Assisted reproductive technology is in a great demand worldwide in light of the growing prevalence of infertility, and up to 1 in 25 babies in western countries is now conceived through assisted reproductive technology.[1,2] Frozen-thawed embryo transfer (FET) has been increasingly adopted in recent years as an important adjunct to in vitro fertilization (IVF) or intracytoplasmic sperm injection. FET can preserve embryos, avoid the supraphysiological levels of hormones in fresh cycles, provide better endometrial receptivity, and reduce the risk of ovarian hyperstimulation syndrome.[3–5] In addition, a recent multicenter randomized controlled trial by Chen et al[6,7] found that FET also increased the live-birth rate in women with polycystic ovary syndrome, leading to the suggestion that all embryos should be frozen prior to use.
However, the pregnancy hopes raised by this efficient treatment are accompanied by potential health concerns for the resulting offspring. Several studies have indicated that non-physiological stimulation during freezing and thawing may affect both genetic and epigenetic stabilities, with possible short- and/or long-term effects on the health and development of the offspring.[8,9] This review aims to summarize the impact of cryopreservation on the embryos, perinatal outcomes, and long-term health of the offspring, to help guide the next steps in designing clinical studies and exploring the potential mechanisms.
Effect of FET on embryos
The effect of cryopreservation on the preimplanted embryo has focused on genetic and epigenetic stabilities, gene expression, and cell structure. There are currently two widely used methods of embryo cryopreservation, namely slow-freezing and vitrification. Animal studies have demonstrated that embryos are frozen and thawed using either method showed significantly increased DNA fragmentation compared with non-cryopreserved embryos,[9,10] with vitrification having a more significant effect than slow-freezing. This DNA damage was positively correlated to the extent of blastocyst expansion during cryopreservation. The damage could be minimized but not eliminated by replacing propanediol in the culture medium with glycerol.
Epigenetic modification is another essential process for normal embryonic development. Both DNA methylation and histone modification could be altered by embryo cryopreservation.[8,12–14] Wang et al reported that methylation in the H19/Igf2 imprinting control region was reduced in 14-day-old mouse fetuses resulting from embryo vitrification. Similarly, promoter methylation of OCT4, NANOG, and CDX2 in mouse blastocysts was reduced after vitrification. However, the results were not consistent with those of Saenz-de-Juano et al, who found that DNA methylation levels of the OCT4 promoter in vitrified rabbit blastocysts were maintained by DNA methyltransferases (DNMTs). Petrussa et al compared the difference in nuclear DNMT3b expression between cryopreserved embryos on day 3 and fresh embryos and showed that DNMT3b expression in the cryopreserved embryos was delayed on days 5–6 and restored on day 6, indicating a time-dependent disturbance of DNMT expression caused by cryopreservation. Changes in histone modifications manifested as increases in H3K27me3 and decreases in H3K4me3 of 20% after slow-freezing of bovine blastocysts. However, these changes may have been attributable to the in vitro culture rather than to vitrification of the embryos. Notably, the above results were all drawn from animal studies, and further research is needed to verify the effects of FET in humans, including exploring the long-term developmental consequences.
Freeze-thawing has also been shown to affect the gene expression profiles of cryopreserved embryos. Seventy differentially expressed genes, including 24 upregulated and 46 downregulated, were identified in 6-day-old frozen rabbit blastocysts compared with in vivo-derived ones. These genes were involved in biological processes such as cellular organization, metabolic processes, and mitochondrial structure, and were considered to be related to decreased birth rates of embryos after cryopreservation. A previous study in mice indicated that gene expression levels were altered in both vitrified and fresh embryos, but to different extents. Expression levels of the Igf2 imprinted gene were decreased more in vitrified compared with fresh in vivo embryos. Shaw et al also confirmed the differential expression of genes critical for preimplantation embryonic development between frozen-thawed and fresh cleavage-stage embryos. However, another study suggested that self-healing of mRNA degradation may occur in these embryos along with extended culture durations.
Some other studies have reported an increase in abnormally shaped spindles in vitrified compared with fresh embryos in both mice and humans.[20,21] This could adversely affect normal cell division, and thus the subsequent development of the embryo. In summary, identifying the effect of cryopreservation on the embryo is critical to maintaining normal preimplantation embryonic development and thus improving embryo quality and pregnancy rates following IVF treatment.
Perinatal outcomes of patients following FET
Several recent meta-analyses have focused on the perinatal complications of FET in singleton pregnancies.[22,23] Compared with babies born after fresh embryo transfer (ET), singleton babies born after FET tended to be larger, manifesting as large-for-gestational age (LGA), high birth weight (> 4000 g), and very high birth weight (> 4500 g). They were accordingly at lower risks of being small-for-gestational age (SGA), low birth weight (< 2500 g), very low birth weight (< 1500 g), preterm delivery (< 37 weeks), and very preterm birth (< 32 weeks). However, there was no difference in perinatal mortality, congenital anomalies, or neonatal intensive care unit admission. A similar trend was found when FET babies were compared with naturally conceived singletons, indicating a 1.3- to 1.4-fold increase in the incidence of LGA and macrosomia in FET babies.
The above changes were apparently unaffected by the method or stage of embryonic development at cryopreservation. A retrospective cohort study compared perinatal outcomes between 11,644 slow frozen and 19,978 vitrified blastocysts, and found similar risks of preterm delivery, low birth weight, SGA, LGA, and perinatal mortality, consistent with a study on cleavage-stage embryos. Comparison between frozen cleavage-stage embryos and blastocysts also showed similar perinatal outcomes, including in relation to preterm birth, low birth weight, SGA, LGA, perinatal mortality, and congenital anomalies,[26–30] except for 1 meta-analysis that reported a higher risk of LGA in babies born from frozen blastocysts.
More evidence is needed, especially from prospective cohort studies, to determine if these early changes are related to the susceptibility to metabolic diseases in later life.
Long-term health outcomes and development of offspring conceived through FET
Studies on the long-term health outcomes and development of FET-offspring are limited. The main topics covered have been growth, neurodevelopment, chronic and common diseases, cancer, and imprinting-related diseases.
The first report on the growth of children produced via FET was published in 1998 by Wennerholm et al They followed up children born from frozen and fresh embryos and from spontaneous pregnancies for 18 months and found no significant difference in any growth parameters among 3 groups, for either singletons or twins. Likewise, Nakajo et al found that the physical and mental development of offspring from frozen embryos, fresh embryos, and naturally conceived children were all similar after the age of 6 months. However, a recent cross-sectional study with a relatively smaller sample size indicated that children from fresh embryos were taller and had more favorable lipid profiles compared with children from FET pregnancies and naturally conceived controls, whereas FET-offspring showed lower levels of high-density lipoprotein than control children. Further studies including more confounding factors, such as parental anthropometry, multiple births, and diet, are needed to elucidate the growth features in FET children.
Studies on neurodevelopment in children produced by FET have reached controversial conclusions. Most studies found that embryo cryopreservation had no significant effects on intelligence, neurological disorders, learning capability, or mental retardation.[31,34–37] In contrast, a population-based prospective cohort study in Sweden based on a 10-year follow-up of 2.5 million infants found that the use of frozen embryos after intracytoplasmic sperm injection was significantly associated with an increased risk of mental retardation compared with spontaneous conception. However, the incidence was low, and the increase in absolute risk was therefore small.
Chronic and common diseases
Two cohort studies, with follow-up durations of 18 months and 3 years respectively, found that early physical health was similar in children born after FET, fresh ET, and spontaneous pregnancies.[31,39] The incidence of common disorders, including upper and lower respiratory diseases, asthma and allergies, otitis, gastroenteritis and colitis, congenital or chromosomal disease, and neurological disease, did not differ among the groups.[31,39] There was no significant difference in the risk of hospital admission associated with embryo cryopreservation among IVF patients, but the risk was slightly increased in FET children compared with their naturally conceived counterparts during a 3-year follow-up. Regarding cancer, children born from cryopreserved embryos had no increased risk in general, but were associated with a small but significantly increased risk of histiocytosis.[35,40]
As mentioned above, cryopreservation might cause epigenetic changes in the embryos, and it is, therefore, essential to evaluate the risks of imprinting-related diseases to determine the safety of the technique. A national population-based cohort study in Denmark demonstrated no significant difference in the prevalence of birth imprinting-related diseases, such as Angelman syndrome and Russel-Silver syndrome, between FET children and fresh ET and naturally conceived children.
In general, the available evidence suggests that children born after embryo cryopreservation seem to be comparably healthy to those born after fresh ET or spontaneous conception, except for a slightly increased risk of mental disorders. However, the potential impacts of DNA damage, epigenetic changes, and gene expression profile alterations in FET embryos still raise the concern of possible adverse outcomes in later life. Well-designed prospective cohort studies including full consideration of related confounding factors, such as both paternal and maternal information, pregnancy complications, genetic background, lifestyle, and other environmental factors, are needed to evaluate the long-term safety of embryo cryopreservation.
SZ and LC contributed to the conception and design of the manuscript. ZJC revised the manuscript. All authors gave their final approval of the version to be published.
Conflicts of interest
The authors declare no conflicts of interest.
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