Improved pregnancy outcomes with single day 5 blastocyst transfer from post-thawed cleavage embryos in the first transfer cycle: a retrospective propensity score-matched cohort study

Improved pregnancy outcomes with single day 5 blastocyst transfer from post-thawed cleavage embryos in the first transfer cycle: a retrospective propensity score-matched cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improved pregnancy outcomes with single day 5 blastocyst transfer from post-thawed cleavage embryos in the first transfer cycle: a retrospective propensity score-matched cohort study Wanli Yang, Jingwen Lang, Liying Peng, Pengcheng Kong, Xiuxian Zhu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8390695/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 16 You are reading this latest preprint version Abstract Background We aimed to test the hypothesis that transferring blastocysts derived from extended culture of cleavage-stage embryos for single embryo transfer in the first transfer cycle would result in better clinical outcomes than transferring frozen-thawed blastocysts, without affecting neonatal outcomes. Methods We conducted a retrospective propensity score-matched cohort study at a single reproductive medicine center. After excluding transfer cycles that did not meet the inclusion criteria, we classified the subjects into two groups based on the blastocyst transfer strategies. The group receiving blastocysts derived from the extended culture of the frozen-thawed cleavage-stage embryos was labeled the F3T5 group, while the group receiving frozen-thawed blastocysts was labeled the F5T5 group. The primary outcomes of this study were the implantation rate and the ongoing pregnancy rate. Secondary outcomes included the biochemical pregnancy rate, miscarriage rate, clinical pregnancy rate, and neonatal outcomes. Results After propensity score matching, a total of 811 blastocyst transfer cycles were included in the analysis. 307 cycles in the F3T5 group and 504 cycles in the F5T5 group. The results revealed that the F3T5 group had significantly higher rates of biochemical pregnancy (78.83% vs 66.07%, P < 0.001), clinical pregnancy (70.36% vs 59.72%, P = 0.029), implantation (70.36% vs 59.72%, P = 0.036), and ongoing pregnancy (64.17% vs 50.99%, P < 0.001) compared to the F5T5 group. There were no statistically significant differences between the two groups in terms of multiple pregnancy rate, ectopic pregnancy rate, miscarriage rate, and neonatal outcomes such as gestational weeks, single birth weight, and pregnancy complications ( P > 0.05). Conclusion Our findings suggested that transferring the single blastocyst derived from the extended culture of the frozen-thawed cleavage embryos in the first transfer cycle might offer a beneficial approach for optimizing assisted reproduction technology success rates in women under 36 years of age without affecting the neonatal outcomes. Blastocyst transfer extended culture frozen-thawed clinical outcome neonatal outcome Figures Figure 1 Figure 2 1. Introduction Since the birth of the first test tube baby in 1978, assisted reproduction technology (ART) has undergone over four decades of development [ 1 ]. One of the key milestones in ART is embryo transfer, which has evolved into two primary methods: fresh embryo transfer and frozen embryo transfer (FET). In fresh embryo transfer, embryos are transferred during the same cycle as oocyte retrieval, while FET involves the transfer of embryos that were previously frozen and then thawed. FET has gained widespread adoption due to its potential advantages, including a reduced risk of ovarian hyperstimulation syndrome (OHSS) [ 2 ], higher cumulative pregnancy rates [ 3 ], and lower incidence of multiple pregnancies [ 4 ]. As a result, the number of FET cycles has progressively increased and has surpassed that of fresh embryo cycles in many countries [ 5 – 7 ]. Traditionally, embryo transfers were performed on day 2 or 3, during the cleavage-stage, when embryos typically comprised four or eight blastomeres. This practice was based on the belief that the uterus provided the optimal environment for embryo survival [ 8 ]. However, with the improvement in laboratory conditions and embryo culture medium, ART has shifted from cleavage-stage transfers to blastocyst transfers [ 9 ], where embryos are cultured to the blastocyst stage on day 5 or 6. The rationale for blastocyst transfer is better to align embryonic development with uterine receptivity, which facilitates the self-selection of viable embryos and has been shown to improve live birth rates [ 9 , 10 ]. In line with the government policies aiming to minimize the risk of multiple gestations by limiting the number of embryos transferred, single blastocyst transfer, whether fresh or via FET, has gained significant popularity due to its potential to improve outcomes and reduce complications. Despite these advantages, concerns have been raised regarding the potential risks of cryopreservation. Some studies suggested that blastocysts might be more susceptible to damage during the vitrification and thawing process compared to cleavage-stage embryos [ 11 ]. This is attributed to the challenge of adequately permeating cryoprotectants into the blastocyst, which may result in cellular injury [ 12 ]. To address this concern, some researchers have proposed extending the culture of thawed cleavage-stage embryos to allow them to develop into blastocysts before transfer, potentially minimizing the risks associated with blastocyst cryopreservation while still achieving the benefits of blastocyst transfer [ 13 – 15 ]. However, the clinical outcomes of this strategy, when compared to frozen blastocyst transfer, remained debated. Some studies have shown improved implantation rates and clinical pregnancy outcomes in the extended culture group [ 12 , 16 ], while others find no significant difference between the two approaches [ 17 ]. These discrepancies highlight the need for further research to fully assess the clinical benefits of this approach, including its effect on embryo implantation rates, live birth outcomes, and miscarriage rates. In the current study, we aimed to test the hypothesis that transferring a single blastocyst derived from the extended culture of cleavage-stage embryos would result in better clinical outcomes compared with transferring the frozen-thawed blastocysts in the first transfer cycle, with the goal of providing novel insights into optimizing embryo transfer strategies in ART. 2 Materials and methods 2.1 Study Population and Design This retrospective cohort study was conducted at the Reproductive Medicine Center of Shanghai First Maternity and Infant Hospital from January 1st, 2019 to October 31st, 2024. We investigated the clinical and neonatal outcomes of women undergoing FET. We included patients who underwent a single Day 5 blastocyst transfer, considering only the first transfer cycle for those with multiple cycles. After excluding patients who underwent more than two transfer cycles, transfer multiple blastocysts, were over 35 years of age, received day 6/7 blastocysts or the early stage blastocysts transfer, underwent embryo transfer from preimplantation genetic testing (PGT) cycles, had an endometrial thickness less than 7 mm on the transfer day, had a history of recurrent spontaneous abortion (RSA), or were lost to follow-up, the remaining cycles were classified into two groups: the extended culture group (F3T5) and the frozen-thawed blastocyst group (F5T5). Finally, a total of 346 blastocyst transfer cycles in the F3T5 group, with a corresponding number of 1,407 transfer cycles in the F5T5 group. This study was approved by the ethics committee of the Shanghai First Maternity and Infant Hospital, and informed consent was waived for individual patients due to the retrospective design of the study. 2.2 Analysis We compared the clinical outcomes between F3T5 and F5T5 blastocyst transfer groups using the propensity score matched (PSM) method. PSM analysis was performed using the MatchIt R package (v.4.5.5), with nearest neighbor matching (caliper 0.1) and a matching ratio of 1:2. The matching factor included patient characteristics at the time of transfer, such as maternal age, body mass index (BMI), endometrial thickness, infertility years, endometrial preparation regimen, infertility factors, type of infertility, and number of high-quality blastocysts transferred. After PSM matching, 307 blastocyst transfer cycles in the F3T5 group and 504 blastocyst transfer cycles in the F5T5 group were included for further analysis. 2.3 Embryo Vitrification Freezing, Thawing, and Extending culture Vitrification was used as the embryo cryopreservation method for cleavage-stage embryos and blastocysts. Freezing and thawing of cleavage-stage embryos and blastocysts were carried out according to the standard protocols of the Kitazato vitrification kit (Kitazato, Japan). For embryo freezing, grade I and grade II cleavage-stage embryos with cytoplasmic fragments less than 20% were exposed to equilibration solution (ES) for 7 minutes at room temperature, while blastocysts were exposed to ES for 9 minutes and then transferred to vitrification solution (VS) for 45–60 seconds, and subsequently placed on the Cryotop strip (Kitazato, Japan). The strip, together with its protective sleeve, was then promptly immersed in the liquid nitrogen for cryopreservation. For embryo thawing, after removing the protective sleeve of the strip and directly immersed in 300µL of thawing solution (TS) at 37°C for 1 minute. Embryos were transferred to 200µL diluent solution (DS) for 3 minutes at room temperature and washed twice using washing solution for 5 minutes each time at room temperature. Finally, the embryos were stored in the G2-plus medium (Vitrolife, Sweden) and cultured at 37°C with a 6% CO 2 and 5% O 2 atmosphere until transfer. The post-thawed embryos with more than 50% intact blastomeres were considered viable and eligible for transfer or extended culture [ 2 ]. The thawed cleavage-stage embryos were cultured in G2-plus medium (Vitrolife, Sweden) for 48 or 72 hours at 37°C in an atmosphere with 6% CO 2 and 5% O 2 . According to the Gardner scoring system, high-quality blastocysts are defined as those at stage 3 or higher, with neither the inner cell mass nor trophectoderm scores assigned a grade of C. 2.4 Endometrial Preparation and FET Endometrial preparation for FET was carried out using a natural cycle, ovarian induction cycle, or hormone replacement therapy (HRT), depending on the patient’s condition or the physicians’ preference, as our previously reported [ 18 , 19 ]. Ultrasound-guided blastocyst transfer was conducted either on the fifth day after ovulation or on the sixth day after progesterone administration. Progesterone supplementation was administrated to all patients as post-transfer luteal support. The serum β-hCG levels were assessed two weeks after embryo transfer to confirm pregnancy at our center. For patients with a positive pregnancy test, a transvaginal ultrasonographic was performed four weeks after embryo transfer to detect the presence of gestation sac and fetal heartbeat. 2.5 Outcome Measurement The primary outcomes of this study were the implantation rate and ongoing pregnancy rate for each transferred cycle. Secondary outcomes included the biochemical pregnancy rate, miscarriage rate, clinical pregnancy rate, and neonatal outcomes. The implantation rate was calculated by dividing the number of gestational sacs detected via transvaginal ultrasonography by the number of embryos transferred. Biochemical pregnancy was considered positive if the serum beta-human chorionic gonadotropin (β-hCG) concentration exceeded 10 mIU/mL, measured 14 days after embryo transfer. Clinical pregnancy was defined as the presence of a gestational sac in the uterine cavity, with or without fetal heart activity, as well as the occurrence of ectopic pregnancies. Ectopic pregnancy was diagnosed when at least one ectopic pregnancy sac was detected by ultrasound. Ongoing pregnancy was defined as a pregnancy that had reached at least 12 weeks of gestation. Multiple pregnancy was defined as the presence of two or more gestational sacs identified through ultrasound. Miscarriage was defined as the loss of a clinical pregnancy within 28 weeks of confirming. For neonatal outcomes, preterm birth was defined as delivery before 37 weeks. 2.6 Statistical All data were analyzed using R ( http://www.R-project.org ) (v.4.2.3) with P < 0.05 considered statistically significant. Continuous variables were presented as mean ± standard deviation (SD), while categorical variables were presented as numbers with percentages. Comparisons of categorical variables were made using Fisher’s exact test or Chi-square test. The baseline characteristics and clinical outcomes of the PSM matched data were compared using the Student’s t-test for continuous variables and Fisher’s exact test or Chi-squared test for categorical variables. We used the logistic regression analysis to investigate the association between different endometrial preparation regimens and the clinical outcomes. The multivariable logistic regression model was adjusted for maternal age, BMI, endometrial thickness, infertility years, infertility factors, type of infertility, and number of high-quality blastocysts transferred. The associations of the univariate logistic regression model and multivariate logistic regression model were presented as odds ratios (OR) with a 95% confidence interval (CI). 3 Results 3.1 Baseline Characteristics of Participants The flowchart of the study is presented in Fig. 1 . A total of 1,753 FET cycles were analyzed, including 346 cycles in the F3T5 group and 1,407 cycles in the F5T5 group. In the F5T5 group, out of 1,453 blastocysts, 1,444 (99.38%) survived in the F5T5 group. In the F3T5 group, 947 out of 950 cleavage-stage embryos survived with at least half of the blastomeres intact (99.68%). From the 947 cleavage-stage embryos in the F3T5 group, 765 blastocysts were formed, yielding a blastocyst formation rate of 80.78%. Of these, 640 blastocysts were available, resulting in an availability rate of 83.66%, and 294 blastocysts were re-cryopreserved after transfer (346 transfer cycle). After establishing a balanced cohort using PSM, 307 transfer cycles in the F3T5 group were matched with 504 transfer cycles in the F5T5 group. Figure 2 shows the distributions of propensity scores and standard differences both before and after matching, illustrating the balance between the two comparing groups. The baseline characteristics of the two groups were presented in Table 1 , with most characteristics comparable ( P > 0.05) except the endometrial preparation regimen ( P < 0.001). Table 1 Baseline characteristics of patients in F3T5 and F5T5 groups following propensity score matching Variables F3T5 F5T5 P Cycles, n 307 504 Maternal age, y 30.69 ± 2.50 30.57 ± 2.63 0.52 Body mass index, kg/m 2 22.27 ± 3.42 22.19 ± 3.71 0.74 Infertility duration, y 2.72 ± 1.83 2.76 ± 1.89 0.76 Type of infertility, n 0.82 Primary infertility 199 331 Secondary infertility 108 173 Infertility diagnosis, n 0.052 Female factor 231 366 Male factor 56 78 Combination of factors 14 34 Unexplained 6 26 Endometrial preparation regimen, n < 0.001 Natural cycle 76 60 Ovarian induction cycle 197 252 Hormone replacement therapy 34 192 Endometrial thickness, mm 11.48 ± 2.31 11.32 ± 2.32 0.33 No. of high-quality blastocysts transferred, n (%) 166 (52.35%) 274 (54.19%) 0.99 n: number; y: years; mm: millimeter; F3T5: extending culture group; F5T5: Frozen-thawed blastocyst transfer group. 3.2 Clinical and Neonatal Outcomes Comparisons between the Two Groups Table 2 summarizes the clinical and neonatal outcomes following blastocyst transfer in the F3T5 group, compared with those after the transfer of frozen-thawed blastocysts in the F5T5 group. The results demonstrated that the biochemical pregnancy rate (78.83% vs 66.07%), clinical pregnancy rate (70.36% vs 59.72%), implantation rate (70.36% vs 59.72%), and ongoing pregnancy rate (64.17% vs 50.99%) were significantly higher in the F3T5 group compared with the F5T5 group ( P 0.05). A total of 172 cycles in the F3T5 group and 202 cycles in the F5T5 group were included in the neonatal outcomes. The incidence of twin births and preterm births was similar between the two groups ( P > 0.05). Additionally, in terms of birthweight for singletons, gestational weeks of birth, and pregnancy complications, both groups were similar ( P > 0.05). Those findings collectively suggest that transferring the blastocysts derived from the extended culture of frozen-thawed cleavage-stage embryos for single embryo transfer in the first transfer cycle may yield better clinical outcomes than frozen-thawed blastocyst transfer without a significant difference in neonatal outcomes. Table 2 Pregnancy and neonatal outcome of the propensity matched data Variables F3T5 F5T5 P Pregnancy outcomes Cycles, (n) 307 504 Biochemical pregnancy rate, n (%) 242 (78.83) 333 (66.07) < 0.001 Clinical pregnancy rate, n (%) 216 (70.36) 301 (59.72) 0.0029 Multiple pregnancy rate, n (%) 7 (3.24) 6 (1.99%) 0.54 Implantation rate, n (%) 216/307 (70.36) 301/504 (59.72) 0.0036 Ectopic pregnancy rate, n (%) 3 (1.39) 5 (1.66) 1 Miscarriage rate, n (%) 23 (10.65) 46 (15.28) 0.16 Ongoing pregnancy rate, n (%) 197 (64.17) 257 (50.99) < 0.001 Neonatal outcomes 0.31 Single newborn, n 166 199 Twin newborns, n 6 3 Live birth cycles 0.77 Preterm delivery, n 16 16 Term deliver, n 156 186 Week of birth, w 38.52 ± 1.73 38.46 ± 1.78 0.73 Single birth weight, g 3259.91 ± 495.27 3356.33 ± 532.16 0.074 Pregnancy complications, n (%) 6 (3.49) 9 (4.46) 0.83 n: number; w: weeks; g: gram; F3T5: extending culture group; F5T5: Frozen-thawed blastocyst transfer group. 3.3 Association between Different Endometrial preparation Regimens and Clinical Outcomes in the two Groups Given the retrospective nature of our study, we used PSM to balance the baseline characteristics between the two groups. Despite this, we still observed differences in the endometrial preparation regimen. To further investigate the association between the endometrial preparation regimen and the clinical outcomes, we performed a logistic regression analysis. The univariate logistic regression model indicated that different endometrial preparation regimens had no significant impact on the biochemical pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, and implantation rate ( P > 0.05) (Table 3 ). These findings remained consistent after adjusting the potential confounders in the multivariate logistic regression model ( P > 0.05) (Table 3 ). This suggested that the improved clinical outcomes observed in the F3T5 group were not influenced by the variations in the endometrial preparation regimens. Table 3 The logistic regression analyses of the association between different endometrial preparation regimens and clinical outcomes Variables Groups OR (95% CI) P Adjusted OR (95% CI) P Biochemical pregnancy rate F5T5 Reference F3T5 0.91 (0.74–1.20) 0.38 0.84 (0.67–1.03) 0.10 Clinical pregnancy rate F5T5 Reference F3T3 1.01 (0.83–1.22) 0.94 0.94 (0.77–1.14) 0.52 Implantation rate F5T5 Reference F3T5 1.01 (0.83–1.22) 0.94 0.94 (0.77–1.14) 0.52 Ongoing pregnancy rate F5T5 Reference F3T5 1.01 (0.84–1.22) 0.90 0.93 (0.77–1.13) 0.47 4. Discussion In the current study, we conducted a retrospective PSM analysis to evaluate the clinical and neonatal outcomes after the transfer of a single day 5 blastocyst from the extended culture of frozen-thawed cleavage embryos or from frozen-thawed blastocysts in women under 36 years of age. To the best of our knowledge, this is the first analysis of this issue in the certain population. Our findings suggested that the extended culture of frozen-thawed cleavage embryos to the blastocyst for single embryo transfer in the first transfer cycle could improve pregnancy outcomes in women under 36 years of age without affecting the neonatal outcomes. The above findings implicate a potential strategy for optimizing embryo selection and transfer protocols in this population. FET plays an important role in ART. Since vitrification freezing was first introduced in China in 2003, this technology has advanced over the past 20 years [ 20 ]. Vitrification is a reliable and safe embryo cryopreservation method that has become widely utilized in ART due to its numerous clinical advantages [ 2 , 3 , 21 ]. Currently, numerous studies have examined the clinical outcomes of frozen-thawed blastocysts and cleavage-stage embryos [ 22 ]. However, the clinical outcomes of transferring blastocysts derived from the extended culture of frozen-thawed cleavage embryos or frozen-thawed blastocysts remain a topic of ongoing debate [ 12 , 16 , 17 ]. In our study, after strictly controlling for potential confounders using the PSM method, we observed an improved biochemical pregnancy rate, clinical pregnancy rate, implantation rate, and ongoing pregnancy rate after transferring blastocysts derived from the extended culture of frozen-thawed cleavage embryos in women under 36 years, with no impact on neonatal outcomes. The blastocyst formation rate after extended culture of frozen-thawed cleavage embryos was 80.53%, consistent with previous findings [ 23 ]. Therefore, we do not attribute the improvement in clinical outcomes to an enhancement in the embryos’ developmental potential, but rather to the following reasons. Compared to transferring frozen-thawed blastocysts, transferring blastocysts derived from the extended culture of the frozen-thawed cleavage-stage embryos more closely resemble a fresh cycle. This helps mitigate the negative effects associated with the true fresh embryo transfer cycle, such as the adverse impact of supra-physiological steroid hormones from ovarian superovulation on the endometrium, elevated estrogen levels, and the general anesthesia for oocyte retrieval, all of which could hinder embryo implantation [ 24 – 27 ]. In addition, inadequate permeation of cryoprotectant into the blastocoel can lead to ice crystal formation and ultrastructural cellular damage to expanded blastocysts. Cryopreserving those blastocysts presents technical challenges, as they are more vulnerable to cryo-damage due to their advanced development and larger size [ 12 , 28 ]. The extended culture of frozen-thawed cleavage embryos might help repair the potential damage caused by vitrification [ 11 ]. Based on the above theory, we propose the hypothesis that transferring blastocysts derived from the extended culture of frozen-thawed cleavage embryos will result in better clinical outcomes compared to directly transferring frozen-thawed blastocysts. These findings were further supported by evidence showing a higher live birth rate after fresh embryo transfer compared to frozen embryo transfer in oocyte donor recipients who did not undergo ovarian stimulation [ 29 ]. In conjunction with our neonatal outcomes, our study highlights that the extended culture of frozen-thawed cleavage embryos to blastocysts would improve clinical outcomes, particularly in women under 36 years. Advancing maternal age negatively impacts the success of ART [ 30 , 31 ]. As women age, both the quantity and quality of the oocytes decline, leading to lower fertilization rates, poor embryo development, and a reduced likelihood of implantation [ 32 ]. Research has shown that embryo implantation rates remain stable until the age of 35, after which they decrease significantly, with a linear decline of 2.77% per year ( P < 0.001, R 2 = 0.975) [ 32 ]. To minimize the impact of maternal age on clinical outcomes, we included patients under 36 years of age. By focusing on this age group, we aimed to reduce variability and ensure that the data was not unduly influenced by age-related factors. This allows for a more accurate assessment of the pregnancy and neonatal outcomes in the F3T5 and F5T5 groups, providing a clearer understanding of the effectiveness of different embryo transfer strategies. After controlling for maternal age, we found that the F3T5 group demonstrated advantages in the biochemical pregnancy rate, clinical pregnancy rate, implantation rate, and ongoing pregnancy rate. These results highlight the importance of optimizing embryo transfer strategy to maximize pregnancy success. Currently, transfer blastocysts are divided into groups of 5, 6, and 7 days according to their developmental days. The implantation rate, clinical pregnancy rate, and live birth rate of day 7 blastocysts were significantly reduced compared to day 5 and day 6 blastocysts [ 33 ]. Compared to day 6 blastocysts, day 5 blastocysts transfer generally result in better clinical outcomes [ 34 – 36 ]. This suggested that delayed blastocyst formation, which extended embryo culture time in vitro , might lead to a decline in embryo quality, causing epigenetic alterations or a reduction in mitochondrial DNA content [ 36 – 38 ]. In this study, we exclusively included the day 5 blastocyst transfer cycles, allowing for a more focused examination of the clinical and neonatal outcomes of different blastocyst transfer strategies. Our results demonstrated that the F3T5 group yielded improved clinical outcomes compared to the F5T5 group, without affecting the neonatal outcomes. This indicated that the F3T5 strategy could be a viable alternative for enhancing ART success rates. Future studies could further investigate the long-term effects of the embryo transfer strategy, including potential impacts on maternal health and the development of the offspring. Endometrial preparation is a key step for successful embryo implantation in FET. Currently, three commonly used regimens for endometrial preparation include the natural cycle, HRT, and ovulation induction cycle. However, the clinical outcomes of different endometrial preparation regimens remain controversial. Some studies suggested that the natural cycle resulted in a higher clinical pregnancy rate compared to the HRT cycle [ 39 ], while others indicated that the different endometrial regimens did not affect implantation rate, clinical pregnancy rate, and live birth rate [ 40 , 41 ]. In this study, despite using the PSM method to balance the baseline data of the included subjects, we still found statistical differences between the two groups regarding the endometrial preparation regimen. To account for the potential influence of different regimens on the study conclusions, we conducted a logistic regression analysis. Both univariate and multivariate logistic regression analyses confirmed that the endometrial preparation regimens did not influence the conclusions of the current study. This was in line with the previous study that the different endometrial regimens had no impact on the clinical outcomes [ 40 , 41 ]. This indicated that the improvement in clinical outcomes in the F3T5 group was not significantly related to the type of endometrial preparation regimen used. Our finding further suggested that different blastocyst transfer strategies may have a more significant impact on improving clinical outcomes. The miscarriage rate associated with the two blastocyst transfer strategies has not been conclusively determined. Some studies with smaller cohorts have suggested that transferring blastocysts derived from extended culture of frozen-thawed cleavage embryos were associated with a lower miscarriage rate compared to transferring the frozen-thawed blastocysts ( P < 0.05) [ 12 , 17 ]. However, other studies have shown that two blastocyst transfer strategies did not significantly impact miscarriage rates, although the data indicate that the miscarriage rate in the extended culture group is slightly lower [ 13 ]. In our study, after rigorously controlling for the age of the participants, we observed a slightly lower miscarriage rate in the F3T5 group (10.65% vs 15.28%, P = 0.16), which provided additional evidence supporting the potential advantage of an extended culture of frozen-thawed cleavage embryo to the blastocyst stage in reducing miscarriage rates. However, it remains uncertain whether the reduction in miscarriage rate is related to the repair of embryo damage during the vitrification and thawing process. While studies have shown that prolonged embryo freezing time is significantly negatively correlated with implantation and clinical pregnancy rates after blastocyst transfer, no significant correlation has been found regarding the miscarriage rate [ 20 ]. Larger-scale studies are still needed in future research to confirm these findings and draw definitive conclusions. However, our study still has some limitations. First, the current study is limited to a single-center retrospective study. Future studies may assess the clinical and neonatal outcomes of different blastocyst transfer strategies in multicenter, large-scale studies. Additionally, due to the retrospective nature of our study, some potential factors that may have influenced embryo implantation were not considered. Future research should include prospective studies to better investigate the impact of different blastocyst transfer strategies on clinical and neonatal outcomes. Furthermore, although some studies have confirmed that no significant correlation between the status of day 3 cleavage embryos and clinical outcomes after blastocyst transfer, our current study did not conduct a stratified analysis of the day 3 cleavage embryos’ status on the clinical outcomes. Future prospective studies should conduct a stratified analysis of the day 3 cleavage embryo status to further validate the conclusions of this study. 5. Conclusion In summary, our current study demonstrated that in the first transfer cycle, transferring blastocysts derived from the extended culture of frozen-thawed cleavage-stage embryos for single embryo transfer improved the biochemical pregnancy, clinical pregnancy rate, ongoing pregnancy rate, and implantation rate compared to the transferring of frozen-thawed blastocysts in women under 36 years of age, without affecting neonatal outcomes. Our findings suggested that extended culture of the frozen-thawed embryos might offer a beneficial approach for optimizing ART success rates in the potential populations. Declarations Author contributions Y.L.F.: Conceptualization, Supervision, and revision of the manuscript; W.L.Y.: data curation, acquisition, analysis and interpretation of data, and drafting and revision of the manuscript; J.W.L.: data curation, analysis and interpretation of data, and revision of the manuscript; L.Y.P.: data curation and drafting the manuscript; P.C.K.: data curation; X.X.Z.: conceptualization and revision of the manuscript. Acknowledgements We would like to thank the staff of the Center for Reproductive Medicine at Shanghai First Maternity and Infant Hospital for their cooperation and support. We would also like to thank the patients for participated in this study. Funding This study was partly supported by the Shanghai First Maternity and Infant Hospital, affiliated with Tongji University School of Medicine (grant numbers: 2025B23 to W.L.Y., 2023B13 to J.W.L., 2023B03 to Y.L.F., 2023B18 to X.X.Z., and 2020RC02 to Y.L.F.), Shanghai Pudong Science & Technology Development Foundation (grant number: PKJ2024-Y11 to Y.L.F.). Data availability statement The data underlying this article will be shared on reasonable request to the corresponding author. Competing interests Ethics approval and consent to participate The study was conducted in accordance with the Helsinki Principles, and the study protocol was revised and approved by the ethical committee and the institutional research board (NO: KS23230). Informed consent was waived for individual patients due to the retrospective design of the study. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References P.C. Steptoe, R.G. 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Lyu, Y. Kuang, Fertility and neonatal outcomes of embryos achieving blastulation on Day 7: are they of clinical value?, Human Reproduction 33 (2018) 1038–1051. https://doi.org/10.1093/humrep/dey092. J. Haas, J. Meriano, C. Laskin, Y. Bentov, E. Barzilay, R.F. Casper, K. Cadesky, Clinical pregnancy rate following frozen embryo transfer is higher with blastocysts vitrified on day 5 than on day 6, J Assist Reprod Genet 33 (2016) 1553–1557. https://doi.org/10.1007/s10815-016-0818-x. M. Bourdon, K. Pocate-Cheriet, A. Finet De Bantel, V. Grzegorczyk-Martin, A. Amar Hoffet, E. Arbo, M. Poulain, P. Santulli, Day 5 versus Day 6 blastocyst transfers: a systematic review and meta-analysis of clinical outcomes, Human Reproduction 34 (2019) 1948–1964. https://doi.org/10.1093/humrep/dez163. Y. Li, J. Wang, T. Sun, M. Lv, P. Ge, H. Li, D. Zhou, Pregnancy outcomes after day 5 versus day 6 blastocyst‐stage embryo transfer: A systematic review and meta‐analysis, J of Obstet and Gynaecol 46 (2020) 595–605. https://doi.org/10.1111/jog.14188. A.M. Klimczak, L.E. Pacheco, K.E. Lewis, N. Massahi, J.P. Richards, W.G. Kearns, A.F. Saad, J.R. Crochet, Embryonal mitochondrial DNA: relationship to embryo quality and transfer outcomes, J Assist Reprod Genet 35 (2018) 871–877. https://doi.org/10.1007/s10815-018-1147-z. U.-B. Wennerholm, C. Bergh, Perinatal outcome in children born after assisted reproductive technologies, Upsala Journal of Medical Sciences 125 (2020) 158–166. https://doi.org/10.1080/03009734.2020.1726534. H. Yarali, M. Polat, S. Mumusoglu, I. Yarali, G. Bozdag, Preparation of endometrium for frozen embryo replacement cycles: a systematic review and meta-analysis, J Assist Reprod Genet 33 (2016) 1287–1304. https://doi.org/10.1007/s10815-016-0787-0. E.R. Groenewoud, A.E.P. Cantineau, B.J. Kollen, N.S. Macklon, B.J. Cohlen, What is the optimal means of preparing the endometrium in frozen–thawed embryo transfer cycles? A systematic review and meta-analysis, Human Reproduction Update 19 (2013) 458–470. https://doi.org/10.1093/humupd/dmt030. Z. Kalem, M.N. Kalem, B. Bakirarar, E. Kent, T. Gurgan, Natural cycle versus hormone replacement therapy cycle in frozen-thawed embryo transfer, SMJ 39 (2018) 1102–1108. https://doi.org/10.15537/smj.2018.11.23299. Additional Declarations No competing interests reported. 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16:38:56","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148206,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8390695/v1/9ad286009e11b5f440f103aa.html"},{"id":99287424,"identity":"40ba3919-dcf5-4018-8c03-649bed408317","added_by":"auto","created_at":"2025-12-31 09:37:45","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":633318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe flowchart of the study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8390695/v1/1b2b837ad64b2647ab3a8db5.jpg"},{"id":99287427,"identity":"d03ad495-d1ca-40fe-b82e-b93d719aae63","added_by":"auto","created_at":"2025-12-31 09:37:45","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":741538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePropensity score matching for F3T5 group versus F5T5 group. A. \u003c/strong\u003ePropensity score distribution of F5T5 group before and after matching; \u003cstrong\u003eB. \u003c/strong\u003ePropensity score distribution of F3T5 group before and after matching; \u003cstrong\u003eC.\u003c/strong\u003eDistribution of standardized differences before and after matching. The results indicated the balance between the compared cohorts. F3T5: cycles involving blastocyst transfer derived from the extended culture of frozen-thawed cleavage-stage embryo; F5T5: cycles involving frozen-thawed blastocyst transfer.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8390695/v1/d3eb7fbe806209930ec5e35e.jpg"},{"id":99323941,"identity":"37c25d5a-f4e4-4930-8d7a-2f10f2d99ba5","added_by":"auto","created_at":"2025-12-31 16:46:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2264100,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8390695/v1/610c5b85-5080-470a-99f1-f0779e82c78b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improved pregnancy outcomes with single day 5 blastocyst transfer from post-thawed cleavage embryos in the first transfer cycle: a retrospective propensity score-matched cohort study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSince the birth of the first test tube baby in 1978, assisted reproduction technology (ART) has undergone over four decades of development [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. One of the key milestones in ART is embryo transfer, which has evolved into two primary methods: fresh embryo transfer and frozen embryo transfer (FET). In fresh embryo transfer, embryos are transferred during the same cycle as oocyte retrieval, while FET involves the transfer of embryos that were previously frozen and then thawed. FET has gained widespread adoption due to its potential advantages, including a reduced risk of ovarian hyperstimulation syndrome (OHSS) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], higher cumulative pregnancy rates [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and lower incidence of multiple pregnancies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As a result, the number of FET cycles has progressively increased and has surpassed that of fresh embryo cycles in many countries [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditionally, embryo transfers were performed on day 2 or 3, during the cleavage-stage, when embryos typically comprised four or eight blastomeres. This practice was based on the belief that the uterus provided the optimal environment for embryo survival [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, with the improvement in laboratory conditions and embryo culture medium, ART has shifted from cleavage-stage transfers to blastocyst transfers [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], where embryos are cultured to the blastocyst stage on day 5 or 6. The rationale for blastocyst transfer is better to align embryonic development with uterine receptivity, which facilitates the self-selection of viable embryos and has been shown to improve live birth rates [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In line with the government policies aiming to minimize the risk of multiple gestations by limiting the number of embryos transferred, single blastocyst transfer, whether fresh or via FET, has gained significant popularity due to its potential to improve outcomes and reduce complications.\u003c/p\u003e \u003cp\u003eDespite these advantages, concerns have been raised regarding the potential risks of cryopreservation. Some studies suggested that blastocysts might be more susceptible to damage during the vitrification and thawing process compared to cleavage-stage embryos [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This is attributed to the challenge of adequately permeating cryoprotectants into the blastocyst, which may result in cellular injury [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To address this concern, some researchers have proposed extending the culture of thawed cleavage-stage embryos to allow them to develop into blastocysts before transfer, potentially minimizing the risks associated with blastocyst cryopreservation while still achieving the benefits of blastocyst transfer [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, the clinical outcomes of this strategy, when compared to frozen blastocyst transfer, remained debated. Some studies have shown improved implantation rates and clinical pregnancy outcomes in the extended culture group [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], while others find no significant difference between the two approaches [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These discrepancies highlight the need for further research to fully assess the clinical benefits of this approach, including its effect on embryo implantation rates, live birth outcomes, and miscarriage rates.\u003c/p\u003e \u003cp\u003eIn the current study, we aimed to test the hypothesis that transferring a single blastocyst derived from the extended culture of cleavage-stage embryos would result in better clinical outcomes compared with transferring the frozen-thawed blastocysts in the first transfer cycle, with the goal of providing novel insights into optimizing embryo transfer strategies in ART.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study Population and Design\u003c/h2\u003e \u003cp\u003eThis retrospective cohort study was conducted at the Reproductive Medicine Center of Shanghai First Maternity and Infant Hospital from January 1st, 2019 to October 31st, 2024. We investigated the clinical and neonatal outcomes of women undergoing FET. We included patients who underwent a single Day 5 blastocyst transfer, considering only the first transfer cycle for those with multiple cycles. After excluding patients who underwent more than two transfer cycles, transfer multiple blastocysts, were over 35 years of age, received day 6/7 blastocysts or the early stage blastocysts transfer, underwent embryo transfer from preimplantation genetic testing (PGT) cycles, had an endometrial thickness less than 7 mm on the transfer day, had a history of recurrent spontaneous abortion (RSA), or were lost to follow-up, the remaining cycles were classified into two groups: the extended culture group (F3T5) and the frozen-thawed blastocyst group (F5T5). Finally, a total of 346 blastocyst transfer cycles in the F3T5 group, with a corresponding number of 1,407 transfer cycles in the F5T5 group. This study was approved by the ethics committee of the Shanghai First Maternity and Infant Hospital, and informed consent was waived for individual patients due to the retrospective design of the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Analysis\u003c/h2\u003e \u003cp\u003eWe compared the clinical outcomes between F3T5 and F5T5 blastocyst transfer groups using the propensity score matched (PSM) method. PSM analysis was performed using the MatchIt R package (v.4.5.5), with nearest neighbor matching (caliper 0.1) and a matching ratio of 1:2. The matching factor included patient characteristics at the time of transfer, such as maternal age, body mass index (BMI), endometrial thickness, infertility years, endometrial preparation regimen, infertility factors, type of infertility, and number of high-quality blastocysts transferred. After PSM matching, 307 blastocyst transfer cycles in the F3T5 group and 504 blastocyst transfer cycles in the F5T5 group were included for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Embryo Vitrification Freezing, Thawing, and Extending culture\u003c/h2\u003e \u003cp\u003eVitrification was used as the embryo cryopreservation method for cleavage-stage embryos and blastocysts. Freezing and thawing of cleavage-stage embryos and blastocysts were carried out according to the standard protocols of the Kitazato vitrification kit (Kitazato, Japan). For embryo freezing, grade I and grade II cleavage-stage embryos with cytoplasmic fragments less than 20% were exposed to equilibration solution (ES) for 7 minutes at room temperature, while blastocysts were exposed to ES for 9 minutes and then transferred to vitrification solution (VS) for 45\u0026ndash;60 seconds, and subsequently placed on the Cryotop strip (Kitazato, Japan). The strip, together with its protective sleeve, was then promptly immersed in the liquid nitrogen for cryopreservation.\u003c/p\u003e \u003cp\u003eFor embryo thawing, after removing the protective sleeve of the strip and directly immersed in 300\u0026micro;L of thawing solution (TS) at 37\u0026deg;C for 1 minute. Embryos were transferred to 200\u0026micro;L diluent solution (DS) for 3 minutes at room temperature and washed twice using washing solution for 5 minutes each time at room temperature. Finally, the embryos were stored in the G2-plus medium (Vitrolife, Sweden) and cultured at 37\u0026deg;C with a 6% CO\u003csub\u003e2\u003c/sub\u003e and 5% O\u003csub\u003e2\u003c/sub\u003e atmosphere until transfer. The post-thawed embryos with more than 50% intact blastomeres were considered viable and eligible for transfer or extended culture [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The thawed cleavage-stage embryos were cultured in G2-plus medium (Vitrolife, Sweden) for 48 or 72 hours at 37\u0026deg;C in an atmosphere with 6% CO\u003csub\u003e2\u003c/sub\u003e and 5% O\u003csub\u003e2\u003c/sub\u003e. According to the Gardner scoring system, high-quality blastocysts are defined as those at stage 3 or higher, with neither the inner cell mass nor trophectoderm scores assigned a grade of C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Endometrial Preparation and FET\u003c/h2\u003e \u003cp\u003eEndometrial preparation for FET was carried out using a natural cycle, ovarian induction cycle, or hormone replacement therapy (HRT), depending on the patient\u0026rsquo;s condition or the physicians\u0026rsquo; preference, as our previously reported [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Ultrasound-guided blastocyst transfer was conducted either on the fifth day after ovulation or on the sixth day after progesterone administration.\u003c/p\u003e \u003cp\u003eProgesterone supplementation was administrated to all patients as post-transfer luteal support. The serum β-hCG levels were assessed two weeks after embryo transfer to confirm pregnancy at our center. For patients with a positive pregnancy test, a transvaginal ultrasonographic was performed four weeks after embryo transfer to detect the presence of gestation sac and fetal heartbeat.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Outcome Measurement\u003c/h2\u003e \u003cp\u003eThe primary outcomes of this study were the implantation rate and ongoing pregnancy rate for each transferred cycle. Secondary outcomes included the biochemical pregnancy rate, miscarriage rate, clinical pregnancy rate, and neonatal outcomes. The implantation rate was calculated by dividing the number of gestational sacs detected via transvaginal ultrasonography by the number of embryos transferred. Biochemical pregnancy was considered positive if the serum beta-human chorionic gonadotropin (β-hCG) concentration exceeded 10 mIU/mL, measured 14 days after embryo transfer. Clinical pregnancy was defined as the presence of a gestational sac in the uterine cavity, with or without fetal heart activity, as well as the occurrence of ectopic pregnancies. Ectopic pregnancy was diagnosed when at least one ectopic pregnancy sac was detected by ultrasound. Ongoing pregnancy was defined as a pregnancy that had reached at least 12 weeks of gestation. Multiple pregnancy was defined as the presence of two or more gestational sacs identified through ultrasound. Miscarriage was defined as the loss of a clinical pregnancy within 28 weeks of confirming. For neonatal outcomes, preterm birth was defined as delivery before 37 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical\u003c/h2\u003e \u003cp\u003eAll data were analyzed using R (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.R-project.org\u003c/span\u003e\u003cspan address=\"http://www.R-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (v.4.2.3) with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Continuous variables were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), while categorical variables were presented as numbers with percentages. Comparisons of categorical variables were made using Fisher\u0026rsquo;s exact test or Chi-square test. The baseline characteristics and clinical outcomes of the PSM matched data were compared using the Student\u0026rsquo;s t-test for continuous variables and Fisher\u0026rsquo;s exact test or Chi-squared test for categorical variables. We used the logistic regression analysis to investigate the association between different endometrial preparation regimens and the clinical outcomes. The multivariable logistic regression model was adjusted for maternal age, BMI, endometrial thickness, infertility years, infertility factors, type of infertility, and number of high-quality blastocysts transferred. The associations of the univariate logistic regression model and multivariate logistic regression model were presented as odds ratios (OR) with a 95% confidence interval (CI).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Baseline Characteristics of Participants\u003c/h2\u003e \u003cp\u003eThe flowchart of the study is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A total of 1,753 FET cycles were analyzed, including 346 cycles in the F3T5 group and 1,407 cycles in the F5T5 group. In the F5T5 group, out of 1,453 blastocysts, 1,444 (99.38%) survived in the F5T5 group. In the F3T5 group, 947 out of 950 cleavage-stage embryos survived with at least half of the blastomeres intact (99.68%). From the 947 cleavage-stage embryos in the F3T5 group, 765 blastocysts were formed, yielding a blastocyst formation rate of 80.78%. Of these, 640 blastocysts were available, resulting in an availability rate of 83.66%, and 294 blastocysts were re-cryopreserved after transfer (346 transfer cycle). After establishing a balanced cohort using PSM, 307 transfer cycles in the F3T5 group were matched with 504 transfer cycles in the F5T5 group. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the distributions of propensity scores and standard differences both before and after matching, illustrating the balance between the two comparing groups. The baseline characteristics of the two groups were presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, with most characteristics comparable (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) except the endometrial preparation regimen (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of patients in F3T5 and F5T5 groups following propensity score matching\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF3T5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF5T5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCycles, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaternal age, y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.69\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody mass index, kg/m\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.27\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.19\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInfertility duration, y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of infertility, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimary infertility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSecondary infertility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInfertility diagnosis, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.052\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale factor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCombination of factors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnexplained\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometrial preparation regimen, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNatural cycle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOvarian induction cycle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHormone replacement therapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndometrial thickness, mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of high-quality blastocysts transferred, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e166 (52.35%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e274 (54.19%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003en: number; y: years; mm: millimeter; F3T5: extending culture group; F5T5: Frozen-thawed blastocyst transfer group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Clinical and Neonatal Outcomes Comparisons between the Two Groups\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the clinical and neonatal outcomes following blastocyst transfer in the F3T5 group, compared with those after the transfer of frozen-thawed blastocysts in the F5T5 group. The results demonstrated that the biochemical pregnancy rate (78.83% \u003cem\u003evs\u003c/em\u003e 66.07%), clinical pregnancy rate (70.36% \u003cem\u003evs\u003c/em\u003e 59.72%), implantation rate (70.36% \u003cem\u003evs\u003c/em\u003e 59.72%), and ongoing pregnancy rate (64.17% \u003cem\u003evs\u003c/em\u003e 50.99%) were significantly higher in the F3T5 group compared with the F5T5 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The multiple pregnancy rate and miscarriage rate were similar between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). A total of 172 cycles in the F3T5 group and 202 cycles in the F5T5 group were included in the neonatal outcomes. The incidence of twin births and preterm births was similar between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Additionally, in terms of birthweight for singletons, gestational weeks of birth, and pregnancy complications, both groups were similar (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Those findings collectively suggest that transferring the blastocysts derived from the extended culture of frozen-thawed cleavage-stage embryos for single embryo transfer in the first transfer cycle may yield better clinical outcomes than frozen-thawed blastocyst transfer without a significant difference in neonatal outcomes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePregnancy and neonatal outcome of the propensity matched data\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF3T5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF5T5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePregnancy outcomes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCycles, (n)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiochemical pregnancy rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e242 (78.83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e333 (66.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClinical pregnancy rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e216 (70.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e301 (59.72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0029\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMultiple pregnancy rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (3.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (1.99%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImplantation rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e216/307 (70.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e301/504 (59.72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEctopic pregnancy rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (1.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 (1.66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMiscarriage rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23 (10.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46 (15.28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOngoing pregnancy rate, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e197 (64.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e257 (50.99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeonatal outcomes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingle newborn, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e166\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e199\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwin newborns, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLive birth cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePreterm delivery, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTerm deliver, n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeek of birth, w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSingle birth weight, g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3259.91\u0026thinsp;\u0026plusmn;\u0026thinsp;495.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3356.33\u0026thinsp;\u0026plusmn;\u0026thinsp;532.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.074\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePregnancy complications, n (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (3.49)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9 (4.46)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003en: number; w: weeks; g: gram; F3T5: extending culture group; F5T5: Frozen-thawed blastocyst transfer group.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Association between Different Endometrial preparation Regimens and Clinical Outcomes in the two Groups\u003c/h2\u003e \u003cp\u003eGiven the retrospective nature of our study, we used PSM to balance the baseline characteristics between the two groups. Despite this, we still observed differences in the endometrial preparation regimen. To further investigate the association between the endometrial preparation regimen and the clinical outcomes, we performed a logistic regression analysis. The univariate logistic regression model indicated that different endometrial preparation regimens had no significant impact on the biochemical pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, and implantation rate (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings remained consistent after adjusting the potential confounders in the multivariate logistic regression model (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This suggested that the improved clinical outcomes observed in the F3T5 group were not influenced by the variations in the endometrial preparation regimens.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe logistic regression analyses of the association between different endometrial preparation regimens and clinical outcomes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOR (95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAdjusted OR (95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBiochemical pregnancy rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF5T5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF3T5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.91 (0.74\u0026ndash;1.20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.84 (0.67\u0026ndash;1.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClinical pregnancy rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF5T5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF3T3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.01 (0.83\u0026ndash;1.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.94 (0.77\u0026ndash;1.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eImplantation rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF5T5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF3T5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.01 (0.83\u0026ndash;1.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.94 (0.77\u0026ndash;1.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOngoing pregnancy rate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF5T5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF3T5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.01 (0.84\u0026ndash;1.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.93 (0.77\u0026ndash;1.13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the current study, we conducted a retrospective PSM analysis to evaluate the clinical and neonatal outcomes after the transfer of a single day 5 blastocyst from the extended culture of frozen-thawed cleavage embryos or from frozen-thawed blastocysts in women under 36 years of age. To the best of our knowledge, this is the first analysis of this issue in the certain population. Our findings suggested that the extended culture of frozen-thawed cleavage embryos to the blastocyst for single embryo transfer in the first transfer cycle could improve pregnancy outcomes in women under 36 years of age without affecting the neonatal outcomes. The above findings implicate a potential strategy for optimizing embryo selection and transfer protocols in this population.\u003c/p\u003e \u003cp\u003eFET plays an important role in ART. Since vitrification freezing was first introduced in China in 2003, this technology has advanced over the past 20 years [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Vitrification is a reliable and safe embryo cryopreservation method that has become widely utilized in ART due to its numerous clinical advantages [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Currently, numerous studies have examined the clinical outcomes of frozen-thawed blastocysts and cleavage-stage embryos [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, the clinical outcomes of transferring blastocysts derived from the extended culture of frozen-thawed cleavage embryos or frozen-thawed blastocysts remain a topic of ongoing debate [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In our study, after strictly controlling for potential confounders using the PSM method, we observed an improved biochemical pregnancy rate, clinical pregnancy rate, implantation rate, and ongoing pregnancy rate after transferring blastocysts derived from the extended culture of frozen-thawed cleavage embryos in women under 36 years, with no impact on neonatal outcomes. The blastocyst formation rate after extended culture of frozen-thawed cleavage embryos was 80.53%, consistent with previous findings [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, we do not attribute the improvement in clinical outcomes to an enhancement in the embryos\u0026rsquo; developmental potential, but rather to the following reasons. Compared to transferring frozen-thawed blastocysts, transferring blastocysts derived from the extended culture of the frozen-thawed cleavage-stage embryos more closely resemble a fresh cycle. This helps mitigate the negative effects associated with the true fresh embryo transfer cycle, such as the adverse impact of supra-physiological steroid hormones from ovarian superovulation on the endometrium, elevated estrogen levels, and the general anesthesia for oocyte retrieval, all of which could hinder embryo implantation [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, inadequate permeation of cryoprotectant into the blastocoel can lead to ice crystal formation and ultrastructural cellular damage to expanded blastocysts. Cryopreserving those blastocysts presents technical challenges, as they are more vulnerable to cryo-damage due to their advanced development and larger size [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The extended culture of frozen-thawed cleavage embryos might help repair the potential damage caused by vitrification [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Based on the above theory, we propose the hypothesis that transferring blastocysts derived from the extended culture of frozen-thawed cleavage embryos will result in better clinical outcomes compared to directly transferring frozen-thawed blastocysts. These findings were further supported by evidence showing a higher live birth rate after fresh embryo transfer compared to frozen embryo transfer in oocyte donor recipients who did not undergo ovarian stimulation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In conjunction with our neonatal outcomes, our study highlights that the extended culture of frozen-thawed cleavage embryos to blastocysts would improve clinical outcomes, particularly in women under 36 years.\u003c/p\u003e \u003cp\u003eAdvancing maternal age negatively impacts the success of ART [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. As women age, both the quantity and quality of the oocytes decline, leading to lower fertilization rates, poor embryo development, and a reduced likelihood of implantation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Research has shown that embryo implantation rates remain stable until the age of 35, after which they decrease significantly, with a linear decline of 2.77% per year (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.975) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To minimize the impact of maternal age on clinical outcomes, we included patients under 36 years of age. By focusing on this age group, we aimed to reduce variability and ensure that the data was not unduly influenced by age-related factors. This allows for a more accurate assessment of the pregnancy and neonatal outcomes in the F3T5 and F5T5 groups, providing a clearer understanding of the effectiveness of different embryo transfer strategies. After controlling for maternal age, we found that the F3T5 group demonstrated advantages in the biochemical pregnancy rate, clinical pregnancy rate, implantation rate, and ongoing pregnancy rate. These results highlight the importance of optimizing embryo transfer strategy to maximize pregnancy success.\u003c/p\u003e \u003cp\u003eCurrently, transfer blastocysts are divided into groups of 5, 6, and 7 days according to their developmental days. The implantation rate, clinical pregnancy rate, and live birth rate of day 7 blastocysts were significantly reduced compared to day 5 and day 6 blastocysts [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Compared to day 6 blastocysts, day 5 blastocysts transfer generally result in better clinical outcomes [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This suggested that delayed blastocyst formation, which extended embryo culture time \u003cem\u003ein vitro\u003c/em\u003e, might lead to a decline in embryo quality, causing epigenetic alterations or a reduction in mitochondrial DNA content [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In this study, we exclusively included the day 5 blastocyst transfer cycles, allowing for a more focused examination of the clinical and neonatal outcomes of different blastocyst transfer strategies. Our results demonstrated that the F3T5 group yielded improved clinical outcomes compared to the F5T5 group, without affecting the neonatal outcomes. This indicated that the F3T5 strategy could be a viable alternative for enhancing ART success rates. Future studies could further investigate the long-term effects of the embryo transfer strategy, including potential impacts on maternal health and the development of the offspring.\u003c/p\u003e \u003cp\u003eEndometrial preparation is a key step for successful embryo implantation in FET. Currently, three commonly used regimens for endometrial preparation include the natural cycle, HRT, and ovulation induction cycle. However, the clinical outcomes of different endometrial preparation regimens remain controversial. Some studies suggested that the natural cycle resulted in a higher clinical pregnancy rate compared to the HRT cycle [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], while others indicated that the different endometrial regimens did not affect implantation rate, clinical pregnancy rate, and live birth rate [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this study, despite using the PSM method to balance the baseline data of the included subjects, we still found statistical differences between the two groups regarding the endometrial preparation regimen. To account for the potential influence of different regimens on the study conclusions, we conducted a logistic regression analysis. Both univariate and multivariate logistic regression analyses confirmed that the endometrial preparation regimens did not influence the conclusions of the current study. This was in line with the previous study that the different endometrial regimens had no impact on the clinical outcomes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This indicated that the improvement in clinical outcomes in the F3T5 group was not significantly related to the type of endometrial preparation regimen used. Our finding further suggested that different blastocyst transfer strategies may have a more significant impact on improving clinical outcomes.\u003c/p\u003e \u003cp\u003eThe miscarriage rate associated with the two blastocyst transfer strategies has not been conclusively determined. Some studies with smaller cohorts have suggested that transferring blastocysts derived from extended culture of frozen-thawed cleavage embryos were associated with a lower miscarriage rate compared to transferring the frozen-thawed blastocysts (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, other studies have shown that two blastocyst transfer strategies did not significantly impact miscarriage rates, although the data indicate that the miscarriage rate in the extended culture group is slightly lower [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our study, after rigorously controlling for the age of the participants, we observed a slightly lower miscarriage rate in the F3T5 group (10.65% \u003cem\u003evs\u003c/em\u003e 15.28%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.16), which provided additional evidence supporting the potential advantage of an extended culture of frozen-thawed cleavage embryo to the blastocyst stage in reducing miscarriage rates. However, it remains uncertain whether the reduction in miscarriage rate is related to the repair of embryo damage during the vitrification and thawing process. While studies have shown that prolonged embryo freezing time is significantly negatively correlated with implantation and clinical pregnancy rates after blastocyst transfer, no significant correlation has been found regarding the miscarriage rate [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Larger-scale studies are still needed in future research to confirm these findings and draw definitive conclusions.\u003c/p\u003e \u003cp\u003eHowever, our study still has some limitations. First, the current study is limited to a single-center retrospective study. Future studies may assess the clinical and neonatal outcomes of different blastocyst transfer strategies in multicenter, large-scale studies. Additionally, due to the retrospective nature of our study, some potential factors that may have influenced embryo implantation were not considered. Future research should include prospective studies to better investigate the impact of different blastocyst transfer strategies on clinical and neonatal outcomes. Furthermore, although some studies have confirmed that no significant correlation between the status of day 3 cleavage embryos and clinical outcomes after blastocyst transfer, our current study did not conduct a stratified analysis of the day 3 cleavage embryos\u0026rsquo; status on the clinical outcomes. Future prospective studies should conduct a stratified analysis of the day 3 cleavage embryo status to further validate the conclusions of this study.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our current study demonstrated that in the first transfer cycle, transferring blastocysts derived from the extended culture of frozen-thawed cleavage-stage embryos for single embryo transfer improved the biochemical pregnancy, clinical pregnancy rate, ongoing pregnancy rate, and implantation rate compared to the transferring of frozen-thawed blastocysts in women under 36 years of age, without affecting neonatal outcomes. Our findings suggested that extended culture of the frozen-thawed embryos might offer a beneficial approach for optimizing ART success rates in the potential populations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.L.F.: Conceptualization, Supervision, and revision of the manuscript; W.L.Y.: data curation, acquisition, analysis and interpretation of data, and drafting and revision of the manuscript; J.W.L.: data curation, analysis and interpretation of data, and revision of the manuscript; L.Y.P.: data curation and drafting the manuscript; P.C.K.: data curation; X.X.Z.: conceptualization and revision of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the staff of the Center for Reproductive Medicine at Shanghai First Maternity and Infant Hospital for their cooperation and support. We would also like to thank the patients for participated in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partly supported by the Shanghai First Maternity and Infant Hospital, affiliated with Tongji University School of Medicine (grant numbers: 2025B23\u0026nbsp;to W.L.Y., 2023B13 to J.W.L., 2023B03 to Y.L.F., 2023B18\u0026nbsp;to X.X.Z., and 2020RC02 to Y.L.F.), Shanghai Pudong Science \u0026amp; Technology Development Foundation (grant number: PKJ2024-Y11 to Y.L.F.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data underlying this article will be shared on reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Helsinki Principles, and the study protocol was revised and approved by the ethical committee and the institutional research board (NO: KS23230). Informed consent was waived for individual patients due to the retrospective design of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eP.C. Steptoe, R.G. Edwards, Birth after the reimplantation of a human embryo, Lancet 2 (1978) 366. https://doi.org/10.1016/s0140-6736(78)92957-4.\u003c/li\u003e\n\u003cli\u003eY.-T. Wu, C. Li, Y.-M. Zhu, S.-H. Zou, Q.-F. Wu, L.-P. Wang, Y. Wu, R. Yin, C.-Y. Shi, J. Lin, Z.-R. Jiang, Y.-J. Xu, Y.-F. Su, J. Zhang, J.-Z. Sheng, W.D. Fraser, Z.-W. Liu, H.-F. 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Truong Dang, Pregnancy Outcomes of Frozen-Thawed Blastocysts versus Blastocysts Derived from Frozen-Thawed Cleavage Embryos: A Retrospective Study, Int J Fertil Steril 19 (2025) 264\u0026ndash;270. https://doi.org/10.22074/ijfs.2024.2036618.1739.\u003c/li\u003e\n\u003cli\u003eC. Roeca, R.L. Johnson, T. Truong, N.E. Carlson, A.J. Polotsky, Birth outcomes are superior after transfer of fresh versus frozen embryos for donor oocyte recipients, Human Reproduction 35 (2020) 2850\u0026ndash;2859. https://doi.org/10.1093/humrep/deaa245.\u003c/li\u003e\n\u003cli\u003eR.J. Van Kooij, C.W.N. Looman, J.D.F. Habbema, M. Dorland, E.R. Te Velde, Age-dependent decrease in embryo implantation rate after in vitro fertilization, Fertility and Sterility 66 (1996) 769\u0026ndash;775. https://doi.org/10.1016/S0015-0282(16)58634-8.\u003c/li\u003e\n\u003cli\u003eP. Devroey, H. Godoy, J. Smitz, M. Camus, H. Tournaye, M.P. Derde, A. Van Steirteghem, Female age predicts embryonic implantation after ICSI: a case-controlled study, Human Reproduction 11 (1996) 1324\u0026ndash;1327. https://doi.org/10.1093/oxfordjournals.humrep.a019380.\u003c/li\u003e\n\u003cli\u003eS.D. Spandorfer, P.H. Chung, I. Kligman, H.-C. Liu, O.K. Davis, Z. Rozenwaks, An Analysis of the Effect of Age on Implantation Rates, J Assist Reprod Genet 17 (2000) 303\u0026ndash;306. https://doi.org/10.1023/A:1009422725434.\u003c/li\u003e\n\u003cli\u003eT. Du, Y. Wang, Y. Fan, S. Zhang, Z. Yan, W. Yu, Q. Xi, Q. Chen, B.W. Mol, Q. Lyu, Y. Kuang, Fertility and neonatal outcomes of embryos achieving blastulation on Day 7: are they of clinical value?, Human Reproduction 33 (2018) 1038\u0026ndash;1051. https://doi.org/10.1093/humrep/dey092.\u003c/li\u003e\n\u003cli\u003eJ. Haas, J. Meriano, C. Laskin, Y. Bentov, E. Barzilay, R.F. Casper, K. 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Crochet, Embryonal mitochondrial DNA: relationship to embryo quality and transfer outcomes, J Assist Reprod Genet 35 (2018) 871\u0026ndash;877. https://doi.org/10.1007/s10815-018-1147-z.\u003c/li\u003e\n\u003cli\u003eU.-B. Wennerholm, C. Bergh, Perinatal outcome in children born after assisted reproductive technologies, Upsala Journal of Medical Sciences 125 (2020) 158\u0026ndash;166. https://doi.org/10.1080/03009734.2020.1726534.\u003c/li\u003e\n\u003cli\u003eH. Yarali, M. Polat, S. Mumusoglu, I. Yarali, G. Bozdag, Preparation of endometrium for frozen embryo replacement cycles: a systematic review and meta-analysis, J Assist Reprod Genet 33 (2016) 1287\u0026ndash;1304. https://doi.org/10.1007/s10815-016-0787-0.\u003c/li\u003e\n\u003cli\u003eE.R. Groenewoud, A.E.P. Cantineau, B.J. Kollen, N.S. Macklon, B.J. Cohlen, What is the optimal means of preparing the endometrium in frozen\u0026ndash;thawed embryo transfer cycles? A systematic review and meta-analysis, Human Reproduction Update 19 (2013) 458\u0026ndash;470. https://doi.org/10.1093/humupd/dmt030.\u003c/li\u003e\n\u003cli\u003eZ. Kalem, M.N. Kalem, B. Bakirarar, E. Kent, T. Gurgan, Natural cycle versus hormone replacement therapy cycle in frozen-thawed embryo transfer, SMJ 39 (2018) 1102\u0026ndash;1108. https://doi.org/10.15537/smj.2018.11.23299.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"middle-east-fertility-society-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mefj","sideBox":"Learn more about [High Temperature Corrosion of Materials](https://www.springer.com/journal/43043)","snPcode":"43043","submissionUrl":"https://submission.nature.com/new-submission/43043/3","title":"Middle East Fertility Society Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Blastocyst transfer, extended culture, frozen-thawed, clinical outcome, neonatal outcome","lastPublishedDoi":"10.21203/rs.3.rs-8390695/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8390695/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eWe aimed to test the hypothesis that transferring blastocysts derived from extended culture of cleavage-stage embryos for single embryo transfer in the first transfer cycle would result in better clinical outcomes than transferring frozen-thawed blastocysts, without affecting neonatal outcomes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe conducted a retrospective propensity score-matched cohort study at a single reproductive medicine center. After excluding transfer cycles that did not meet the inclusion criteria, we classified the subjects into two groups based on the blastocyst transfer strategies. The group receiving blastocysts derived from the extended culture of the frozen-thawed cleavage-stage embryos was labeled the F3T5 group, while the group receiving frozen-thawed blastocysts was labeled the F5T5 group. The primary outcomes of this study were the implantation rate and the ongoing pregnancy rate. Secondary outcomes included the biochemical pregnancy rate, miscarriage rate, clinical pregnancy rate, and neonatal outcomes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAfter propensity score matching, a total of 811 blastocyst transfer cycles were included in the analysis. 307 cycles in the F3T5 group and 504 cycles in the F5T5 group. The results revealed that the F3T5 group had significantly higher rates of biochemical pregnancy (78.83% \u003cem\u003evs\u003c/em\u003e 66.07%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), clinical pregnancy (70.36% \u003cem\u003evs\u003c/em\u003e 59.72%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029), implantation (70.36% \u003cem\u003evs\u003c/em\u003e 59.72%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036), and ongoing pregnancy (64.17% \u003cem\u003evs\u003c/em\u003e 50.99%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the F5T5 group. There were no statistically significant differences between the two groups in terms of multiple pregnancy rate, ectopic pregnancy rate, miscarriage rate, and neonatal outcomes such as gestational weeks, single birth weight, and pregnancy complications (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings suggested that transferring the single blastocyst derived from the extended culture of the frozen-thawed cleavage embryos in the first transfer cycle might offer a beneficial approach for optimizing assisted reproduction technology success rates in women under 36 years of age without affecting the neonatal outcomes.\u003c/p\u003e","manuscriptTitle":"Improved pregnancy outcomes with single day 5 blastocyst transfer from post-thawed cleavage embryos in the first transfer cycle: a retrospective propensity score-matched cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-31 09:37:41","doi":"10.21203/rs.3.rs-8390695/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-21T08:56:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T19:28:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-09T03:09:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277362337521342346301971000082224213805","date":"2026-01-04T06:27:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T21:15:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"43148995218071926037626334990808549760","date":"2025-12-31T14:04:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T09:38:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"209879955069963749491521120374667812809","date":"2025-12-30T12:16:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154583734065838595639085046602750797941","date":"2025-12-30T00:54:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5972509259900714038139072273955605362","date":"2025-12-29T12:20:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-29T12:15:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4669319825694866585478163277592000511","date":"2025-12-29T11:40:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-29T11:26:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-22T16:51:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-22T16:31:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Middle East Fertility Society Journal","date":"2025-12-18T03:36:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"middle-east-fertility-society-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mefj","sideBox":"Learn more about [High Temperature Corrosion of Materials](https://www.springer.com/journal/43043)","snPcode":"43043","submissionUrl":"https://submission.nature.com/new-submission/43043/3","title":"Middle East Fertility Society Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5926d2f-8e62-4b0a-99bb-ac53dcb94cf1","owner":[],"postedDate":"December 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-18T09:56:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-31 09:37:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8390695","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8390695","identity":"rs-8390695","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}