layers, i.e., cardiomyocytes. A conventional mouse strain often utilized in reproductive laboratories was capable of producing ESCs with similar patte...

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layers, i.e., cardiomyocytes. A conventional mouse strain often utilized in reproductive laboratories was capable of producing ESCs with similar patterns of differentiation to the permissive 129 strain. Supported by: Institutional

P-819 Retention of Primary Follicles and Response to Exogenous Human Gonadotropins for Squirrel Monkey Ovarian Fragments as Xenotransplants in NOD/SCID Mice. J. C. Snead, A. Spiekerman, T. J. Kuehl. Scott and White Clinic, Temple, TX. OBJECTIVE: This study evaluated the potential for maintenance of primary follicles in xenotransplants of squirrel monkey ovarian fragments to subcutaneous sites in the NOD/SCID mouse. DESIGN: A prospective cohort of NOD/SCID mice received xenografts which were examined after 3 and 8 weeks and 6 months. MATERIALS AND METHODS: Two trials were conducted. In one, six small (1 mm3) fragments of ovaries from female squirrel monkeys were transplanted subcutaneously into the right flank of 12 male NOD/SCID mice. In a second trial, an equivalent volume of ovarian tissue was transplanted as one larger fragment into each of 12 mice. Mice with smaller fragments were euthanized at either 3 or 8 weeks and mice with the larger fragments were euthanized at 6 months. Mice received intra-peritoneal injections of 1 IU hMG 3 times per week during the engraftment intervals. At the end of the engraftment intervals, mice received an ovarian stimulation protocol consisting of daily injections of 12 IU hMG for 9 days. On the evening of Day 9, each mouse was given 50 IU hCG to initiate oocyte maturation. At 16 hours after hCG injection on Day 10, mice were euthanatized for collection of blood and xenotransplants for analyses of plasma reproductive steroid levels and tissue histology. RESULTS: Three mice died prematurely: one from pneumonia and two from injection accidents. Xenotransplants implanted and were maintained for recovery in 20/21 mice. Primary follicles were found in 12 of 20 transplants. Antral follicles were found in 1/6 mice at 3 weeks, 2/6 mice at 8 weeks, and 4/9 at 6 months. The largest antral follicle was 3mm diameter with oocyte visible in cumulus mass. Estradiol (p⫽0.27) and progesterone (p⫽0.09) levels did not differ between the three groups based on duration and nature of xenotransplants. Animals with primary or antral follicles did not differ from those without for either estradiol (p ⫽ 0.72) or progesterone (p ⫽ 0.49). Ovarian fragments survived the engraftment intervals with limited numbers of primary follicles and interstitial cells. Blood vessels were visible within the xenotransplants coming from surrounding mouse tissue (skin in 18/20 mice and muscle in 2/20 mice) at all engraftment intervals. Antral follicles had fewer layers of granulosa and thecal cells than those of intact ovaries. CONCLUSION: Squirrel monkey ovarian xenotransplants equivalent to 1/6th of an ovary survive and maintain a small fraction of primary follicles seen in original tissue. Mice receiving hMG during the engraftment interval and hMG and hCG immediately prior to tissue collection have blood levels of estradiol and progesterone that do not vary in relationship to duration of study interval or success at maintaining any primary or antral follicles. However, in this trial we demonstrated survival of ovarian follicles from fresh ovarian fragments and development of antral follicles. Supported by: Support from the Noble Centennial Endowment.

P-820 The Effect of Poor Fertilization vs. Poor Oocyte Recruitment on Successful Outcomes. J. Zenker, D. Nogueira, J. R. Trimarchi, R. Hackett, G. Celia, D. L. Keefe. Women and Infants’ Hospital, Brown University, Providence, RI. OBJECTIVE: We intended to determine the chance of IVF success for cycles in which few embryos are available due to limited oocyte yield or poor fertilization. There are two general situations that result in the availability of few embryos at the time of transfer: 1. Retrieval of few oocytes followed by relatively good fertilization. 2. Retrieval of many oocytes followed by poor fertilization. We sought to determine whether the cause of a low number of embryos available for transfer provides an indicator of outcome and therefore could be used to adjust the number of embryos transferred.


DESIGN: Retrospective analysis of data from 658 IVF/ICSI cycles that occurred from January 2002 to February 2005 at a large IVF program. Cycles using donor oocytes or preimplantation genetic diagnosis were excluded. MATERIALS AND METHODS: Clinical pregnancy rate (PR) and implantation rate (IR) were calculated for the following groups; 1) patients with 1-4 oocytes at retrieval with ⱕ 25% fertilization; 2) patients with 1-4 oocytes at retrieval with ⱖ 70% fertilization; 3) patients with 8-20 oocytes at retrieval with ⱕ 25% fertilization; and 4) patients with 8-20 oocytes at retrieval with ⱖ 70% fertilization. Fertilization rates were calculated as the number of 2 pronuclei on day 1 divided by the number of oocytes inseminated. Data was analyzed by Chi-square and Fisher’s exact test as appropriate. RESULTS: PR and IR did not differ significantly among groups. Groups 1 & 2 (ⱕ 4 oocytes) had low IR’s independent of fertilization rate. IR’s for groups 3 & 4 (high oocyte yield) varied, depending on % fertilization (P ⬍ 0.0001). IR’s for groups in which few embryos were available (groups 1-3) did not differ, indicating that the number of embryos available, regardless of whether arising from few oocytes or poor fertilization, provides a low implantation rate. As expected, group 4 had the highest PR and IR (P ⬍ 0.001).


Different letters denote significant differences (P ⬍ 0.001)

CONCLUSION: For the individual patient undergoing IVF, the number of embryos transferred must be adjusted to maximize pregnancy and minimize multiple gestation rates. We determined that regardless of the cause, whether from poor ovarian reserve or poor fertilization, a limited cohort of embryos signals a poor prognosis for IVF success. This effect is not limited to PR, but also includes reduced IR. The clinician and patient may wish to consider increasing the number of embryos transferred to overcome reduced IR for cases in which an adequate number of oocytes are retrieved but fertilization is poor. Supported by: None. P-821 Comparison of Automated and Manual Sperm TUNEL Assay Classification. M. L. Sanderson, B. R. Emery, D. T. Carrell. University of Utah School of Medicine, Salt Lake City, UT. OBJECTIVE: The TUNEL assay has been shown to be as effective as SCSA in detecting sperm chromatin damage and is used clinically to evaluate sperm chromatin integrity in samples of patients undergoing fertility evaluation. Inter-technician variability in classification of TUNEL reacted sperm poses a concern in providing consistent results. We have tested a Metasystems© designed classification program using the Zeiss Axioplan 2 Imaging© fluorescent microscope to evaluate sperm TUNEL results compared to manual data obtained from a single technician. DESIGN: A prospective comparison of an automated sperm TUNEL assay classification system versus manual classification. MATERIALS AND METHODS: Semen samples from 34 patients were evaluated for sperm chromatin damage using the TUNEL assay. Samples were evaluated in their fresh state, and after undergoing density gradient centrifugation. 500 sperm were evaluated manually by a trained technician and by Metasystems© designed classification program using the Zeiss Axioplan 2 Imaging© fluorescent microscope. Sperm cells were determined using 2.5 ␮m2 -100 ␮m2 as a parameter for size . TUNEL reaction was determined by taking the total pixel intensity in the area contour measured divided by integration time. An arbitrary measure of above and below 40 was used to determine reacted and non-reacted sperm respectively. The