Expression And Imprinting Analyses Of Growth And Development Related Imprinted Genes CDKN1C, PHLDA2 And IGF2 In Placentas Derived From Assisted Reproductive Technology

Since the birth of Louise Brown in 1978, assisted reproductive technology (ART), as a worldwidely accepted technology, has enabled many infertile couples to enjoy parenthood in the past 30 years. The babies conceived by ART have accounted for 1-4% of all births in developed countries now and gradually become an important part of the world population. ART induces an increasing worry about its risk in reproductive genetics because of its artificial character and the absence of sufficient risk evaluation before clinical use. Till now no evidence has found that ART is directly related to obvious malformation in offspring. But a series of epidemiological studies have revealed that ART babies tend to have health problems, such as growth and development problems. However, little is known about the exact underlying mechanisms. It is also unclear whether the genetic disorders of the infertile parents or the manipulations of ART raise the risk.Epigenetics means changes in phenotype that are heritable but do not involve DNA mutation, including DNA methylation, genomic imprinting, histone modifications and so on. Genomic imprinting is an epigenetic process by which the male and female germ line are confered a sex-specific mark (imprint) on certain chromosomal regions. As a consequence, the imprinted gene is expressed from only one of the paternal or maternal chromosomes while the other copy is silent. There are only a few genes in human genome found to be imprinted, but they play important roles in growth and development and the abnormality will induce imprinting defects or tumors. Genomic imprinting is reversible and is vulnerable to environmental stress which will lead to heritable disturbance and change the phenotype of offspring. Genomic imprinting will undergo erasing and re-establishment during gamete and preimlantation embryo development. It is high time that ART manipulations take place. A series of studies have found that ART would lead to aberrant imprinted genes expression, DNA methylation, and so on. A number of reports have suggested that ART might be associated with an increased risk of imprinting defects, such as Beckwith-Wiedemann syndrome (BWS). The previous research of our team also found aberrant imprinted genes expression and DNA methylation in the umbilical blood of ART offspring. Therfore, ART probably influence the growth and development potential of offspring by disturbing the genomic imprinting.Trophectoderm cells might be more sensitive to environmental stress than inner cell mass, so that the risk of epigenetic changes is higher in placenta than in fetus. There are many imprinted genes in placenta known to be crucial for placental and fetal growth. In general, the paternally expressed (maternally imprinted) genes enhance fetal growth and the maternally expressed (paternally imprinted) genes restrict fetal growth. Loss or aberrant activation of them will cause abnormalities in fetal growth and development.Do the growth and development problems of ART babies have something to do with the aberrant genomic imprinting in placenta caused by ART? How seriously can it be disturbed by ART? What are the underlying mechanisms?In the present study, we focused on paternally imprinted growth restricting genes CDKN1C (cyclin-dependent kinase inhibitor 1C), PHLDA2 (pleckstrin homology-like domain, family A, member 2) and maternally imprinted growth promoting gene IGF2 (insulin-like growth factor 2) in placenta. We conducted reverse transcription (RT)-Real time PCR, Westernblot, direct sequencing, PCR-polyacrylamide gel electrophoresis and bisulfite sequencing (BSP) to investigate the expression and imprinted status of these three genes and DNA methylation patterns of their impinting control regions and promoters in term and singleton placentas derived from ART and spontaneous pregnancy in order to elucidate the underlying epigenetic mechanisms for the growth and development problems of the ART conceived babies and evaluate the epigenetic risk of ART.Part I The expression of imprinted genes CDKN1C, PHLDA2 and IGF2 changed in human placentas derived from ARTObjective:To investigate the mRNA and protein expression differences of CDKN1C, PHLDA2 and IGF2 between ART and spontaneously conceived placentas.Methods:We collected 39 ART conceived, term and singleton placentas and 40 comparative spontaneously conceived placentas. We compared the birth weights of babies between two groups, used RT-Real time PCR to analyze the mRNA expression levels and used Westernblot to analyze the protein expression levels of the target genes.Results:The average birth weight of ART group was lower than that of control group, but without significant difference. In ART conceived placenta, CDKN1C and IGF2 were significantly up-regulated in mRNA levels and PHLDA2 was significantly up-regulated in protein level. There were no significant differences in PHLDA2 mRNA and CDKN1C, IGF2 protein expressions between two groups. Conclusion:The expression of several growth and development related imprinted genes changed in ART conceived placentas. It may influence the growth and development potential of ART babies.Partâ…¡DNA methylation analysis of imprinting control regions and promoters of imprinted genes CDKN1C, PHLDA2 and IGF2 in human placentas derived from ARTObjective:To investigate the change of DNA methylation patterns in imprinting control regions and promoters of CDKN1C, PHLDA2 and IGF2 between ART and spontaneously conceived placentas, and elucidate the relationship between aberrant DNA methylation and abnormal gene expression.Methods:Six ART conceived and 5 spontaneously conceived term and singleton placentas were subjected to bisulfite sequencing. DNA methylation patterns in CpG islands of imprinting control regions KvDMRl, H19 DMR and promoters of CDKN1C and PHLDA2 were analyzed.Results:All spontaneously conceived placentas were differentially methylated in KvDMR1 and H19 DMR. The methylation rates were both around 50%. The average methylation rate of every CpG site in KvDMRl was lower in ART group and there was a significant decrease in methylation at CpG site 9. One case of ICSI conceived placentas was extremely hypomethylated in this region. The average methylation rates of most CpG sites in H19 DMR were higher in ART group and there was a significant increase in methylation at CpG site 7. One case of IVF conceived placentas was extremely hypermethylated in this region. Both ART and control group were hypomethylated in the promoters of CDKN1C and PHLDA2. The methylation rates were 0-23%. However, in ART group, there was a significant decrease in methylation at CpG site 7-11 in CDKN1C promoter while a significant increase at CpG site 1,2,7, 10,11 and a significant decrease at CpG site 5 in PHLDA2 promoter.Conclusion:The aberrant DNA methylation patterns in the imprinting control regions and promoters may contribute to the abnormal expressions of imprinted genes in ART conceived placentas.Partâ…¢Imprinted status analysis of imprinted genes CDKN1C, PHLDA2 and IGF2 in human placentas derived from ARTObjective:To investigate the change of allele-specific expression of CDKN1C, PHLDA2 and IGF2 and parent-specific expression of CDKN1C and IGF2 in ART conceived placentas and elucidate the relationship among imprinted status, aberrant DNA methylation and abnormal gene expression.Methods:Thirty-nine ART conceived and 40 spontaneously conceived term and singleton placentas were involved. PCR-polyacrylamide gel electrophoresis, PCR-direct sequencing and RT-PCR-direct sequencing were applied to detect the imprinted status of target genes in placentas.Results:CDKN1C, PHLDA2 and IGF2 maintained monoallelic expression in all samples analyzed, so that they are all imprinted genes in placenta. CDKN1C is a maternally expressed and paternally imprinted gene. IGF2 is a paternally expressed and maternally imprinted gene. We found no alteration in imprinted status of target genes in ART conceived placentas.Conclusion:We found aberrant DNA methylation patterns in imprinting control regions and promoters of ART conceived placentas but detect no instability of imprinted status in target genes. It suggests that other modifications besides DNA methylation may also contribute to the genomic imprinting in placenta.