Date of Award

5-2017

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Lee H. Lee

Committee Member

Nathan R. Treff

Committee Member

John Gaynor

Abstract

Early embryo development in humans is a complex process. Due to recent advances in research, many improvements have been made in in vitro-fertilization (IVF) outcomes. Success has been credited to the application of comprehensive chromosome screening (CCS) however, not every euploid embryo will always implant due to various outside factors (1). More reproductive markers would need to be evaluated to optimize the success of implantation of euploid embryos.

MiRNAs are emerging as important regulators of cellular differentiation and may be involved in the human pre-implantation process. Any dysregulation of microRNA expression may play an important role in the progression or to other factors involving infertility. MiRNAs may have the potential to be a maker for embryonic reproductive competence. This study aims to develop and validate a new methodology in which both miRNA and CCS can be evaluated from the same trophectoderm biopsy.

In this study, two miRNA complimentary DNA (cDNA) synthesis strategies (the Megaplex Synthesis kit and the TaqMan Advanced miRNA cDNA synthesis kit) were tested on 5- cell or 7-cell samples from two human fibroblast cell line (GM00323) and (GM01359) to establish the validity of the methods. RNA purified from large amount of cells was also tested to serve as a gold standard. MicroRNA expression was assessed by Real-time quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). CCS was performed simultaneously by previously established platforms (targeted NGS and PCR24).

The miRNA expression from 5-cell samples showed inconsistent results to purified RNA by using the Megaplex Synthesis kit and qPCR. The short length and similarity of miRNAs sequence make the alignment of NGS reads difficult for bioinformatics analysis. Better results were obtained using the TaqMan Advanced miRNA cDNA synthesis kit, which also have the advantage to detect novel miRNA in future. The synthesized cDNA was preamplified with miRNA primers and previously validated CCS primers. The expression of the three miRNAs was detected in 7-cell samples and bulk total RNA. PCR24 CCS showed 100% consistency with the expected karyotypes. More samples will be needed to be processed to improve the consistency. Once this method for simultaneous CCS and miRNA analysis has been established, it can provide a foundation for the development of miRNA biomarkers of reproductive potential.

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