Effects of assisted reproductive technology on DNA methylation at the IGF2/H19 gene locus and the phenotype of newborns

Assisted reproductive technology (ART) refers to treatments used for infertile couples to achieve pregnancy in vitro. The main technology of ART is in vitro fertilization (IVF), which may also include intracytoplasmic sperm injection (ICSI) and/or embryo cryopreservation and frozen embryo transfer (FET). ART treatments are well-accepted in Western countries and there is an increasing number of children being conceived in that way. Even though, majority of ART derived newborns appear healthy, they have been associated with increased risks of adverse perinatal outcomes, especially, alterations in birth size as well as higher frequencies of imprinting disorders and alterations in epigenetic modifications, such as in DNA methylation, of imprinted genes. Epigenetically regulated imprinted genes have crucial roles in fetal and placental growth during development and they are known to be affected by environmental factors. Since ART takes place in the early embryo in vulnerable time-period of epigenetic reprogramming, ART has been suggested to impact on epigenetic profiles of the embryo, consequently, affecting the phenotype of newborns, and therefore potentially causing long-term health effects. This thesis aimed to study whether ART has effects on DNA methylation in the placenta and whether ART has effects on the phenotype of newborns. To study these effects, this thesis focused on the sixth binding sequence of CTCF (CTCF6) of H19 ICR1 of the growth-related imprinted IGF2/H19 gene locus. The aim was also to study whether the possible changes associate with the rs10732516 G/A polymorphism locating at CTCF6 of H19 ICR1. DNA methylation levels of placental tissue as well as white blood cells in umbilical cord blood of ART derived, and spontaneously conceived newborns were explored by mass spectrometry-based Sequenom MassARRAY® EpiTYPER® method and traditional bisulfite sequencing. To study the effects of ART on the phenotype of newborns, the birth weight, length and head circumference of ART and control newborns were explored using international growth standards. Moreover, placental weights were compared. The results of this thesis showed slightly, but consistently decreased DNA methylation levels at H19 ICR1 in the paternal allele of ART derived placentas in rs10732516 patA/matG genotype, but not in patG/matA genotype. Thus, the results suggest that the changes in DNA methylation at IGF2/H19 in the placenta are genotype-specific and associate with the rs10732516 polymorphism. Similar decreased methylation levels in the paternal allele of patA/matG genotype was not detected in white blood cells suggesting that the effects on DNA methylation levels are also cell type-specific. The effects of ART on the phenotype also associated with the rs10732516 polymorphism. Fresh embryo transfer derived newborns with A/A genotype were seen to have smaller birth weight than newborns with G/G genotype. Moreover, in A/A genotype, frozen embryo transfer derived newborns were demonstrated to be heavier and to have heavier placentas than fresh embryo transfer derived newborns. The findings of this thesis suggest that ART has effects on DNA methylation in the placenta and on the phenotype of newborns, and the effects associate with the rs10732516 G/A polymorphism. This underlines the significance of the polymorphism when studying the effects of ART. However, further investigations are needed to confirm these findings and to discern whether the changes are due to the ART procedures or underlying infertility.