O-061 Comparative study of sperm deoxyribonucleic acid (DNA) content and damage between fertile and infertile men using flow cytometry and gel electrophoresis

due, and D 1 deletion could further restrict DHP binding. A more extensive deletion encompassing the IVS3 DHP binding site and D1 region has been identified in one cDNA clone. Antibodies detecting the a l subunit in the postacrosome region of the h u m a n sperm head identify two antigenic species, - 1 8 0 kDa and 60 kDa, on Western blots of membrane proteins. Conclusions: The 3 splice variants of the h u m a n testis VDCC a l subunit may represent different alleles of the a l gene, as the RNA template was prepared from 20 pooled testes. Differential expression of a shortened a l subunit, missing D1 or IVS3 and D1, may help explain variations in the ability to undergo a progesterone-stimulated AR observed in motile sperm populations from different men and will provide a new tool for the evaluation and diagnosis of h u m a n male infertility. No direct counterpart of the h u m a n a l diversity was observed in rat testes cDNA nor has been reported in somatic tissues. Likewise, a 60 kDa protein sharing a l subunit antigenic epitopes has not been described. The altered DHP binding sites in the a l subunit are targets for non-hormonal pharmaceutical and contraceptive vaccine development. An advantage of this approach to h u m a n male contraception is its ready reversibility as a result of the need to target only free cells, rather than to induce a proliferative state change in an organized tissue as required with current hormonal protocols. (Supported by ASRM/Organon Grant in Reproductive Medicine to LOG and NIH Grant No. ES 06100 to SB.)

O-061 Comparative Study of Sperm Deoxyribonucleic Acid (DNA) Content and Damage B e t w e e n Fertile and Infertile Men Using Flow Cytometry and Gel Electrophoresis. 1S. Jacob, 2M. O'Donoghue, 1D. M. Jenkins, 2j. K. Collins, 3M. M. Cole, 2R. O'Shea. 1Dept. of OB/ GYN, 2Dept. of Microbiology, 3Dept. of Statistics. University College Cork, Ireland. Objectives: (1) To compare the sperm DNA profile of infertile men with that of fertile controls using DNA flow cytometry. (2) To study sperm DNA damage by agarose gel electrophoresis. Design: A prospective case control study. Setting: Infertility Clinic at University Teaching Hospital. Subjects: Eleven men from infertile relationships and 10 fertile men whose partners had recently delivered. Methods: The sperm DNA profile was analysed by Propidium Iodide stained DNA flow cytometry using a Coulter Epics Elite Fluorescence-Activated Cell Sorting (FACS) machine. The machine was standardized by running standard beads (Standard Brite, Coulter) at random during the procedure. The histograms obtained were superimposed on one another to get standardized graphs. The frequency of subhaploid sperms in infertile cases were compared with those of fertile men using the Mann Whitney test. Evidence of apoptosis or necrosis in sperm was determined by agarose gel electrophoresis. Results: The mean fluorescence of the standard beads

did not vary significantly during the procedure. The DNA profiles of the 10 fertile controls were homogenous with a mean peak fluorescence at 197.47 (SD = 47.4). Those of the infertile cases were heterogeneous with mean peak at 175.27 (SD = 64.1). In the infertile men there was a significant increase in the sperm population in the subhaploid DNA region (p = 0.0242 Mann Whitney test). Three of these patients had apoptosis and 4 patients had necrosis on Agarose gel electrophoresis. Neither was seen in the fertile controls. Conclusions: The sperm DNA profiles of infertile men are heterogeneous with significantly increased numbers of subhaploid sperms compared to fertile controls. There is increased frequency of sperm apoptosis and necrosis in infertile men. These findings may allow for improved screening of spermatozoa for use in assisted Reproductive Technology. A further randomized double blind study using larger sample size is proposed.

0-062 Immunolocalization of Ejaculated Spermatids and Prediction of Testicular Pathology and Sperm Extraction in Non-Obstructive Azoospermia Using Semen Characteristics. 1U. I. Ezeh, 2M. Martin, 1j. St. John, 1C. L. R. Barratt, 1'2H. M. D. Moore, 1I. D. Cooke. ~'2University Depts. of OB/GYN and Molecular Biology, Jessop Hospital for Women, Sheffield, UK. Objective: Despite current success in treating men with non-obstructive azoospermia using testicular sperm and ICSI, limiting testicular biopsy to those with a high chance of having testicular sperm has not been possible because of the poor predictive value of the current clinical and laboratory methods. However, there is a good correlation between the number of testicular spermatids and sperm count in men with oligozoospermia. 1 The aim of this study was to correlate testicular pathology and sperm extraction with semen characteristics of men with non-obstructive azoospermia. Design: Controlled prospective study: Semen volume and pH, and visualization of at least one fluorescent stained spermatid in the semen sample were used as pre~ dictors of the recovery of at least one testicular spermatozoon and of histological patterns. Materials and methods: Washed cellular elements in the ejaculate of 18 patients with non-obstructive azoospermia smeared on microscope slides and fixed in 100% methanol, were first incubated with acrosome specific monoclonal antibody (18.6) 2 and then FITC-labelled anti-mouse goat IgG, and examined by epifluorescent microscopy at a wavelength of 495-520 nm. Semen from 5 men with oligozoospermia and 5 men with non-obstructive azoospermia served as positive and negative controls, respectively. Clinical evaluation and testicular biopsy were performed. Results: Both groups were comparable with regards to age, plasma FSH level, and testicular volume. All 8 (47%) patients who had positive immunofluorescence (spermatid present) had spermatozoa retrieved from their testes (5 hypospermatogenesis, 3 focal spermatogenesis), and all 10 patients with negative immunofluorescence (sperAbstracts

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