The Effect Of Blastomeres Loss In Human Early Cleavage Stage Frozen-Thawed Embryo On The Safety Of Offspring

Since the first child after frozen-thawed embryo transfer (FET) was born in Britain in1984, embryo cryopreservation technology has made feasible the achievement of pregnancy from human frozen-thawed zygotes, cleavage stage embryos and blastocysts. The option of storing spare embryos could increase the cumulative pregnancy rate and reduce the risk of adverse outcomes related to multiple gestations by replacing fewer embryos at one time, it also decrease the risk of severe ovarian hyperstimulation syndrome, which establish embryo cryopreservation as an essential part of assisted reproductive technology (ART). At present, the babies conceived by ART have accounted for1-4%of all births, and FET cycles have accounted for almost20%of all ART treatment cycles in Europe. The children conceived from frozen-thawed embryos is one of the most important part in ART-conceived offspring. As ART is a artificial technology, it was introduced into practice without sufficient risk evaluation, increasing worry is focused on the health of ART-ceonceived offspring now. Some studies have investigated short-term outcomes in children born after replacing frozen embryos. Data concerning neonatal outcome after embryo freezing was always reassuring, which indicated a better or at least as good outcome compared with babies conceived from fresh embryos, including heavier birthweight of singletons, reduced risks of preterm birth (PTB) and low birthweight (LBW).Following freezing and thawing, a proportion of cleavage stage embryos will be fully intact, some will survive partially as lysis or necrosis of several blastomeres, and some will degenerate. Since individual blastomere of early cleavage stage embryo has been shown to be undifferentiated and totipotent, a frozen embryo with blastomeres loss still has the ability to develop into a health baby. The embryos with more than50%surviving blastomeres and keeping cleaved were considered optimal for transfer. Blastomere loss is a common occurrence in cyrothawed cleavage-stage embryos, almost50%of embryos will partially damage or degenerate after cryopreservation. The previous focus is the implantational ability of such cryodamaged embryo. Human or mouse frozen embryos with blastomeres loss are currently considered to have a lower viability than intact ones. If the necrotic cells are removed from the partially damaged embryos, the implantation rate and pregnancy rate will be significantly improved.The preimplantation stage is one of the most challenging phases in embryonic development. During this time an embryo must initiate some important processes, including genome activation, maintaining gene imprinting and a second wave of demethylation and gradual remethylation of non-imprinted gene. Some animal experiments showed bad events in the embryonic preimplantation stage, such as in vitro culture, embryo freezing and blastomere biopsy, could not only influence fetal and postnatal development but also extend to long-term susceptibility to disease in later life. Apart from conventionally cellular morphologic and metabolic changes during freezing and thawing process, frozen embryo with one or more blastomeres loss suffer from reduction of embryonic material. And a possible toxic effect of damaged cells on the remaining healthy ones has been evoked, as necrotic material may disrupt embryonic implantation and development. However, research about its related safety seems to be neglected. There is no report on long-term outcome in children born from such cryodamaged embryos so far.In the present study, we focused on the effect of blastomeres loss in human early cleavage stage frozen-thawed embryo on the safety of offspring. We first analysis the early pregnancy outcome and neonatal health of singletons after partially damaged frozen-thawed embryo transfer, using data from pregnanies conceived from fully intact frozen embryo as a reference. Then we evaluated growth and intellectual development of young singletons between the two groups. Thus, the safety of offspring born from partially damaged frozen embryos was evaluated in neonates and young children.Part Ⅰ Comparison of early pregnancy outcome and neonatal outcome of singletons after partially damaged and fully intact frozen-thawed embyro transfer in FET cyclesObjective:To determine whether blastomeres loss in human early cleavage stage frozen-thawed embryo has an influence on early pregnancy outcome and neonatal health of singletons.Patients and methods:A retrospective analysis was performed on220pregnancies conceived from partially damaged frozen-thawed embryo transfer and687pregnancies conceived from fully intact embryo transfer between January2000and April2010at the Reproduction Center of Women’s Hospital, Zhejiang University. The early pregnancy outcome and neonatal outcome of live born singletons were compared between the two groups. Forthermore, we further analyzed the variables that could possibly affect birthweight.Results:The frequency of biochemical pregnancy was significantly higher (8.6%versus4.1%, P=0.008), and multiples were less prevalent (28.2%versus48.6%, P<0.001) in blastomeres loss group compared with the controls. After adjusting for the available confounding factors that could affect birthweight,125singletons born after partially damaged frozen-thawed embryo had a significantly lower average birthweight than that of338singletons born after fully intact embryo (3229.6±510.1g versus3338.5±473.8g, P=0.014). However, mean gestational ages were not significantly different, the frequencies of low birthweight (LWB), preterm birth (PTB) and small for gestational age (SGA) were also similar between the two groups.Conclusions:Our study further demonstrated the lower developmental potential of thawed embryo with blastomeres loss, and such cryodamage in human frozen-thawed embryos could affect birthweight of live born singletons. Part II Growth and intellectual development of singleton children born after partially damaged frozen-thawed embyro transfer compared with fully intact frozen-thawed embyro transferObjective:To investigate whether blastomeres loss in human early cleavage stage frozen-thawed embryo has an effect on growth and intellectual development of singleton children.Subject and method:We contacted the parents of singleton conceived by partially damaged or fully intact frozen-thawed embyro transfer between August2002and August2008at the Reproduction Center of Women’s Hospital, Zhejiang University. The children’s physical developmental assessment was done with the height and weight reference published by WHO in2006. Developmental quotient was calculated with Gesell Developmental Schedules, intellectual development was assessed with Chinese Wechsler Young Children Scale of Intellignence or Chinese Wechsler Intelligence Scale for Children. Results: Physical development related parameters were similar between42singletons in the study group and75singletons in the control group. After adjusting for confounders, such as parental educational level, low birthweight and gender,22children in the study group had a singnificantly lower developmental quotient of personal-social behavior compared with48children in the control group. For adaptive behavior, gross motor, fine motor and language, all of the DQs were comparable between the two groups. Forthermore, no statistically significant differences regarding verbal intelligence quotient, performance intelligence quotient and full-scale intelligence quotient were observed between20singleton children in the study group and27singleton children in the control group. Multiple linear regression analysis showed a significant association between maternal educational level and adaptive behavior DQ, while paternal educational level would significantly affect language DQ, personal-social behavior DQ, VIQ, PIQ and FIQ.Conclusions:Our study demonstrated blastomeres loss in human early cleavage stage frozen-thawed embryo hadn’t an adverse effect on growth and intellectual development of singleton children. However, parental cognitive ability is more important in determining the long-term intellectual ability of children.