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Thesis (M.A.)--Bowling Green State University, 2006. / Document formatted into pages; contains v, 83 p. Includes bibliographical references.

Assisted reproductive technologies have become a common method used to treat infertility. These techniques have advanced quickly since the first birth of an in vitro fertilization (IVF) baby in 1978, at the Royal Oldham Hospital in the United Kingdom. Currently, IVF with or without intracytoplasmic sperm injection, is used throughout the world to achieve oocyte retrieval, fertilization, implantation of an embryo, clinical pregnancy, ongoing clinical pregnancy, and a live-born infant. The rationale for selecting one type of fertility treatment over another is multifactorial: the confirmed or unconfirmed cause of infertility, the age of the gamete donor and the recipient, the availability of the type of treatment, and the cost associated with the treatment. The ultimate goal of any fertility treatment is to achieve a successful pregnancy that results in a healthy infant. However, the literature is equivocal on the effects of fertility treatment cycles on the health outcomes of infants and mothers. Presently, there are thirty-six fertility treatment centres across Canada, eighteen of which reside in Ontario. A national, comprehensive database of assisted reproductive technology treatment cycles (Canadian Assisted Reproductive Technologies Register (CARTR) Plus) began collecting data in 2013, and has made the research objectives of this doctoral thesis feasible. Before this data collection system, population-wide studies involving fertility treatments were not possible in Canada. Two understudied issues associated with IVF are the impact of fertility treatments on the maternal serum screening markers used in prenatal screening programs to identify fetal aneuploidies; and the association between fertility treatments and adverse perinatal outcomes, such as preeclampsia and stillbirth. Given the increasing number of women who are using fertility treatments to conceive, it is imperative that studies investigating the association with adverse outcomes are conducted. As the science supporting fertility treatment procedure has advanced, so has prenatal screening. One of the first screening tests that are performed for newly pregnant women, including women who conceived following IVF, is maternal serum screening. The first objective of this doctoral thesis was to systematically review the literature on the association between IVF treatment and maternal serum screening marker levels and nuchal translucency (NT) thickness. After the search and screening of the literature there were 40 studies that were included in this systematic review. A decrease in pregnancy-associated plasma protein A (PAPP-A) and an increase in total human chorionic gonadotropin (hCG) was consistently reported for IVF pregnancies. However, since the levels of the other maternal serum screening markers reported also varied we were unable to generalize about the differences between prenatal screening results in the IVF population. These results led to investigating maternal serum screening marker levels among IVF patients in Ontario, Canada. The second objective of this thesis was three-fold: 1) to investigate the accuracy of IVF identification on the Ontario prenatal screening record, relative to reference standard on the CARTR Plus database; 2) to compare the prenatal screening markers in IVF versus non-IVF pregnancies in the population of Ontario; and 3) to propose updated IVF adjustment factors for prenatal screening in the Ontario population, based on the more accurate coding for IVF status in the CARTR Plus database. Significant differences between IVF and non-IVF groups, based on both the prenatal screening requisition information and CARTR Plus information, were found among the ethnicity adjusted mean multiple of the median (MoM)s for several prenatal screening markers: alpha-fetoprotein (AFP), PAPP-A, unconjugated estriol (µE3), first trimester hCG, total hCG, and dimeric inhibin A (DIA). When we developed the proposed adjustment factors for all CARTR Plus identified pregnancies we found that for PAPP-A, total hCG, and µE3 the mean adjusted marker MoMs were significantly closer to 1.00, as compared to the prenatal screening adjusted or the unadjusted mean marker MoMs. Currently, there is no adjustment made to the other maternal serum screening markers and NT measurement. The third objective was to examine the effect of type of infertility on placental-mediated adverse outcomes (preeclampsia, intrauterine growth restriction, placental abruption, and stillbirth). Type of infertility was classified as male factor (sperm count, poor sperm motility, and abnormal sperm morphology), female factor (ovulation disorders, tubal infertility, and uterine or cervical causes), and unexplained infertility. No significant associations were found between type of conception and the composite outcome, as well as each individual primary outcome. Similarly, the type of infertility was not associated with the composite outcome or any of the individual primary outcomes, except for female factor infertility, which was associated with increased probability of placental abruption. Overall, the results from this doctoral thesis suggest that there are substantial differences seen in maternal serum screening marker MoMs among women who use IVF to conceive, suggesting that appropriate adjustment factors should be employed to ensure accurate results for determining the risk of Down syndrome and trisomy 18. Additionally, although the literature has shown an association between fertility treatment and placental-mediated adverse outcomes no significant associations were found in the population of Ontario. Further studies should be performed to confirm the results of these observational studies.

Capacitation of spermatozoa is essential for fertilization. Rabbit spermatozoa are particularly difficult to capacitate in vitro and require treatment with high-ionic-strength Brackett's defined medium. Spermatozoa treated with platelet activating factor had significantly higher fertilization rates when compared with nontreated (fresh, twice washed) spermatozoa (63% vs 34%). Fertilization rates of spermatozoa treated with platelet activating factor, although higher than those of high-ionic-strength capacitated spermatozoa, were not significantly different (63% vs 57%). Spermatozoa treated with lyso-platelet activating factor, the biologically inactive form of platelet activating factor, were noted to have fertilization rates similar to those of the untreated (noncapacitated) group. These data show that synthetic platelet activating factor treatment of uncapacitatedspermatozoa induces fertilization of rabbit oocytes in vitro in a manner similar to that for spermatozoa capacitated by high-ionic-strength media and significantly higher than that for untreated spermatozoa or after treatment with the biologically inactive form of platelet activating factor (lyso-platelet activating factor).

<p>Ovarian stimulation in assisted reproduction often leads to the production of a high number of oocytes. After fertilization of these oocytes, the resulting embryos can be cryopreserved for later use. Vitrification is a recently introduced method for cryostoring embryos, showing high survival rates for both cleavage stage embryos and blastocysts. Characteristic of vitrification are high concentrations of cryoprotectants and ultra fast freezing which makes the material glassily. A major concern with vitrification has been the direct contact of the cryo-solutions with liquid nitrogen. Therefore, sealed containers have been developed and one of these is the Rapid-i™ made by Vitrolife Sweden AB.</p><p>We evaluated this new device using embryos not suitable for embryo transfer or cryopreservation for clinical purposes. Embryos at cleavage stages were first vitrified and then warmed. Outcome parameters were cryosurvival and development to the blastocyst stage. Blastocysts were randomised between the established VitroLOOP™ and the Rapid-i™ as carriers. Outcome parameters were cryosurvival and further development. Our results show that Rapid-i™ gives good survival rates in vitrification for cleavage stage embryos and blastocysts.</p>

Ovarian stimulation in assisted reproduction often leads to the production of a high number of oocytes. After fertilization of these oocytes, the resulting embryos can be cryopreserved for later use. Vitrification is a recently introduced method for cryostoring embryos, showing high survival rates for both cleavage stage embryos and blastocysts. Characteristic of vitrification are high concentrations of cryoprotectants and ultra fast freezing which makes the material glassily. A major concern with vitrification has been the direct contact of the cryo-solutions with liquid nitrogen. Therefore, sealed containers have been developed and one of these is the Rapid-i™ made by Vitrolife Sweden AB. We evaluated this new device using embryos not suitable for embryo transfer or cryopreservation for clinical purposes. Embryos at cleavage stages were first vitrified and then warmed. Outcome parameters were cryosurvival and development to the blastocyst stage. Blastocysts were randomised between the established VitroLOOP™ and the Rapid-i™ as carriers. Outcome parameters were cryosurvival and further development. Our results show that Rapid-i™ gives good survival rates in vitrification for cleavage stage embryos and blastocysts.

Master of Science / Department of Animal Sciences and Industry / Karol E. Fike / This thesis involves two separate studies that evaluate the effects of different beef cattle management practices on reproduction. The objective of the first study was to determine if feedlot heifers administered melengestrol acetate (MGA) and growth promoting implants could serve as viable oocyte donors for in vitro embryo production. Ovaries from heifers administered MGA and growth promotants (MGA-Implant) and ovaries from heifers not administered either substance (Control) were collected from heifers post-slaughter. Oocytes were harvested and in vitro maturation, in vitro fertilization (IVF), and in vitro culture were completed. Treatment and time interacted to affect the number of oocytes aspirated per ovary (P = 0.07) and the number of zygotes per ovary (P = 0.07). Fertilization (P = 0.90) and cleavage rates (P = 0.80) did not differ between treatments. Blastocyst rates (P = 0.30) and the number of embryos per ovary (P = 0.50) did not differ between treatments. We concluded that beef feedlot heifers fed MGA and implanted with growth promotants seem to be a viable source of oocytes for in vitro embryo production. In the second study, we hypothesized that continuous fenceline exposure of prepubertal beef bulls to cycling beef females would hasten the onset of puberty as well as increase the percentage of bulls passing their initial breeding soundness examination (BSE). Bulls were either exposed to estrous females (exposed) or were not exposed (control). Monthly scrotal circumference (SC) measurements, blood samples, semen evaluations, and bull behavior assessments were conducted. Age at puberty (P = 0.40), SC at puberty (P = 0.50), and weight at puberty (P = 0.30) did not differ between treatments. A similar (P = 0.50) percentage of bulls passed their initial BSE at 363 ± 21.5 d of age (exposed: 87.8%; control: 74.2%). Treatment, month, and stage of the estrous cycle of cows interacted to affect the number of mount attempts (P = 0.05) and the number of flehmen responses (P < 0.001). In conclusion, bulls given continuous fenceline exposure to cycling beef females were neither younger at puberty nor did a greater percentage pass their initial BSE.

Traditional methods of oocyte selection for in vitro studies have proven inefficient with respect to achieving a level of predictability for competency. In this study, a novel method of oocyte selection was implemented that identified a relationship between oocyte morphological parameters (as defined by a ratio of a shape factor (SF) to average fluorescence intensity (AFI) and AFI, followed by in vitro fertilization (IVF) and in vitro culture (IVC) using the Well of Well (WOW) method to evaluate oocyte competency. Specifically, we used non-cytotoxic fluorescent molecular probes and multiphoton microscopy to non-invasively characterize spatial localization and functional activity of mitochondria, mitochondrial membrane potential (Δψm), and intracellular calcium activity ([Ca2+]i) using rhodamine 123, JC-1 and Fluo-4, AM, respectively in bovine and canine in vitro matured (IVM) oocytes. Comparison of morphological grading with fluorescence intensity yielded similar trends between all grades of oocytes for both species with no visually obvious, distinct characteristic staining that would permit classification of each oocyte as a specific morphological grade. Our studies confirmed that oocyte mitochondria were homogeneously distributed but primarily localized to the peri- and sub-cortical regions of the oocyte at MII stage for both species. Further, heterogeneously polarized mitochondria were localized to the peri- and sub- cortical regions of the oocyte for both species. In bovine oocytes labeled with Fluo-4, AM, levels of [Ca2+]i were either unremarkable, or very low and limited to the peri-cortical areas, just beneath the oolema. For canine MII stage oocytes, levels of [Ca2+]i were within the same range of AFI as bovine. Ranges of fluorescence intensity compatible for optimal embryo development for bovine and optimal fertilization for canine oocytes were 30-300 and 20-35, and 20-30 and 20-25.5 for rhodamine 123 and Fluo-4, AM, respectively. The optimal range for bovine oocytes imaged with JC-1 was 1.25-2.25 and <6 for canine.

Introducción: Desde su primera descripción en 1984 las indicaciones de donación de óvulos han ido aumentando, lo que ha provocado un incremento progresivo en el número de ciclos realizados a nivel mundial. Aunque esta técnica de reproducción garantiza una elevada tasa de embarazo en la receptora, los profesionales también se esfuerzan en convertir el tratamiento para la donante en un proceso sencillo y seguro. Durante los últimos diez años los antagonistas de la GnRH, por sus características farmacodinámicas, ha sido el fármaco utilizado para desarrollar protocolos de estimulaciones ováricas sencillos y seguros para las donantes de óvulos. Materiales: La presente tesis doctoral resume las conclusiones de dos artículos publicados recientemente (2010) sobre la utilización de antagonistas de GnRH en la estimulación ovárica de donantes de óvulos. Además se discuten las conclusiones de otros cuatro artículos (publicados en revistas científicas de impacto durante los años 2006 y 2009) estrechamente relacionados a los aquí publicados. Los objetivos de esta tesis son: 1. comparar la eficacia de protocolos de estimulación basados en antagonistas de GnRH en comparación con protocolos basados en agonistas de GnRH a través de una revisión sistemática de la literatura y meta-análisis. 2. ilustrar que los protocolos de estimulación basados en antagonistas de GnRH aumentan la seguridad de la estimulación ovárica para la donante a través de una eliminación completa del riesgo del síndrome de hiperestimulación ovárica (SHO). Resultados: 1. El meta-análisis de de ocho ensayos clínicos llevados al cabo en donantes de óvulos estimuladas con antagonistas de GnRH no han demostrado diferencia significativa en el número de ovocitos obtenidos y las tasas de embarazo evolutivos en las receptoras correspondientes en comparación con agonistas de GnRH. 2. El estudio observacional, prospectivo realizado en donantes de óvulos de alto riesgo ha demostrado la eliminación completa de SHO moderado/severo tras la descarga con bolo de agonista de GnRH. Además se discuten las conclusiones de cuatro otros estudios apoyando las conclusiones mencionados arriba. Conclusiones: En el contexto de la donación de óvulos los protocolos de estimulación ovárica basados en antagonistas de la GnRH son igual de eficaces que los protocolos con agonistas de la GnRH. La inducción final de la maduración ovocitaria se puede llevar a cabo satisfactoriamente con un bolo de agonista de GnRH en vez de hCG, lo que prácticamente elimina el riesgo de SHO moderado/severo. La utilización preferencial de este protocolo de estimulación ovárica es muy aconsejable porque permite un tratamiento más sencillo y aumenta considerablemente la seguridad de la estimulación ovárica en donantes de óvulos. / Background: Since its first description in 1984 the indications of oocyte donation (OD) has widened considerably which has led to a continuous increase in the number of OD treatment cycles performed worldwide. Although this treatment option secured the highest pregnancy rates for the recipients of donor oocytes increased efforts were also made to achieve safer and simpler treament protocols for the oocyte donor. During the last decade with the advent and increased use of the GnRH antagonists this new pharmacological agent was also explored in ovarian stimulation protocols specifically tailored for oocyte donors. Materials: The present doctoral thesis summarizes the findings of two recently published articles (2010) on the application of GnRH antagonists in the ovarian stimulation of oocyte donors. Furthermore the findings of another four strictly related articles (published in high-impact international journals between 2006 and 2009) are also discussed. The primary objectives were: 1. to compare efficiency of GnRH antagonist protocols in comparison with GnRH agonist-based protocols in the context of oocyte donation by means of a systematic review and meta-analysis and 2. to illustrate that GnRH antagonist protocols substantially increase the safety of ovarian stimulation for oocyte donors by reducing or even eliminating the incidence of moderate/severe ovarian hyperstimulation syndrome (OHSS). Results: 1. A meta-analysis of eight randomized clinical trials (RCTs) performed in oocyte donors undergoing stimulation with GnRH antagonists showed no significant difference in the number of retrieved oocytes or recipient ongoing pregnancy rate when compared with GnRH agonists. 2. A prospective, follow-up study of a group of high risk oocyte donors showed that early onset moderate/severe OHSS was completely eliminated after triggering with a GnRH agonist. Furthermore the findings of four studies supporting the above conclusions are also presented. Conclusions: In the context of oocyte donation the GnRH antagonist based ovarian stimulation protocols are equally efficient compared to down regulation by GnRH agonists. The induction of final oocyte maturation can be successfully achieved by a GnRH agonist instead of hCG which practically eliminates early-onset moderate/severe OHSS. The proposed ovarian stimulation protocol should be preferentially used because it permits the simplification and considerably increases the safety of ovarian stimulation for oocyte donors.

Traditional methods of oocyte selection for in vitro studies have proven inefficient with respect to achieving a level of predictability for competency. In this study, a novel method of oocyte selection was implemented that identified a relationship between oocyte morphological parameters (as defined by a ratio of a shape factor (SF) to average fluorescence intensity (AFI) and AFI, followed by in vitro fertilization (IVF) and in vitro culture (IVC) using the Well of Well (WOW) method to evaluate oocyte competency. Specifically, we used non-cytotoxic fluorescent molecular probes and multiphoton microscopy to non-invasively characterize spatial localization and functional activity of mitochondria, mitochondrial membrane potential (Δψm), and intracellular calcium activity ([Ca2+]i) using rhodamine 123, JC-1 and Fluo-4, AM, respectively in bovine and canine in vitro matured (IVM) oocytes. Comparison of morphological grading with fluorescence intensity yielded similar trends between all grades of oocytes for both species with no visually obvious, distinct characteristic staining that would permit classification of each oocyte as a specific morphological grade. Our studies confirmed that oocyte mitochondria were homogeneously distributed but primarily localized to the peri- and sub-cortical regions of the oocyte at MII stage for both species. Further, heterogeneously polarized mitochondria were localized to the peri- and sub- cortical regions of the oocyte for both species. In bovine oocytes labeled with Fluo-4, AM, levels of [Ca2+]i were either unremarkable, or very low and limited to the peri-cortical areas, just beneath the oolema. For canine MII stage oocytes, levels of [Ca2+]i were within the same range of AFI as bovine. Ranges of fluorescence intensity compatible for optimal embryo development for bovine and optimal fertilization for canine oocytes were 30-300 and 20-35, and 20-30 and 20-25.5 for rhodamine 123 and Fluo-4, AM, respectively. The optimal range for bovine oocytes imaged with JC-1 was 1.25-2.25 and <6 for canine.