Effect of Oxidative Stress on Mouse Oocyte Cytoskeleton and Embryo Development

D. Barad, N. Santoro. Montefiore’s Institute for Reproductive Medicine and Health, Hartsdale, NY; Albert Einstein College of Medicine, Bronx, NY. OBJECTIVE: To evaluate embryo quality in a dual media culture system. DESIGN: Retrospective study. MATERIALS AND METHODS: Data from initial IVF cycles only between January 2002 and December 2004 were analyzed. Oocytes were inseminated by IVF or by ICSI and cultured overnight in groups of 2-4. Only cycles with 7 or more zygotes were included to facilitate comparison between media. After confirmation of 2 pronuclei day 1 post-insemination, sibling zygotes from each patient were randomized equally in groups of 2-4 to G1v3 medium ⫹ 10% HSA (Vitrolife) or to Quinn’s Cleavage medium ⫹ 10% HSA (QHTF, Cooper Surgical). Embryos were cultured in 20 °L culture drops under oil with 5.5% CO2, and the pH of both media was kept at 7.2. On day 3 of culture embryos were transferred to the uterus, discarded or moved into sequential media for blastocyst formation. The best embryos for transfer were combined from both media. Univariate analysis was employed to assess the association between media type (G1v3 or QHTF) with number of embryos formed, blastomere number and % fragmentation on day 3. Blastomere number and % fragmentation were averaged per patient per media and compared as single values. A p value of ⬍0.05 was considered statistically significant. RESULTS: A total of 351 cycles (188 ICSI and 163 IVF) of patients aged 24 to 48 years (mean 34.9) were analyzed. Each patient served as their own control for age, suppression protocol and stimulation protocol. The average cell number per embryo (⫹/- SEM) was not significantly different when cultured in G1v3 medium versus QHTF medium. However, the degree of fragmentation of embryos in G1v3 medium was significantly higher than in QHTF medium with a level of significance of pⱕ 0.0001 as determined by ANOVA.

CONCLUSIONS: A dual media system provides maximization of a patient’s embryo quality by controlling for any media variations. This study evaluated the impact of two culture media on embryo development. While the rate of embryo development was not different between the two media, embryo fragmentation was significantly improved in QHTF. Further analysis of embryo development to blastocyst, cryopreservation, survival and pregnancy rates is currently being conducted to fully evaluate the strength of the dual media culture system. Supported by: None.

Monday, October 17, 2005 6:15 p.m. O-45 Effect of Oxidative Stress on Mouse Oocyte Cytoskeleton and Embryo Development. W. Choi, X. Zhang, R. K. Sharma, T. Falcone, A. Agarwal. Cleveland Clinic Foundation, Cleveland, OH. OBJECTIVE: Oxidative stress has been implicated in the pathophysiology of poor oocyte and embryo quality during the in-vitro culture. Microtubule architecture and chromosomal arrangement are important in oocyte meiosis and subsequent development of the embryo. The objective of this study was to 1) evaluate the effect of hydrogen peroxide on oocyte spindle structure (microtubule morphology and chromosomal alignment) and 2) evaluate the embryotoxic effects following exogenous exposure to hydrogen peroxide (H2O2). DESIGN: Prospective in vitro study MATERIALS AND METHODS: Frozen mouse oocytes (metaphase II stage; n ⫽ 199) and embryos (n ⫽ 326) were used (Embryotech Laboratories Inc., Wilmington, MA). Hydrogen peroxide was diluted

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with human tubal fluid (HTF) to give working concentration from 12.5 to 150␮M. Controls were incubated with HTF alone. Immunohistochemical staining was done to evaluate the effects of H2O2 on the oocyte microtubule and staining with propidium iodide for chromosomal alterations. Stained oocytes were scored for alterations in microtubule morphology and chromosomal alignment under a Fluorescent (Leica, Germany) and scanning Confocal microscope (Leica Lasertechnik GmbH, Heidelberg, Germany). Scores of 1-2 were considered as being normal for oocyte microtubule morphology and chromosomal alignment, and 3-4 as abnormal (modified from Saunders and Parks, 1999). For blastocyst development rate (% BDR), embryos were divided into 5 groups and exposed to varying concentrations of H2O2. Embryos were examined after 72h for %BDR. RESULTS: A dose dependent decrease in normal microtubule morphology and chromosomal alignment was seen with increasing concentration of H2O2. Hydrogen peroxide exposure at 25 ␮M caused a significant decrease in the percentage of normal microtubule morphology and chromosomal alignment (Fig. 1A). Similarly, a dose dependent decrease in % BDR was seen with increasing concentrations of H2O2 compared with controls (Fig.1B). A significant increase in embryotoxicity was seen at 50␮M H2O2. Higher concentrations (⬎60 ␮M) were embryotoxic.

Fig. 1: (A) Normal percentage of microtubule (MT) and chromosomal alignment (CH) in metaphase II oocytes and (B) % blastocyst development rate in embryos following exposure to various concentrations of H2O2. CONCLUSIONS: Hydrogen peroxide at 25 ␮M can induce alterations in both microtubule and chromosome alignment. Higher concentrations are embryotoxic. Oxidative stress may be one of the many causes of poor oocyte quality in in-vitro culture system. Use of antioxidants may be beneficial in reducing/reversing these changes. Supported by: None

MENTAL HEALTH PROFESSIONAL GROUP Monday, October 17, 2005 3:45 p.m. O-46 The Bioethics of Parenthood: in Pursuit of the Proper Standard for Gatekeeping in the Clinical Setting. R. F. Storrow. Pennsylvania State University, Carlisle, PA.

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