Pregnancy Outcomes of Frozen-Thawed Blastocysts versus Blastocysts Derived from Frozen-Thawed Cleavage Embryos: A Retrospective Study.

Millions of couples throughout the world struggle with infertility. Fortunately, in vitro fertilization (IVF) followed by the transfer of frozen embryos (FET) has revolutionized assisted reproductive technologies (ART), offering hope to couples facing infertility challenges. Nowadays, embryo cryopreservation is a crucial aspect of IVF ( 1 ). It offers several advantages for couples undergoing fertility treatments. For example, when the embryos are cryopreserved, patients can delay their pregnancy. This flexibility can be beneficial for personal reasons and reduce the risk of ovarian hyperstimulation syndrome (OHSS). Additionally, cryopreservation enables the storage of surplus embryos, providing multiple opportunities for embryo transfer without the need for repeated IVF cycles. Furthermore, cryopreserved embryos can undergo preimplantation genetic testing (PGT), allowing for the selection of normal embryos, reducing the risk of genetic disorders. It is worth noting that the transfer of fresh or frozen embryos offers the same ongoing pregnancy rate and live birth rate ( 2 ). In the early day, cryopreservation and transfer of cleavage embryo on day 3 after fertilization is the most common approach ( 3 ). Consequently, numerous problems occurred where an extensive number of cleavagestage embryos were frozen. In some cases, three or more cleavage embryos were vitrified together on a single cryotop. However, with recent advancements in extended embryo culture, blastocyst cryopreservation and transfer become more popular ( 4 ). Notably, only a small percentage of fertilized eggs develop to the blastocyst stage, approximately 32% ( 5 ). Therefore, culturing and observing embryos over a longer period plays as a natural selection to identify the most competence embryo for transfer. This approach helps reduce the number of vitrified embryos, subsequently lowering costs. Moreover, extended embryo culture aligns with the principles of a single embryo transfer strategy ( 6 ). Indeed, many scientists highlighted the advantages of single blastocyst transfer, including higher pregnancy rate and lower the risk of multiple pregnancy ( 7 ). Blastocyst cryopreservation is not without limitations. For example, the blastocyst cryopreservation process itself can be technically challenging. A study by Mukaida et al. ( 8 ) confirmed that artificial shrinkage is necessary to collapse the expanded blastocysts before vitrification in order to improve the survival (97.2%) and pregnancy rate (60.2%). This conclusion was further confirmed by a meta-analysis by Boyard et al. ( 9 ). Furthermore, the stage of embryo development with the highest cryo-survival rate remains unclear. Limited data are available regarding the vulnerability of embryos at different stages during the vitrification and thawing processes. Notably, Solumets et al. have suggested that embryos at the cleavage stage may experience more damage during the freezing-thawing process compared to those at day 2 ( 10 ). In recent times, an increasing number of patients choose extended culture to enhance their chances of achieving pregnancy. This choice simultaneously addresses concerns related to the extensive freezing of cleavage-stage embryos and associated costs. Additionally, there is a growing inquiry into potential damage to expanded blastocysts during the vitrification procedure ( 9 ). Given these circumstances, the strategy of cryopreserving cleavage-stage embryos, followed by thawing and culturing them to the blastocyst stage, appears to offer promising advantages over the direct transfer of a thawed blastocyst. However, limited data exist regarding the outcomes of frozen-thawed cleavage-stage embryos that undergo extended culture to reach the blastocyst stage. Could this method present an improved approach for frozen embryo transfer? Consequently, our study is designed to address this question by comparing the pregnancy outcomes of two distinct groups. The D5 group involves frozen and thawed blastocysts, while the D3-5 group comprises embryos that were frozen and thawed at the cleavage stage, subsequently extended to the blastocyst stage for transfer.

Table 1 details the characteristics of embryo transfer cycles within the D3-5 group. The primary reason for cycle cancellations was the absence of developed blastocysts for transfer. No cycle cancellations were observed in the D5 group. After propensity score matching in a 1:1 ratio, 58 matched cycles for each group were included in the analysis. Flowchart for patient eligibility. Characteristics of embryo transfer cycle of the D3-5 group Data are presented as n (%). D3-5; Thawed cleavage-stage embryo cultures to blastocyst. Table 2 compares the baseline characteristics of the D3-5 group and the D5 group after propensity-score matching. This method ensures that the groups are statistically comparable, reducing biases and confounding variables that might affect the outcomes. There are no significant differences in age, BMI, AMH, number of oocytes retrieved, number of mature oocytes, infertility reasons, number of blastocysts transferred, and the quality of transferred blastocysts. These results suggest that the baseline characteristics were well-matched. Table 3 provides a detailed comparison of pregnancy outcomes between the D3-5 group and the D5 group. These results indicate that there are no significant differences in the key pregnancy outcomes between transferring blastocysts derived from frozen-thawed cleavage embryos and transferring frozen-thawed blastocysts. Baseline characteristics of the D3-5 and D5 groups after propensity-score matching Data are presented as mean ± SD or n (%). A P<0.05 was considered significant. SD; Standard deviation, D3-5; Thawed cleavage-stage embryo cultures to blastocyst, and D5; Thawed blastocyst. Outcomes of the D3-5 and D5 groups after prospensity-score matching Data are presented as n (%). A P<0.05 was considered significant. D3-5; Thawed cleavage-stage embryo cultures to blastocyst, D5; Thawed blastocyst, hCG; Human chorionic gonadotropin, OR; Odd ratio, and CI; Confidence interval.

In this study, we compared the pregnancy outcomes and efficiency of two approaches: transferring blastocysts derived from frozen-thawed cleavage embryos (D3-5 group) versus transferring frozen-thawed blastocysts (D5 group). Our findings demonstrated that the D5 group had comparable pregnancy outcomes to the D3-5 group, with no significant differences in positive hCG rate, clinical pregnancy rate, ongoing pregnancy rate, live birth rate, or pregnancy failure rate. These results are consistent with previous studies ( 3 , 14 ). However, a notable finding was that a significant proportion of cycles in the D3-5 group (65.3%) were cancelled, primarily due to the absence of developed blastocysts for transfer (85.3%). This aspect of higher cancellation rates is not as extensively discussed in other studies but is crucial for considering the efficiency and patientfriendliness of the procedure. The high cancellation rate observed in the D3-5 group can be attributed to several key factors. In the D5 group, all embryos were cultured to the blastocyst stage prior to freezing, allowing natural selection across the entire embryo population and increasing the likelihood of obtaining a viable blastocyst for transfer. In contrast, the D3-5 approach involves thawing and culturing only a limited number (typically 2 or 3) of cleavage-stage embryos per cycle, selected based on quality indicators at the cleavage stage. However, cleavage-stage morphology has a lower predictive value for blastocyst development than assessments at later stages, introducing variability in outcomes. Additionally, the higher cancellation rate in the D3-5 group is due to differences in the developmental stage at freezing and the additional selection pressure for blastocyst formation. In the D3-5 group, cleavage-stage embryos were frozen, thawed, and cultured for two additional days to reach the blastocyst stage. This process introduces an additional selection step, as only embryos capable of progressing to high-quality blastocysts are ultimately eligible for transfer. The high cancellation rate due to the absence of developed blastocysts suggests that the process of deriving blastocysts from frozen-thawed cleavage embryos may present significant challenges. Patients invest emotional energy, time, and hope into the FET process, and a canceled embryo transfer can evoke feelings of disappointment and stress. In addition, FET cycle preparation can be costly, and the cancellation of a cycle may impose an additional financial burden due to further treatments. Furthermore, patients might need to readjust their schedules, considering the delay and potential changes to future treatment plans. The relatively lower number of actual embryo transfer cycles (34.7%) compared to the total number of participants also points out the need for better selection criteria or enhanced protocols to increase the likelihood of achieving viable blastocysts ready for transfer. This might include optimizing the thawing process, improving culture conditions, or selecting patients with higher chances of success based on their individual characteristics ( 15 - 17 ). The D3-5 approach may increase workload for embryologists. This aspect is also not discussed in the existing literature. According to a recent report, there is a downward trend in the workload that an embryologist can effectively manage ( 18 ). Considering the necessary time for FET preparation, each FET cycle demands 20 minutes ( 19 ). If a patient in the D3-5 group ends up with no viable blastocyst for transfer, the entire preparation process and workload become futile. Concerning the manipulation of blastocysts, the vitrification process alone takes 38.5 minutes ( 13 ). It has been reported that about 40-52% of embryos developed to the blastocyst stage ( 14 ). Consequently, cryopreserving of day-3 embryos may lead to double working time for embryologist. In contrast, the D5 method - transferring frozenthawed blastocysts - presents advantages in terms of reliability and efficiency. Since embryos are cultured to the blastocyst stage before freezing, the entire embryo cohort undergoes selection, reducing the risk of cycle cancellation due to lack of viable blastocysts. This approach is less likely to disrupt the treatment timeline, and with the preparation for blastocyst transfer occurring directly, patients face lower risks of cycle cancellations. However, the D5 method has its own limitations. The cryopreservation of expanded blastocysts can present technical challenges, as blastocysts are more sensitive to cryo-damage due to their advanced development and larger volume. It is noteworthy that some studies also support the utilization of transferring blastocysts derived from frozen-thawed cleavage embryos ( 20 , 21 ). This support may be attributed to overcoming potential damage to the blastocyst during cryopreservation. Briefly, insufficient permeation of cryoprotectant inside the blastocoel can lead to some ice crystal formation and ultrastructural cellular damage to expanded blastocysts ( 22 ). The process of cryopreserving at a cleavage stage, followed by thawing and culturing for two additional days in fresh media, may help avoid potential expanded blastocoelic damage prior to vitrification. In our perspective, despite the growing concern about blastocyst cryopreservation, current solutions such as the artificial shrinkage technique and optimization of vitrification protocols seem capable of overcoming this obstacle ( 8 , 9 ). Our study has several strengths. We provided detailed exploration of various clinical and laboratory parameters, offering a comprehensive understanding of the factors influencing the choice between the two approaches. The use of propensity-score matching helps to create comparable groups and reduce bias. This methodological approach enhances the validity of the comparisons made between the two embryo transfer strategies. However, the study is not without limitations. The sample size, although matched, was relatively small. Although propensity-score matching was used to reduce bias and improve comparability between the D3-5 and D5 groups, larger studies are needed to confirm these findings. Additionally, the retrospective design could introduce selection bias despite the propensity-score matching. The lack of detailed information on other potential confounding factors, such as patient lifestyle factors, is another limitation. Moreover, the study was conducted at a single center, raising concerns about the external validity and the generalizability of the results. Our study suggests that while transferring blastocysts derived from frozen-thawed cleavage embryos is not inferior to transferring frozen-thawed blastocysts in terms of pregnancy outcomes, this approach may not be as efficient or patient-friendly due to the high cancellation rates. This finding has important implications for clinical practice, suggesting that the direct transfer of frozen-thawed blastocysts might be a more reliable and less burdensome option for patients. Future research with larger, multicenter cohorts would enhance the statistical power and offer more generalizable insights into the benefits and limitations of each approach. Additionally, studies should consider long-term follow-up to assess the health outcomes of children born from these different methods. Research exploring patient preferences and experiences with these embryo transfer methods would also be valuable.

Our study suggests that the strategy of culturing frozen-thawed cleavage embryos for two days and transferring them as blastocysts yield comparable pregnancy outcomes to transferring frozen-thawed blastocysts. However, this approach increases the workload for embryologists and poses a significant risk of cycle cancellations. We propose that the direct use of frozen-thawed blastocysts may be a more efficient and patient-friendly option. Given the study’s retrospective design, single-center setting, and relatively small sample size, these findings should be interpreted with caution. Larger, prospective, multicenter studies are needed to confirm these findings and contribute to the refinement of IVF protocols, ultimately optimizing patient outcomes.

This retrospective observational cohort analysis was conducted at Hung Vuong Hospital between January 2022 and December 2023. We received ethical approval from the Committee of Hung Vuong Hospital (CS/HV/24/23). We included patients aged 18-45 who underwent IVF followed by FET cycles and had no history of uterine interventions or significant medical conditions. The D3-5 group comprised embryos that were frozen and thawed at the cleavage stage, subsequently extended two more days of culturing to the blastocyst stage for transfer. The D5 group involved frozen and thawed blastocysts. Patients with uterine factor infertility (e.g., significant uterine anomalies, severe intrauterine adhesions), or a history of uterine surgery were excluded. We also excluded donor cycles or surrogacy, and cycles that involved preimplantation genetic testing. We collected information regarding patients demographic, clinical characteristics, and laboratory parameters. Relevant data included patient age, body mass index (BMI), anti-Mullerian hormone (AMH) levels, infertility reasons, number of oocytes retrieved, number of mature oocytes, number of good-quality embryos, fertilization rate, blastulation rate, and details of the embryo transfer cycles. Embryos were graded at the cleavage and blastocyst stages based on established morphological criteria ( 11 ). Regarding pregnancy outcomes, we compared positive hCG (human chorionic gonadotropin) rate, clinical pregnancy rate, ongoing pregnancy rate, live birth rate, and pregnancy failure rate between the two groups. We employed artificial hormone replacement to prepare the endometrium for the transfer cycle. Specifically, from the second to the third day of the cycle, patients initiated a regimen of 2 mg oral estradiol (Progynova, Bayer, Germany) twice daily. The dosage was increased by 4 mg every five days, with adjustments based on ultrasound and follow-up examinations. A maximum dosage of 16 mg daily was used, tailored to each patient’s needs. Over the subsequent 14-16 days, transvaginal sonography was utilized to evaluate the pattern and thickness of the endometrium. Once the endometrial thickness achieved a range of 8 to 14 mm with a triple-line pattern, luteal support began with the vaginal administration of progesterone, utilizing Utrogestan 200 mg (Capsugel Ploermel, France). The progesterone administration was defined as P+0, and embryo transfer was conducted after a complete five-day of progesterone administration (P+5). We used vitrification method for embryo cryopreservation. The freezing and thawing process were performed using Kitazato medium kit, following manufacture’s instruction ( 12 ). Regarding the D3-5 group, thawed cleavage embryos were cultured for two more days until blastulation and transfer. Regarding the D5 group, blastocysts transfer was performed at least 2 hours after warming. The embryo transfer procedure was performed under abdominal ultrasonographical guidance. Pregnancy outcomes were recorded as follows ( 13 ): The positive hCG rate was defined as the proportion of cases with a positive hCG test relative to the total number of cases. The clinical pregnancy rate was defined as the number of cases in which a viable fetal heartbeat and a crown-rump length (CRL) of ≥7 mm were confirmed at 7 to 8 weeks’ gestation, divided by the total number of cases. The ongoing pregnancy rate was defined as the proportion of pregnancies continuing beyond 12 weeks’ gestation. The live birth rate was calculated as the number of live births relative to the total number of cases. Pregnancy failure was defined as the proportion of cases resulting in pregnancy loss at any stage, divided by the total number of cases. The cancellation rate was defined as the number of cycles in which no embryo transfer occurred, divided by the total number of initiated cases. We used R software to analyze data. Descriptive statistics were used to summarize patient characteristics. Continuous variables were presented as means with standard deviations, and categorical variables as frequencies and percentages. Statistical comparisons between the D3-5 and D5 groups were performed using t test for continuous variables and Chi-squared test for categorical variables. A P<0.05 was considered statistically significant. Propensity-score matching was performed to achieve a balanced cohort by controlling for potential confounding variables. The matching ratio was 1:1. The matching factors included patient characteristics (age, BMI, and AMH), along with the number of blastocysts transferred, the number of high-quality blastocysts transferred, and the number of good-quality embryos. Figure 1 illustrates the flowchart for patient eligibility. A total of 509 patients were included in the study. Of these, the D3-5 group consisted of 167 patients who underwent embryo transfer with frozen-thawed cleavage embryos, which were cultured for 2 days before being transferred as blastocysts. The D5 group included 342 patients who received direct frozen-thawed blastocysts. In the D3-5 group, 109 (65.3%) patients were further excluded.