S. Jaroudi

Expression profiling of DNA repair genes in human oocytes and blastocysts using microarrays

BACKGROUNDnThe early preimplantation embryo relies on mRNA and protein from the oocyte to detect DNA damage and activate DNA repair, cell cycle arrest or apoptosis. Expression of some repair genes has been detected in mammalian oocytes and embryos; however, little is known about DNA repair gene expression in human blastocysts. In this study, DNA repair gene expression was investigated in human oocytes and blastocysts to identify the pathways involved at these stages and detect potential differences in repair mechanisms pre- and post-embryonic genome activation.nnnMETHODSnTriplicate sets of pooled metaphase II oocytes or blastocysts were processed for analysis using the Human Genome Survey Microarrays V2.0 (Applied Biosystems).nnnRESULTSnOf 154 DNA repair genes investigated, 109 were detected in blastocysts and 107 in oocytes. Among differentially expressed DNA repair genes, 40/55 (73%) had lower expression levels in blastocysts compared with oocytes (P < 0.05, fold change >3).nnnCONCLUSIONnDespite experimental limitations due to culture or freezing and thawing of samples, large numbers of repair genes were detected indicating that all DNA repair pathways are potentially functional in human oocytes and blastocysts. The higher mRNA level for most repair genes in oocytes compared with blastocysts ensures sufficient availability of template until embryonic genome activation.

Investigation of gene expression profiles before and after embryonic genome activation and assessment of functional pathways at the human metaphase II oocyte and blastocyst stage

OBJECTIVEnTo compare the oocyte versus the blastocyst transcriptome and provide data on molecular pathways before and after embryonic genome activation.nnnDESIGNnProspective laboratory research study.nnnSETTINGnAn IVF clinic and a specialist preimplantation genetics laboratory.nnnPATIENT(S)nCouples undergoing or having completed IVF treatment donating surplus oocytes or cryopreserved blastocysts after patient consent.nnnINTERVENTION(S)nSets of pooled metaphase II (MII) oocytes or blastocysts were processed for RNA extraction, RNA amplification, and analysis with the use of the Human Genome Survey Microarrays v2.0 (Applied Biosystems).nnnMAIN OUTCOME MEASURE(S)nAssociation of cell type and gene expression profile.nnnRESULT(S)nTotals of 1,909 and 3,122 genes were uniquely expressed in human MII oocytes and human blastocysts respectively, and 4,910 genes were differentially expressed between the two sample types. Expression levels of 560 housekeeping genes, genes involved in the microRNA processing pathway, as well as hormones and hormone receptors were also investigated.nnnCONCLUSION(S)nThe lists of genes identified may be of use for understanding the processes involved in early embryo development and blastocyst implantation, and for identifying any dysregulation leading to infertility.

Functional assessment for elimination of mismatches in nuclear and whole cell extracts obtained from mouse and human blastocysts

ABSTRACT Preimplantation embryos may have an increased risk of having mismatches due to the rates of cell proliferation and DNA replication. Elimination of mismatches in human gametes and embryos has not been investigated. In this study we developed a sensitive functional assay to examine the repair or elimination of mismatches in both commercially available cell extracts and extracts obtained from preimplantation embryos. Heteroduplex molecules were constructed using synthetic oligonucleotides. Efficiency of the repair of mismatches was semi-quantitatively analysed by exposure to nuclear/whole cell extracts (as little as 2.5 µg) and extracts obtained from pooled mouse and human blastocysts to investigate the repair capacity in human embryos. A cell free in vitro assay was successfully developed to analyze the repair of mismatches using heteroduplex complexes. The assay was further optimized to analyze repair of mismatches in cell extracts obtained from oocytes and blastocysts using minute amounts of protein. The efficiency of mismatch repair was examined in both mouse and human blastocysts (2.5 µg). The blastocysts were observed to have a lower repair efficiency compared to commercially available nuclear and whole cell extracts. In conclusion, a sensitive, easy, and fast in vitro technique was developed to detect the repair of mismatch efficiency in embryos.