The Effect Of Blastomere Loss On The Outcome Of Frozen-Thawed Embryo Transfer And Transcriptome Analysis Of Offspring’s Cord Blood Lymphocytes

Frozen-thawed Embryo Transfer (FET) has become an important component of Assisted Reproductive Technology (ART). However, the associated blastomere loss problem is gaining more and more attraction. Previous researches are controversial about the relationship between embryo cryopreservation associated blastomere loss and impaired embryo development. In terms of the outcome of perinatal outcome, most researches compared FET with fresh embryo transfer instead of investigating the impact of blastomere loss. Recently emerging RNA-Seq enables us to collect FET offspring’s genetic information and reveal the underlying molecular mechanism. The aim of this study is assessing the impact of FET and blastomere loss on the perinatal outcome and RNA profiles of FET offspring by transcriptomics and large sample analysis of clinical data. Thus we can provide evidence for the reasonable clinical application of FET.PART I Impact of blastomere loss on FET and offspring perinatal outcomeObjective:Investigate whether the blastomere loss during embryo frozen-thawing would impact the embryo transfer and development as well as the perinatal outcome.Methods:A retrospective cohort analysis was performed on5573FET cycles conducted in4reproductive centers during Jan.2005and Dec.2012. The FET cycles were divided into two groups:damage group (N=1860) including cycles in which all embryos transferred have lost at least one blastomere; intact group (N=3713) including cycles in which all embryos transferred with all their blastomeres intact. FET outcome measures were implantation rate, pregnancy rate, and birth rate.The perinatal outcomes of singleton offspring from damaged group (N=269), intact group (N=687) and spontaneous pregnancy group (N=1762) are compared and analyzed.Results:In the comparison of FET outcomes, the chemical pregnancy rate, clinical pregnancy rate, implantation rate per embryo transferred and birth rate were significantly higher in intact group compared with those in damage group. In terms of the perinatal outcome of singleton offspring, preterm birth rate, cesarean section rate and large for gestational age (LGA) infants rate were both significantly higher in damage group and intact group compared with those in spontaneous pregnancy group. The risks of small for gestational age (SGA) were significantly higher in the damaged group compared with that in intact and spontaneous group.Conclusions:Blastomeres loss during embryo frozen-thawed procedure significantly decreases the embryos’ development potential and increased the offspring’s risk of SGA. Meanwhile, FET increases the risk of LGA for offsprings and its mechanism is yet to be further research. PART Ⅱ Transcriptome studies of offspring from spontaneous pregnancy and frozen-thawed embryo with blastomere loss and intact Objective:Further study the impact of frozen-thawed embryos and blastomere loss on offspring with transcriptomics. Analyze the expression of related genes at RNA level.Methods:Singletons delivered during Jun.2012and May2013after FET in our hospital are studied. The singletons were divided into three groups:damage group including offspring developed from embryo with blastomere loss, intact group including offspring developed from embryo with blastomere intact and reference group including offspring randomly selected from spontaneous pregnancy. We utilized RNA-Seq technology to assess global gene expression profiles of15infants’ umbilical cord blood lymphocytes samples from damaged group, intact group and spontaneous pregnancies respectively.Results:A total of9427transcripts were identified as expressed in lymphocytes of cord blood. Furtherly, we identified92DEGs between damage group and spontaneous pregnancies respectively and2DEGs, IGF2/H19, between intact group and spontaneous pregnancies. And most of these genes related to cell growth. When comparing DEGs between intact group and damage group,132genes showed differential expression at a significant P-value, but none of them remained significant after correcting for genome-wide analysis (FDR<0.05).Conclusions:The global gene expression analysis of cord blood lymphocytes provides new insight into the underlying molecular mechanism involved in the FET and blastomere loss, which may be the altered insulin-like growth factor system including IGF2. The changes of H19and IGF2are also worth to be investigated.