Xin TaoFollow

Date of Award

Fall 9-11-2017

Degree Type

Dissertation

Degree Name

PhD Molecular Bioscience

Department

Biology

Advisor

Tinchun Chu, Ph.D.

Committee Member

Nathan R. Treff, Ph.D.

Committee Member

Jane L. Ko, Ph.D.

Committee Member

Angela V. Klaus, Ph.D.

Committee Member

Daniel B. Nichols, Ph.D

Keywords

IVF, aneuploidy, aging, embryo, oocytes, comprehensive chromosome screening, mitochondrial DNA, NGS, microbiome, 16S rRNA.

Abstract

In humans, early embryo development is a complex process that consists of sequential events: oocyte maturation, fertilization, embryonic growth and implantation. Disruption of these highly regulated processes results in reproductive failure and infertility. This study characterizes and describes embryonic aneuploidy, mitochondrial content level and endometrial microbial environment related to reproductive competence, in particular instances in which failure results.

To examine the molecular underpinnings of mammalian gamete and early embryo chromosome segregation, we established a comprehensive chromosomal screening (CCS) method for mice poly bodies, oocytes and embryos by the application of whole genome amplification (WGA) and next generation sequencing (NGS). First, we validated this approach using single mouse embryonic fibroblasts engineered to have stable trisomy 16. We further validated this method by identifying reciprocal chromosome segregation errors in the products of meiosis I (gamete and polar body) in oocytes from reproductively aged mice. Finally, we applied this technology to investigate the incidence of aneuploidy in IVM- and IVF- derived blastocysts from both young and reproductively aged mice.

It was reported that mtDNA was significantly increased in aneuploid human embryos compared to euploid embryos and also associated with maternal age. In this study, we established the mouse model of mitochondrial DNA (mtDNA) quantitation in reproductive samples based on WGA and NGS. The method was validated on a tumor-derived mouse cell line, and then applied to mouse reproductive samples. Cells in blastocysts from younger mice contained significantly lower amounts of mtDNA compared to aged mice (P

euploid blastocysts (P=0.0045). WGA and NGS provided a reliable method to assess mtDNA content in mouse gametes and embryos. We also established a quantitative method to detect mtDNA copy number in human embryos. The maternal age of the embryos displayed a significant correlation with mtDNA content (p=0.007). This well controlled study demonstrated that mtDNA quantitation provided no additional selection advantage between euploid sibling embryos in a double embryo transfer model.

There is growing interest in the microbiome of the reproductive tract. The vaginal and placental microbiome have been partially characterized and shown to be related to obstetric outcomes. In this study, we assessed the quality of Ion PGM- and Illumina MiSeq-generated data for 16S rRNA metagenomics, established the sensitivity of Illumnia 16S V4 metagenomics, then studied the endometrial microbial environment by analyzing the embryo transfer (ET) tip after embryo transfer. There were a total of 248 genera detected amongst all specimens. Lactobacillus genera were detected in all of the samples. When analyzing fraction of lactobacillus reads, there were no differences (p=0.464) The Shannon Diversity Index did not differ between the two groups (p=0.164). The data in this study showed the microbiome at the time of ET may differ by pregnancy outcome but highlighted the challenge of low bacterial load and read counts when analyzing ET catheter tips alone.

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