Title: Paternal Environmental Exposures and Reproductive Outcomes: A Comparison of in vitro and in vivo Fertilization Statement of the Problem: Nine hundred children with Spina Bifida of Agent Orange exposed Vietnam veterans receive VA presumptive service connected benefits. Developmental endpoints such as structural birth defects, failed pregnancy, low birth weight, increased childhood cancers; and developmental, endocrine, and biochemical abnormalities have been reported in F1 and F2 generations following paternal exposures to pesticides, herbicides, and radiation. Multiple mechanisms of action are implicated in male mediated adverse reproductive events to include: a) toxins in semen that can result in vaginal absorption and in exposure of the embryo post conception, and b) deleterious effects that can disrupt fertility through interaction with the neuroendocrine axis. To our knowledge no studies examine the role of paternal preconception exposure to environmental agents on the reproductive outcomes by separating sperm related factors from seminal fluid factors through a comparison of in vitro and in vivo fertilization. Research Objectives: The primary objective is to characterize the effects of male preconception dioxin exposure on selected parameters of natural mating as well as in vitro fertilization. Murine in vitro fertilization circumvents female seminal fluid contact thereby separating the effect of vaginal absorption of toxins and the exposure of the embryo to these toxins post conception from any genetic effects on the male germ cells. The secondary objective is to determine if aryl hydrocarbon receptor (AHR) activation affects glucose transporter (GLUT) 1 localization in either the blastocyst stage embryo or stromal cell of the uterine environment, thus having a detrimental effect on embryo development and/or implantation respectively. AHR activation has been shown to alter GLUT 1 localization in an embryonic cell line thereby resulting in nutrient deficient cells and providing one explanation of Dioxin's embryo toxic effects. Research Methods: An experimental 2 x 2 block design at T+14 and T+35 days post treatment will be used to characterize the reproductive outcomes that result from in vivo natural mating vs. in vitro fertilization of non exposed females with Dioxin exposed and non exposed males. In vivo reproductive measures include 3.5 day post coitum (dpc): a) blastocyst numbers;b) blastocyst TUNEL, CYP1A1, and GLUT levels and localization;c) RT-PCR of in vivo blastocyst and stromal cell CYP1A1 mRNA, and d) stromal cell decidualization. In vivo and in vitro parameters include: a) 14.5 dpc placental and fetal weights, and crown to rump lengths;b) full term litter size, birth weights, and 5 week growth rates;and c) gross and microscopic structure.
Title: Paternal Environmental Exposures and Reproductive Outcomes: A Comparison of in vitro and in vivo Fertilization At this writing, 900 children with Spina Bifida of Agent Orange exposed Vietnam veterans receive VA presumptive service connected benefits. This underscores the pressing need to explore the intersection of paternal environmental exposures and birth outcomes and corresponds to the expectations of the Agent Orange Act of 1991 (Public Law 102-4) that new scientific information related to the exposures will be generated. This study design is unique in its ability to: a) characterize the impact of the exposure to seminal fluid during in vivo fertilization on the reproductive outcomes resulting from mating with either Dioxin exposed or non exposed males, b) address reproductive outcomes following paternal preconception exposure in the absence of maternal seminal fluid contact through the process of in vitro fertilization, and c) provide evidence for deleterious effects on developing gametes during the process of spermatogenesis. Through the identification of potential biologic mechanisms, progress can be made in the prevention of future adverse reproductive outcomes that result from male preconception exposure to environmental agents, a concern that continues for Gulf War I veterans and OEF/OIF veterans and military personnel today