Molecular and clinical investigations using animal and human systems to improve assisted reproductive technologies

More than 30 years have passed since the birth of the first in vitro fertilization (IVF) baby. Since that time, advances in reproductive technologies have been remarkable. Yet with all that has been achieved so far, there is still much to be learned. To further our understanding, this dissertation investigated several aspects of IVF important to assisted reproductive medicine.;The first area of investigation focused on the effects of gonadotropin-releasing hormone (GnRH) analogs on granulosa cells. Important components of stimulation protocols are the suppression of endogenous gonadotropins with GnRH analogs and the timely administration of exogenous gonadotropins to promote follicular development and final maturation of oocytes for use in IVF. Central to this process is the response of granulosa cells to gonadotropins by regulating the production of steroids and growth factors during folliculogenesis. In this study, GnRH agonist and antagonist protocols were shown to differentially affect the expression of microRNAs (miRNAs) in granulosa cells. This could have important consequences for the function of granulosa cells as these small non-coding RNAs have been shown to post-transcriptionally regulate the expression of numerous genes essential for folliculogenesis, which may affect stimulation response and reproductive success.;The ensuing investigation focused on the effects of globulin proteins in solutions used for embryo cryopreservation. Successful cryopreservation is dependent upon the osmoregulation of water during freeze/thaw to avoid the formation of potentially detrimental ice crystals. Globulin-rich proteins, which have previously been shown to promote embryo development, have been suggested to alter the embryo microenvironment through interactions with water molecules and therefore were tested in cryopreservation solutions to improve post-thaw embryo survival. Results showed more blastocysts survived, as measured by re-expansion following freezing and thawing, in solutions supplemented with globulin-rich proteins compared to human serum albumin. These results indicate inclusion of globulins promotes blastocyst thaw-survival, which may prove beneficial for clinical cryopreservation of embryos.;The final investigation studied the effects of a protein phosphatase inhibitor, calyculin-A, used to induce premature chromosome condensation (PCC) within single embryonic cells to identify a complete chromosome complement for genetic analysis. Traditional methods for analyzing chromosomal abnormalities in embryos utilize interphase fluorescence in situ hybridization (FISH), which is limited by the number of chromosomes that can be analyzed at one time. In an effort to improve these methods, this study exposed single blastomeres from bovine and murine embryos to calyculin-A to induced PCC. Results demonstrated blastomeres underwent rapid chromatin condensation; however, the quality of condensed chromosomes was inconsistent so that application of this technology was unreliable for genetic screening of embryos.