 2). Notably, simplified warming protocols did not impact these outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSubgroup analysis based on developmental stage, morphological quality, and day of blastulation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed subgroup analyses to determine whether a simplified warming protocol for blastocyst-stage embryos is effective across embryos with various characteristics. The equivalence in fetal heartbeat confirmation rates between the three-step, two-step, and one-step protocols was consistently observed across all analyzed subgroups, including those based on developmental stage (full-blastocyst: 43.2%, 39.6%, and 33.7%, p=0.3111; expanded-blastocyst: 49.6%, 49.5%, and 49.1%, p=0.9950), morphological quality (poor-grade: 27.5%, 23.5%, and 23.4%, p=0.7636; good-grade: 52.1%, 51.6%, and 48.4%, p=0.6390), and day of blastulation (Day 5: 52.7%, 52.9%, and 50.3%, p=0.8234; Day 6: 34.4%, 28.5%, and 27.4%, p=0.4088) (Table 3\u0026ndash;5). Furthermore, a subgroup analysis based on patient age at blastocyst vitrification revealed that the simplified warming protocol had no adverse effects, even on embryos derived from advanced maternal age patients (Supplemental table 1) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSurvival rate and clinical outcomes of cleavage-stage embryos\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the analysis of 150 cleavage-stage embryos after warming, there were no differences in patient backgrounds among the groups (Table 6). The blastomere number, embryo grade, and day of vitrification were similar across all protocols. All warming protocols showed 100% survival rate after warming, with no observed loss of blastomeres. A comparable proportion of embryos in each protocol progressed with cell division during the recovery culture period of at least 2 hours before transfer: 21.0%, 15.6%, and 24.3% for the three-, two-, and one-step protocols, respectively (p = 0.6693). The primary clinical outcomes were similar regardless of the warming protocol used. The rates of fetal heartbeat confirmation were 11.11%, 12.50%, and 10.81% for the three-, two-, and one-step protocols, respectively, with no significant differences (p = 0.9717). However, there was a significantly higher proportion of pregnancy losses in the two simplified protocol groups (p = 0.0284), though the low event rate suggests that this finding should be interpreted with caution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Background of blastocyst-stage embryos used for embryo transfer and their survival and clinical outcomes\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"699\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eThree-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003eTwo-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eOne-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eNo. of warmed embryos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e661\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eAge (years, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e35.95 \u0026plusmn; 0.18\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e35.08 \u0026plusmn; 0.22\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e35.28 \u0026plusmn; 0.29\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0062\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eAMH (ng/mL, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e3.47 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e3.74 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e3.39 \u0026plusmn; 0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.2338\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e21.88 \u0026plusmn; 0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e21.78 \u0026plusmn; 0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e21.97 \u0026plusmn; 0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.7945\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eLAH performed at the time of warming, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e638 (96.52)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e401 (94.13)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e220 (84.62)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eEmbryo backgrounds, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 260px;\"\u003e\n \u003cp\u003eFull-blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e199 (30.11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e139 (32.40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e89 (34.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.4574\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 260px;\"\u003e\n \u003cp\u003eExpanded-blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e462 (69.89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e290 (67.60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e172 (65.90)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 260px;\"\u003e\n \u003cp\u003ePoor grade (Gardner criteria \u0026lt;BB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e120 (18.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e82 (19.11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e48 (18.39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.9223\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 260px;\"\u003e\n \u003cp\u003eGood grade (Gardner criteria \u0026ge;BB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e541 (81.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e347 (80.89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e213 (81.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 260px;\"\u003e\n \u003cp\u003eDay 5 blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u0026nbsp;478 (72.31)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e310 (72.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e187 (71.65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.9782\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 260px;\"\u003e\n \u003cp\u003eDay 6 blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e183 (27.69)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e119 (27.74)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e74 (28.35)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eSurvival rate, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e661 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e426 (99.30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e260 (99.62)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.1112\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eNo. of transferred blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e661\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eGestational sac, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e351 (53.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e226 (53.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e129 (49.62)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.6032\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003eFetal heartbeat, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e315 (47.66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e197 (46.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e114 (43.85)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.5765\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 274px;\"\u003e\n \u003cp\u003ePregnancy loss, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e75 (21.37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e51 (22.57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e33 (25.58)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.6187\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults are shown as mean \u0026plusmn; standard deviation or percentages when appropriate. Age, AMH level, and BMI are shown for female patients. Poor-grade indicated by grades CC, BC, CB, AC, and CA blastocysts; good-grade indicated by grades BB, AB, BA, and AA blastocysts. Categorical data were analyzed by Chi-square test, and other backgrounds were analyzed by one-way ANOVA test. Gestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed. P values in bold typeface indicate statistical significance. AMH, anti-M\u0026uuml;llerian hormone; BMI, body mass index; LAH, laser assisted hatching\u003c/p\u003e\n\u003cp\u003eTable 2. Logistic regression analysis of variables affecting clinical outcome in vitrified warm embryo transfer cycles\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"936\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 198px;\"\u003e\n \u003cp\u003eGestational sac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 203px;\"\u003e\n \u003cp\u003eFetal heartbeat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 212px;\"\u003e\n \u003cp\u003ePregnancy loss\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003eaOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e95%CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003eaOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e95%CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003eaOR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e95%CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eAge (years, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.906\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.881\u0026ndash;0.932\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.908\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.882\u0026ndash;0.933\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.095\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.046\u0026ndash;1.148\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eAMH (ng/mL, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e1.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e0.965\u0026ndash;1.048\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.8100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e1.021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.981\u0026ndash;1.063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.3132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.969\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.903\u0026ndash;1.035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.3646\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e0.992\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e0.960\u0026ndash;1.024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.6015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.959\u0026ndash;1.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.5622\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.970\u0026ndash;1.071\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4374\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eLAH performed at the time of warming, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e0.833\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e0.515\u0026ndash;1.346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.4553\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.738\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.460\u0026ndash;1.184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.2080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e2.319\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.990\u0026ndash;5.430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0528\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eDevelopmental stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003eFull-blastocyst (reference)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003eExpanded-blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.785\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.379\u0026ndash;2.310\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.494\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.156\u0026ndash;1.932\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0022\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e1.094\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.714\u0026ndash;1.678\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.6787\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eMorphological\u0026nbsp;quality\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003ePoor grade (reference)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003eGood grade\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.474\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.772\u0026ndash;3.455\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.218\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.573\u0026ndash;3.126\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.594\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.334\u0026ndash;1.058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0768\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eDay of blastulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003eDay 6 (reference)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003eDay 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.74\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.302\u0026ndash;2.327\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.635\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.219\u0026ndash;2.193\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.516\u0026ndash;1.336\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.4426\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 293px;\"\u003e\n \u003cp\u003eWarming protocols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003eThree-step (reference)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003eTwo-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e0.916\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e0.702\u0026ndash;1.196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.5197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.664\u0026ndash;1.128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.2845\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e1.241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.811\u0026ndash;1.897\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.3195\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 279px;\"\u003e\n \u003cp\u003eOne-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e0.824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e0.600\u0026ndash;1.132\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e0.2326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e0.796\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.580\u0026ndash;1.092\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.1575\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e1.553\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.944\u0026ndash;2.554\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e0.0831\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAdjusted odd ratios for gestational sac, fetal heartbeat, and pregnancy loss were calculated using a logistic regression model containing terms for patient age, AMH level, BMI, LAH performed during the warming process, developmental stage, morphological quality, day of blastulation, and warming protocols. P values in bold typeface indicate statistical significance. aOR, adjusted odd ratio; 95%CI, 95% confidence interval\u003c/p\u003e\n\u003cp\u003eTable 3. Blastocyst survival and clinical outcomes classified by developmental stage\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 233px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eThree-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eTwo-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eOne-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 233px;\"\u003e\n \u003cp\u003eFull-blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eNo. of warmed blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eSurvival rate, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e199 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e139 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e89 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eNo. of transferred blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eGestational sac, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e90 (45.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e63 (45.32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e34 (38.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.4896\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eFetal heartbeat, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e86 (43.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e55 (39.57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e30 (33.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.3111\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003ePregnancy loss, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e17 (18.89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e15 (23.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e9 (26.47)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.5988\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 233px;\"\u003e\n \u003cp\u003eExpanded-blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eNo. of warmed blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e290\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eSurvival rate, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e462 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e287 (98.97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e171 (99.42)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.1037\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eNo. of transferred blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e171\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eGestational sac, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e261 (56.49)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e163 (56.79)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e95 (55.56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.9659\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003eFetal heartbeat, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e229 (49.57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e142 (49.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e84 (49.12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.9950\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 220px;\"\u003e\n \u003cp\u003ePregnancy loss, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e58 (22.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e36 (22.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e24 (25.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.8090\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed.\u003c/p\u003e\n\u003cp\u003eTable 4. Blastocyst survival and clinical outcomes classified by morphological quality\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"714\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 314px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eThree-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eTwo-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eOne-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 314px;\"\u003e\n \u003cp\u003ePoor-grade (Gardner criteria \u0026lt;BB) blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eNo. of warmed blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eSurvival rate, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e120 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e81 (98.78)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e47 (97.92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.3420\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eNo. of transferred blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eGestational sac, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e37 (30.83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e23 (28.40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e12 (25.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.7849\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eFetal heartbeat, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e33 (27.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e19 (23.46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e11 (23.40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.7636\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003ePregnancy loss, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e13 (35.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e10 (43.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e3 (25.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.5492\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 314px;\"\u003e\n \u003cp\u003eGood-grade (Gardner criteria \u0026ge;BB) blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eNo. of warmed blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e347\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eSurvival rate, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e541 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e343 (99.42)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e213 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.1134\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eNo. of transferred blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e345\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eGestational sac, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e314 (58.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e203 (58.84)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e117 (54.93)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.6443\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003eFetal heartbeat, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e282 (52.13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e178 (51.59)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e103 (48.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.6390\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 300px;\"\u003e\n \u003cp\u003ePregnancy loss, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e62 (19.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e41 (20.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e30 (25.64)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.3873\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003ePoor-grade indicated by grades CC, BC, CB, AC, and CA blastocysts; good-grade indicated by grades BB, AB, BA, and AA blastocysts according to Gardner criteria. Gestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed.\u003c/p\u003e\n\u003cp\u003eTable 5. Blastocyst survival and clinical outcome classified by day of blastulation\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"631\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 231px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eThree-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eTwo-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003eOne-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 231px;\"\u003e\n \u003cp\u003eDay 5 blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eNo. of warmed blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eSurvival rate, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e478 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e310 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e187 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eNo. of transferred blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eGestational sac, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e279 (58.37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e186 (60.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e106 (56.68)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.7622\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eFetal heartbeat, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e252 (52.72)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e164 (52.90)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e94 (50.27)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.8234\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003ePregnancy loss, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e53 (19.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e38 (20.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e27 (25.47)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.3727\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 231px;\"\u003e\n \u003cp\u003eDay 6 blastocyst\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eNo. of warmed blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eSurvival rate, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e183 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e116 (97.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e73 (98.65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.4438\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eNo. of transferred blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e183\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e116\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eGestational sac, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e72 (39.34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e40 (34.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e23 (31.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.4438\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003eFetal heartbeat, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e63 (34.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e33 (28.45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e20 (27.40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.4088\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 217px;\"\u003e\n \u003cp\u003ePregnancy loss, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e22 (30.56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e13 (32.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e6 (26.09)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.8665\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eGestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed.\u003c/p\u003e\n\u003cp\u003eTable 6.\u0026nbsp;Background of cleavage-stage embryos used for embryo transfer and their survival and clinical outcomes\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"737\" class=\"fr-table-selection-hover\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eVariables\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eThree-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eTwo-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003eOne-step\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eNo. of warmed embryos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eAge (years, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e39.64 \u0026plusmn; 0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e38.78 \u0026plusmn; 0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e39.89 \u0026plusmn; 0.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e0.6259\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eAMH (ng/mL, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e1.80 \u0026plusmn; 0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e1.82 \u0026plusmn; 0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e2.23 \u0026plusmn; 0.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e0.6121\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e, mean \u0026plusmn; SD)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e22.31 \u0026plusmn; 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e21.67 \u0026plusmn; 0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e22.41 \u0026plusmn; 0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e0.6229\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eEmbryo backgrounds, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 249px;\"\u003e\n \u003cp\u003e2\u0026ndash;4-cell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e53 (65.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e23 (71.88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e29 (78.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.5682\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 249px;\"\u003e\n \u003cp\u003e5\u0026ndash;6-cell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e20 (24.69)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e6 (18.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e7 (18.92)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 249px;\"\u003e\n \u003cp\u003e\u0026gt;6-cell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e8 (9.88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e3 (9.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e1 (2.70)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 249px;\"\u003e\n \u003cp\u003ePoor grade (Veeck criteria 3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e57 (70.37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e19 (59.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e17 (45.95)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.1559\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 249px;\"\u003e\n \u003cp\u003eFair grade (Veeck criteria 2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e16 (19.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e8 (25.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e13 (35.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 249px;\"\u003e\n \u003cp\u003eGood grade (Veeck criteria 1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e8 (9.88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e5 (15.63)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e7 (18.92)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 249px;\"\u003e\n \u003cp\u003eDay 2 embryo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e72 (88.89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e30 (93.75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e36 (97.30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.2713\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 249px;\"\u003e\n \u003cp\u003eDay 3 embryo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e9 (11.11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e2 (6.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e1 (2.70)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eSurvival rate, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e81 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e32 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e37 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eNo. of transferred blastocysts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eGestational sac, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e9 (11.11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e5 (15.63)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e5 (13.51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e0.7968\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003eFetal heartbeat, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e9 (11.11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e4 (12.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e4 (10.81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e0.9717\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" style=\"width: 263px;\"\u003e\n \u003cp\u003ePregnancy loss, n(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0 (0)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1 (20.00)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3 (60.00)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 119px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0284\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults are shown as mean \u0026plusmn; standard deviation or percentages when appropriate. Age, basal AMH level, and BMI are shown for female patients. Categorical data were analyzed by Chi-square test, and other backgrounds were analyzed by one-way ANOVA test. Gestational sac and fetal heartbeat were confirmed by transvaginal ultrasound at 4 and 8 weeks after embryo transfer. Pregnancy loss was defined as the failure of a continuing pregnancy after a gestational sac had been confirmed. P values in bold typeface indicate statistical significance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides comprehensive evidence supporting that simplified warming protocols do not compromise outcomes for either blastocyst- or cleavage-stage embryos. Our primary finding is that the post-warming survival rate and subsequent embryo transfer outcomes were comparable across three different protocols: a conventional three-step, an intermediate two-step, and a highly simplified one-step protocol. Notably, this equivalence was consistently observed even in various embryo cohorts, including different embryo morphological and developmental stages. These results suggest that vitrified human embryos can tolerate sudden osmotic changes resulting from a simplified warming procedure.\u003c/p\u003e\u003cp\u003eIn cryobiology, it has been theoretically established that stepwise rehydration is essential for cryopreserved cells to avoid cryoinjury [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, recent clinical evidence has questioned this theory in vitrified human blastocysts. Liebermann et al. demonstrated that a one-step rehydration protocol not only achieves equivalent post-warming survival rates but also improves ongoing pregnancy rates and reduces miscarriage rates compared to conventional multi-step rehydration protocols [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Similarly, retrospective analysis by Jiang et al. and Licata et al. confirmed that a simplified warming protocol achieves equivalent live birth rates and clinical outcomes compared to a conventional protocol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In our study, we analyzed 1351 blastocyst transfers and found that the simplified protocol achieved equivalent post-warming survival rate and embryo transfer outcomes as the conventional protocol. Shioya et al. previously demonstrated that a simplified warming protocol does not impair key indicators of blastocyst viability, including re-expansion potential, cell survival rate assessed by Hoechst33258/Propidium iodide staining, and trophoblast outgrowth using discarded embryos [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The consistency between this previous in-vitro finding and the clinical results of the present study supports the safety of omitting the stepwise dilution process. The sudden osmotic pressure changes from a hypertonic warming solution (TS; 1656 mOsm/kg) to an isotonic culture medium (265 mOsm/kg), as in simplified protocols, can cause significant hypotonic stress. In fact, Ezoe et al. reported that the osmotic shift from TS to culture medium causes cell membrane collapse due to over-rehydration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Theoretically, this could result in a rapid influx of water into the blastocoel, inner cell mass, and trophectoderm, potentially leading to cell membrane rupture and impaired embryo viability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Contrary to these theoretical concerns, Chan et al. concluded that a direct rehydration protocol for vitrified embryos without warming solution not only resulted in 100% survival, but also allowed repeated vitrification, warming, and biopsy, so that stepwise rehydration with careful attention to osmotic pressure was not necessary [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA key finding of this study is the consistent efficacy of the simplified warming protocol across all embryonic qualities and developmental stages. Previous literature has highlighted that blastocysts with certain pre-vitrification features are less tolerant to traditional cryopreservation protocols; Desai et al. showed a 5% decrease in post-warming survival rate in a standard warming protocol for day 6 blastocysts compared to day 5 blastocysts, indicating that the number of days required for blastulation influences embryo survival [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, Cimadomo et al. reported that, in addition to the developmental speed to the blastocyst stage, the morphological quality before vitrification is an important factor affecting post-warming survival [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In delayed-development or poor quality blastocysts, the energy required for responding to physiological stresses and blastocoel re-expansion through the vitrification-warming process may be depleted, which may be the cause of reduced cryosurvival. Additionally, Mukaida et al. suggested that highly expanded-blastocysts are at greater risk due to their large fluid volume, which increases the chance of ice crystal formation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Considering the variety of embryos subjected to warming, traditional warming protocols are designed to carefully rehydrate embryos that are particularly vulnerable to sudden osmotic stress. Rapid influx of water may damage poor-quality cellular structures and potentially lead to reduced survival rates. Thus, it was necessary to determine whether these characteristics of vitrified blastocysts affect viability with simplified warming. However, there were no significant differences in embryo survival and ET outcomes among the three protocols, either in poor quality, delayed development, or advanced expansion stage embryos, and the effectiveness of simplified warming was not affected by embryo characteristics. These unexpected results suggest that the tolerance of embryos to hypotonic stress and the resumption of metabolic activity from the vitrified state may not depend on embryo quality in simplified warming. On the other hand, the simplified warming protocol reported by Liebermann et al. showed better clinical outcomes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We speculate that the adverse effects of prolonged in vitro handling in a multi-step protocol may outweigh the risks associated with rapid osmotic changes. For embryos with limited energy reserves, it may be important to minimize the time spent outside the incubator. Therefore, our findings indicate that clinicians and embryologists do not need to limit the use of warming protocols based on age or embryo characteristics, enabling the universal application of a safe and efficient warming protocol.\u003c/p\u003e\u003cp\u003eThis study provides clinical data on the application of a simplified warming protocol for cleavage-stage embryos. While blastocyst-stage embryo transfer is becoming a common strategy, cleavage-stage transfer remains a necessary clinical option in some countries. The results of the present study showed that the survival rate of cleavage-stage embryos was 100% in all protocols, and the fetal heartbeat confirmation rate was also equivalent. However, it is important to address the statistically significant differences in pregnancy loss rates between these groups. Due to the small number of pregnancies, these results should be interpreted with caution. Further investigation with a larger sample size is necessary to assess these specific outcomes more clearly.\u003c/p\u003e\u003cp\u003eThis study has several limitations. First, as this is a retrospective, single-center study, there is an inherent risk of selection bias. Second, the warming kits were obtained from two different manufacturers. Although the main components of the solutions are similar, slight differences in their composition may be confounding factors. However, commercialized vitrification/warming kits are available from various manufacturers, and their composition does not appear to affect embryo survival and pregnancy rates [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Kato et al. used three commercially available vitrification/warming kits for simplified warming, and their different compositions made no difference in blastocyst re-expansion rates and clinical outcomes [31]. Therefore, the use of different warming solutions across the protocols does not detract from the significance of our findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, a simplified warming protocol is a safe and effective method for warming vitrified human embryos. This approach is applicable to both blastocyst- and cleavage-stage embryos, regardless of embryo characteristics. The broader implication of this research supports the adoption of more efficient, evidence-based standard operating procedures in IVF laboratories. Implementing simplified protocols can significantly reduce procedure times, minimize embryo handling, and lower operational time, thereby improving the overall quality and efficiency of assisted reproductive technology without compromising the chances of a successful pregnancy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis retrospective cohort study was conducted with the approval of the Institutional Review Board at the study institution. Patient consent for data use was obtained through an opt-out approach.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003ch2\u003eTrial registration number:\u003c/h2\u003e\n\u003cp\u003eN/A.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by M.S., M.O.-K., K.K., M.F., and K.T. The first draft of the manuscript was written by M.S. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank the staff of Takahashi Women’s Clinic.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBourdon M, Maignien C, Pocate-Cheriet K, Plu Bureau G, Marcellin L, Patrat C, Chapron C, Santulli P. The freeze-all strategy after IVF: which indications? Reprod Biomed Online 2021;42:529-545.\u003c/li\u003e\n\u003cli\u003eBosch E, De Vos M, Humaidan P. The Future of Cryopreservation in Assisted Reproductive Technologies. Front Endocrinol 2020;11:67.\u003c/li\u003e\n\u003cli\u003eCasciani V, Monseur B, Cimadomo D, Alvero R, Rienzi L. Oocyte and embryo cryopreservation in assisted reproductive technology: past achievements and current challenges. Fertil Steril 2023;120:506-520. \u003c/li\u003e\n\u003cli\u003eGallardo M, Saenz J, Risco R. Human oocytes and zygotes are ready for ultra-fast vitrification after 2\u0026thinsp;minutes of exposure to standard CPA solutions. Sci Rep 2019;9:15986.\u003c/li\u003e\n\u003cli\u003eSciorio R, Tramontano L, Campos G, Greco PF, Mondrone G, Surbone A, Greco E, Talevi R, Pluchino N, Fleming S. Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review. Front Cell Dev Biol 2024;12:1398049. \u003c/li\u003e\n\u003cli\u003eSchiewe MC, Reichelderfer R, Wozniak K, De Romana C, Nordbak M, Baek K, Chung K. Ultra-fast vitrification and rapid elution of human oocytes: part I. germinal vesicle model validation. Reprod Biomed Online 2024;49:104691. \u003c/li\u003e\n\u003cli\u003eWozniak K, Reichelderfer R, Ghaemi S, Hupp D, Fuzesi P, Ringler G, Marrs RP, Schiewe MC. Ultra-fast vitrification and rapid elution of human oocytes: Part II - verification of blastocyst development from mature oocytes. Reprod Biomed Online 2024;49:104690\u003c/li\u003e\n\u003cli\u003eCosta-Borges N, Matia-Algu\u0026eacute; Q, Coello A, Mestres E, Acacio M, Flores-Saiffe Farias A, Castello C, Gallardo M, Chavez-Badiola A, Marco-Jim\u0026eacute;nez F et al. Preclinical validation of fast oocyte vitrification and warming protocols with comparable efficiencies to a standard method. Hum Reprod 2025;40:1066-1076. \u003c/li\u003e\n\u003cli\u003eLiebermann J, Brohammer R, Wagner Y, Smith R, Even K, Hirshfeld-Cytron J, Uhler ML. Fast and furious: successful survival and resumption of meiosis in immature human oocytes vitrified and warmed using a short protocol. Reprod Biomed Online 2024;49:103976.\u003c/li\u003e\n\u003cli\u003eRall WF and Fahy GM. Ice-free cryopreservation of mouse embryos at -196 degrees C by vitrification. Nature 1985;313:573-575. \u003c/li\u003e\n\u003cli\u003eMazur P, Schneider U. Osmotic responses of preimplantation mouse and bovine embryos and their cryobiological implications. Cell Biophys 1986;8:259\u0026ndash;285. \u003c/li\u003e\n\u003cli\u003ePedro PB, Zhu SE, Makino N, Sakurai T, Edashige K, Kasai M. Effects of hypotonic stress on the survival of mouse oocytes and embryos at various stages. Cryobiology 1997;35:150\u0026ndash;158.\u003c/li\u003e\n\u003cli\u003eKasai M. Advances in the cryopreservation of mammalian oocytes and embryos: development of ultrarapid vitrification. Reprod Med Biol 2002;1:1\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eVanderzwalmen P, Ectors F, Panagiotidis Y, Schuff M, Murtinger M, Wirleitner B. The Evolution of the Cryopreservation Techniques in Reproductive Medicine\u0026mdash;Exploring the Character of the Vitrified State Intra- and Extracellularly to Better Understand Cell Survival after Cryopreservation. Reproductive Medicine 2020;1:142-157. \u003c/li\u003e\n\u003cli\u003eManns JN, Katz S, Whelan J III, Patrick JL, Holt T, Merline AM, Taylor TH. Ultra-fast blastocyst, warming technique reduces warming times to 1 min and yields similar survival and re-expansion compared to blastocysts warmed using a standard method. Fertil Steril 2021;116:e165. \u003c/li\u003e\n\u003cli\u003eLiebermann J, Hrvojevic K, Hirshfeld-Cytron J, Brohammer R, Wagner Y, Susralski A, Jasulaitis S, Chan S, Takhsh E, Uhler M. Fast and furious: pregnancy outcome with one-step rehydration in the warming protocol for human blastocysts. Reprod Biomed Online 2024;48:103731. \u003c/li\u003e\n\u003cli\u003eBartolacci A and Albertini DF. The new ice age: the promise and challenges of rapid oocyte warming protocols. J Assist Reprod Genet 2024;41:2969-2971.\u003c/li\u003e\n\u003cli\u003eJiang VS, Cherouveim P, Naert MN, Dimitriadis I, Souter I, Bormann CL. Live birth outcomes following single-step blastocyst warming technique - optimizing efficiency without impacting live birth rates. J Assist Reprod Genet 2024;41:1193-1202.\u003c/li\u003e\n\u003cli\u003eLicata E, VerMilyea M, Ducote B, Ferguson T, Bianco C, Gallo M, Paciotti G, Passerini R, Meneghini C, Fabiani C et al. A diluted one-step warming protocol: survival of vitrified blastocyst. J Assist Reprod Genet 2025;42:1101-1107.\u003c/li\u003e\n\u003cli\u003eShioya M, Hashizume R, Okabe-Kinoshita M, Kojima K, Nishi S, Nakano S, Koga K, Fujita M, Takahashi K. One-step warming of vitrified human cleavage and blastocyst stage embryos does not adversely impact embryo survivability and subsequent developmental potential. Hum Reprod 2025;40:261-269.\u003c/li\u003e\n\u003cli\u003eVeeck LL. An atlas of human gametes and conceptuses. An illustrated reference for assisted reproductive technology; 1999.\u003c/li\u003e\n\u003cli\u003eGardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73:1155-1158.\u003c/li\u003e\n\u003cli\u003eEzoe K, Miki T, Fujiwara N, Kato K. Influence of the shortened warming protocol on human blastocyst viability: an in-vitro experimental study. Reprod Biomed Online 2025;50:104454.\u003c/li\u003e\n\u003cli\u003eChan DYL, Xu M, Wu W, Fung KK,Zhang L, Wan HM, Chan CPS, Chung JPW. Live birth resulting from the direct warming and direct rehydration of vitrified human blastocyst in embryo culture medium: a pilot study. Fertil Reprod 2025;7:73\u0026ndash;79. \u003c/li\u003e\n\u003cli\u003eDesai N, Ploskonka S, Goodman L, Attaran M, Goldberg JM, Austin C, Falcone T. Delayed blastulation, multinucleation, and expansion grade are independently associated with live-birth rates in frozen blastocyst transfer cycles. Fertil Steril 2016;106:1370-1378. \u003c/li\u003e\n\u003cli\u003eCimadomo D, Capalbo A, Levi-Setti PE, Soscia D, Orlando G, Albani E, Parini V, Stoppa M, Dovere L, Tacconi L et al. Associations of blastocyst features, trophectoderm biopsy and other laboratory practice with post-warming behavior and implantation. Hum Reprod 2018;33:1992-2001.\u003c/li\u003e\n\u003cli\u003eMukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M, Takahashi K. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003;18:384-391.\u003c/li\u003e\n\u003cli\u003eLopes AS, Frederickx V, Van Kerkhoven G, Campo R, Puttemans P, Gordts S. Survival, re-expansion and cell survival of human blastocysts following vitrification and warming using two vitrification systems. J Assist Reprod Genet 2015;32:83-90.\u003c/li\u003e\n\u003cli\u003eGunst J, Vynck M, Hostens K, Standaert V, Roggeman S, van de Vijver A. Comparative Assessment of Survival and Clinical Outcome Between Two Commercial Vitrification Kits with Different Warming Protocols After Blastocyst Culture: Potential Perspectives Toward Simplified Warming Procedures. Reprod Sci 2023;30:3212-3221. \u003c/li\u003e\n\u003cli\u003eKato R, Itoi F, Tajima A, Oono N, Mori K, Watanabe S, Tanaka R, Akita M, Touyama H, Ibata M et al. One-step warming technique for vitrified human blastocysts using commercially available thawing solutions. Reprod Biomed Online 2025 (in press).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"blastocyst, cleavage, cryopreservation, embryo warming, embryo vitrification, one-step warming, two-step warming, osmotic stress, rehydration, dilution","lastPublishedDoi":"10.21203/rs.3.rs-7611070/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7611070/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose:\u003c/h2\u003e\u003cp\u003eTo determine whether a simplified warming protocol for vitrified human embryos is effective regardless of embryo characteristics such as developmental stage, morphological quality, and day of blastulation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003e: This is a retrospective cohort study conducted at a single IVF center. A total of 1501 single vitrified-warmed embryo transfer cycles, involving 1351 blastocyst- and 150 cleavage-stage embryos, performed from January to December 2024, were analyzed. Post-warming survival rate and embryo transfer outcomes were compared between a conventional three-step warming protocol (thawing solution [TS] for 1 minute, dilution solution [DS] for 2 minutes, and washing solution [WS] for 3 minutes, n\u0026thinsp;=\u0026thinsp;741) and simplified warming protocols, including a two-step (TS for 1 minute, followed by DS for 2 minutes, n\u0026thinsp;=\u0026thinsp;461) and a one-step (TS for 1 minute, n\u0026thinsp;=\u0026thinsp;298). The analysis primarily focused on blastocyst-stage embryos, for which detailed subgroup analyses were performed based on developmental stage, morphological quality, and day of blastulation. In addition, a separate analysis was conducted for cleavage-stage embryos.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eFor blastocyst-stage embryos, post-warming survival rates were excellent and comparable among the three-step, two-step, and one-step protocols (100%, 99.3%, 99.6%; p\u0026thinsp;=\u0026thinsp;0.1112). There were also no statistically significant differences in the fetal heartbeat confirmation rates (47.7%, 46.2%, 43.9%; p\u0026thinsp;=\u0026thinsp;0.5765). Furthermore, this equivalence was consistently observed across all subgroups, including those based on developmental stage (full-blastocyst: 43.2%, 39.6%, 33.7%, p\u0026thinsp;=\u0026thinsp;0.3111; expanded-blastocyst: 49.6%, 49.5%, 49.1%, p\u0026thinsp;=\u0026thinsp;0.9950), morphological quality (poor-grade: 27.5%, 23.5%, 23.4%, p\u0026thinsp;=\u0026thinsp;0.7636; good-grade: 52.1%, 51.6%, 48.4%, p\u0026thinsp;=\u0026thinsp;0.6390), and day of blastulation (day 5: 52.7%, 52.9%, 50.3%, p\u0026thinsp;=\u0026thinsp;0.8234; day 6: 34.4%, 28.5%, 27.4%, p\u0026thinsp;=\u0026thinsp;0.4088). For cleavage-stage embryos, all protocols resulted in 100% post-warming survival with comparable fetal heartbeat confirmation rates (11.1%, 12.5%, 10.8%; p\u0026thinsp;=\u0026thinsp;0.9717).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eA simplified one- or two-step warming protocol is a safe and effective protocol for warming of vitrified human embryos. This approach is applicable to a variety of embryos, regardless of their characteristics.\u003c/p\u003e","manuscriptTitle":"Simplified warming protocol for vitrified human embryos: a comprehensive analysis across various developmental stages and morphological qualities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 11:34:37","doi":"10.21203/rs.3.rs-7611070/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"39008cd4-0698-4129-b6cd-653ae02845ea","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-28T16:54:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 11:34:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7611070","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7611070","identity":"rs-7611070","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}