Jaroudi, S.; (2010) Investigation of DNA repair in human oocytes and preimplantation embryos. Doctoral thesis , UCL (University College London).

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Abstract

DNA repair genes are expressed in mammalian embryos and in human germinal vesicles, however, little is known about DNA repair in human preimplantation embryos. This project had three aims: 1) to produce a DNA repair profile of human MII oocytes and blastocysts using expression arrays and identify repair pathways that may be active before and after embryonic genome activation; 2) to design an in vitro functional assay that targeted mismatch repair and which could be applied to human oocytes and embryos; 3) to investigate the effect of germline mutations in DNA repair genes on early human embryonic development by initiating a preimplantation genetic diagnosis (PGD) service for these genes. Microarray profiling showed that all DNA repair pathways were potentially functional in human oocytes and blastocysts. Higher mRNA levels were detected for most repair genes in oocytes compared to blastocysts. The functional assay for insertion/deletion loops (IDL) and mismatch repair was more sensitive than available methods. Repair was detected for 3, 21 and 24-nucleotide IDLs and single base mismatches. Optimisation is necessary to improve sensitivity of the assay before it can be applied to nuclear extracts from pooled oocytes or embryos. PGD protocols were developed for two BRCA1 mutations (c.68-69delAG and c.3339T>G) and one MSH2 mutation (c.1277-?_1386+?del). One BRCA1 protocol was clinically applied resulting in the birth of a healthy child. Morphological assessment of embryos from this treatment cycle showed that the presence of the germline mutation adversely affected development between days 3 and 5 post fertilisation. The other two PGD protocols were licensed for treatment by the HFEA. The availability of donated oocytes and embryos from this PGD service will allow the investigation of repair pathways by real time PCR to examine specific genes selected from the microarray expression profiles and by the application of the functional assay.

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