In recent years, embryo cryopreservation has played an increasingly important role in assisted reproductive technologies. This technique preserves embryos to increase the cumulative pregnancy rate of a single ovarian stimulation cycle, helps avoid unfavorable embryo transfer timings due to poor endometrial receptivity and high risk of ovarian hyperstimulation, and improves the pregnancy rate of a single transplant. Embryo cryopreservation has been used for 38 years and has been widely recognized by domestic and foreign reproductive centers [1]. Frozen-thawed embryo transfer (FET) cycles can achieve similar pregnancy and perinatal outcomes, and similar offspring health status as fresh embryo transfer cycles [2, 3]. Currently, vitrification is the most widely adopted embryo cryopreservation technique.

However, in clinical practice, resuscitated embryos are occasionally frozen again on the same day to be thawed and transferred into the uterus in the subsequent cycle due to special patient circumstances such as sudden diarrhea, fever, and vaginitis. Currently, there are limited and controversial data on FET outcomes of embryos undergoing repeated cryopreservation-warming cycles, especially cleavage-stage embryos. Therefore, the impact of repeated cryopreservation-warming procedures on clinical pregnancy and perinatal outcomes needs to be further elucidated. In this study, we retrospectively analyzed the pregnancy and perinatal outcomes of repeated cryopreservation-warming single cleavage-stage embryo transfers at our center from 2017 to 2021.

Embryo cryopreservation is an important part of assisted reproductive therapy. The earliest method for cryopreservation of human embryos was “slow freezing” to ensure the safety of the embryo through gradual cooling. However, this process can form crystals that may damage the cell structure, resulting in a poor embryo survival rate. Vitrification, also known as rapid freezing, is based on rapid cooling ($>$20,000 ${}^{\circ }$C/min) combined with a high concentration of cryoprotectant. In this process, the water in cells is quickly removed, and the cell components instantly turn into highly viscous solid amorphous glassy substances, which prevents the formation of ice crystals in cells and greatly reduces the possibility of cell damage. Vitrification has greatly increased the safety of embryo cryopreservation and is a simple, time-saving operation with a good resuscitation effect unlike the slow freezing method. Current evidence indicates that vitrification-based FET cycles have success rates similar to those of fresh embryo transfer cycles [5]. Therefore, vitrification is gradually becoming the mainstream method of embryo cryopreservation worldwide.

Repeated freezing-thawing is often necessary to avoid wasting human embryos. However, the developmental potential of embryos subjected to repeated freezing and thawing could be impaired despite the use of vitrification, a safe cryopreservation protocol. At present, most studies have observed that the pregnancy rate of twice-vitrified embryo transfer is comparable to the implantation and pregnancy rates of once-vitrified embryo transfer, which supports the safety and reliability of repeated vitrification [6, 7, 8, 9, 10]. In contrast, Aluko et al. [11] have observed that in the PGT-A (preimplantation genetic testing for aneuploid cycle), biopsied blastocysts subjected to double vitrification had lower clinical pregnancy and live birth rates than once-vitrification blastocysts. Wang et al. [12] have shown that the implantation, clinical pregnancy, and live birth rates of twice-vitrified embryos were significantly lower than those of once-vitrified embryos, and the miscarriage rate was slightly higher. Additionally, their logistic regression analysis showed that repeated cryopreservation could increase the risk of embryo implantation failure (OR ratio = 1.79) [12].

These contradictory findings suggest that further studies are needed to verify the effects of repeated vitrification on the reproductive potential of embryos. Furthermore, a majority of the current research focuses on the blastocyst FET cycle. In this study, we assessed the effect of repeated vitrification on the outcome of FET in cleavage-stage embryos and found that the clinical pregnancy, early miscarriage, and live birth rates after the transfer of twice-vitrified cleavage embryos were similar to those of once-vitrified cleavage embryos. Our study suggests that repeated cryopreservation of cleavage-stage embryos may not reduce the success rate of single embryo transfer. Nevertheless, there is evidence suggesting that cryopreservation may impair the physiological function of embryos; studies have suggested that high concentrations of cryoprotectants used in vitrification may cause toxic effects and osmotic shock in embryos [13]. Wang et al. [12] observed that embryos were morphologically intact after repeated cryopreservation; however, they suggested that the ultrastructure of the embryo cells might be damaged. Shaw et al. [14] observed differences in gene expression between frozen and fresh embryos. Animal studies have also shown that repeated freezing-thawing procedures can lead to an abnormal increase in chromosomal ploidy in oocytes or embryos [15, 16]. To date, the safety of repeated vitrification of embryos has not been fully established, and its impact on embryonic developmental potential remains to be verified. Therefore, the decision for repeated cryopreservation of cleavage-stage embryos must be made cautiously after informing the patient of the benefits and disadvantages of the procedure.

We noted that in the twice-cryopreserved group, the survival rate of the initial cryopreservation–warming cycles was slightly lower than that of the second cryopreservation–warming cycles (96.8% vs 100.0%), although the difference was not statistically significant. In a study based on blastocyst transfer cycles, it was also observed that the survival rate of the initial cryopreservation–warming cycles was lower than that of the second ones (94.6% vs 98.9%) [11]. This may be because the most vulnerable embryos may not survive the first thawing. Twice freezing-thawing cycles may have led to twice viability screening on the embryos, which made us wonder whether this mitigated the negative effects of cryopreservation on pregnancy outcomes in this group. This is an interesting question that deserves further exploration.

Additionally, offspring safety is crucial in the field of assisted reproductive technology. Therefore, perinatal outcomes are an important measure of the safety of repeated cryopreservation procedures. In this study, a total of 46 singletons were successfully delivered, of which, 11 were from the twice-cryopreserved group. We analyzed sex, gestational age, birth weight, cesarean section, and neonatal complications, including preterm birth, low-birth-weight, and birth defect rates, and found no significant differences between the two groups. Our results are consistent with previous studies suggesting that repeated cryopreservation does not negatively affect neonatal outcomes [12, 17, 18]. However, these studies are limited and have small sample sizes; therefore, studies with larger sample sizes are needed to determine whether repeated cryopreservation is a perinatal risk factor for neonatal health.

Notably, we observed a significant incidence of pregnancy complications/clinical pregnancy in the twice-cryopreserved group, such as pregnancy-induced hypertension, gestational diabetes mellitus, and premature rupture of membranes in the twice-cryopreserved group, although not statistically significant. Evidence from published studies currently does not support the association of embryo cryopreservation with the risk of gestational diabetes mellitus and premature rupture of membranes, but the relation between cryopreservation and Pregnancy-induced hypertension has been well established [19, 20, 21]. Opdahl et al. [22] compared the prenatal outcomes of 6444 singleton pregnancies after FET and 268,599 singleton pregnancies after natural pregnancy, and found that the risk of preeclampsia was significantly increased in the cryopreservation group (Odds ratio 1.41, 95% confidence interval (CI) 1.27–1.56). A multicenter randomized controlled trial based on a Chinese population also showed a significantly increased risk of preeclampsia in FET patients (Risk ratio 3.13, 95% CI 1.06–9.30, p = 0.029) [23]. The reasons for the increased risk of gestational hypertension in FET transplant cycles are unclear. Some scholars put forward some hypotheses for this: (1) the absence of corpus luteum in hormone replacement cycles may lead to a deficiency of vasoactive products such as relaxin, leading to an increased risk of abnormal maternal cardiovascular adaptation to pregnancy and subsequent increased risk of preeclampsia [24]; (2) the trophectoderm may be damaged during vitrification and warming, leading to abnormal placenta formation and thus an increased risk of preeclampsia [25]; (3) prematurely elevated estradiol may inhibit trophectoderm invasion of the uterine spiral arteries, thereby increasing the risk of preeclampsia during FET [26]; (4) embryo freezing-thawing procedure may cause the embryonic epigenetic/transcriptomic alterations and reduce DNA integrity, contributing to the happening of preeclampsia [27, 28, 29]. Whether the risk of pregnancy-induced hypertension increases with the times of embryo freezing-thawing cycles is an interesting question, which deserves further confirmation.

Currently, there is some evidence that some assisted reproduction procedures, such as PGT and blastocyst transfer, may increase the possibility of producing male offspring [30, 31]. Therefore, it is necessary to explore the effect of repeated cryopreservation on the sex ratio. The study by Nagata et al. [32] showed that fresh or frozen embryo transfer did not affect the sex ratio of IVF live births. In our study, we observed that there is a higher number of males in the twice-cryopreserved group, although not statistically significant. Unfortunately, there were too few live births in our study to draw reliable conclusions (only 11 live births in the twice-cryopreserved group), and no other studies have explored the effect of repeated cryopreservation on sex ratios. Whether the Y chromosome can help the survival of those embryos after repeated cryopreservation cycles are worth further exploration in future studies.

Previous studies on repeated cryopreservation of embryos mostly focused on blastocyst transfer cycles. However, our study revealed that for cleavage-stage embryos, which are more fragile than blastocysts, repeated cryopreservation may not reduce the implantation potential of embryos, and there was no significant negative impact on perinatal outcomes. However, because repeated cryopreservation of embryos is a relatively rare event in clinical practice, the conclusions of this study are limited by the small sample size of the twice-cryopreserved group and the fact that the study is retrospective. Moreover, we were unable to distinguish some confounding factors related to pregnancy outcomes, including infertility type, infertility cause, embryo morphology score, insemination method, endometrial preparation protocol, and the duration of cryopreservation. Large-scale prospective studies with a long-term follow-up are needed in the future to verify the feasibility and safety of repeated cryopreservation and explore its long-term impact on offspring health outcomes.

This study suggests that repeated cryopreservation may not negatively affect the pregnancy and perinatal outcomes of single cleavage embryo transfer and may be a safe option for resuscitated cleavage embryos that have been canceled for transfer. However, the reliability of the conclusions of this study is limited by the small sample size and its retrospective nature. Hence, the results need to be confirmed in future studies.

YW—Conceptualization, Formal Analysis, Project Administration, Resources, Writing – Review & Editing; DC—Data Curation, Investigation, Methodology, Validation, Writing – Original Draft Preparation; BC—Writing – Review & Editing; DH—Data Curation, Formal Analysis; YX—Supervision; CD—Supervision, Writing – Review & Editing. All authors read and approved the final manuscript.

This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University and conducted in strict accordance with the Helsinki Declaration (No 2022481). The patient data required for this study were obtained from the center database and analyzed anonymously. The requirement for informed consent from the involved couples was waived because of the retrospective nature of the study.

The authors thank Taylor and Francis Editing Services for English language editing.

This research received no external funding.

The authors declare no conflict of interest.