Incidence and post-operative outcomes of accidental ligation of the testicular artery during microsurgical varicocelectomy.

Tuesday, October 23, 2001 2:30 P.M. O-128 Incidence and post-operative outcomes of accidental ligation of the testicular artery during microsurgical varicocelectomy. P. T. Chan, E. J. Wright, M. Goldstein. Cornell Institute for Reproductive Medicine and Dept of Urology, Weill Medical Coll of Cornell Univ, New York, NY. Objective: Varicocelectomy is the most commonly performed surgical treatment for male infertility. Microsurgical varicocelectomy enhances the ability to identify and preserve the testicular artery. The objective of this study was to determine the incidence of and evaluate the outcomes after accidental ligation of the testicular artery during microsurgical varicocelectomy. Design: Retrospective analysis in a tertiary infertility referral center. Materials/Methods: From 1984 to 2001, 2048 men who had microsurgical varicocelectomy performed by a single surgeon were evaluated. Accidental artery ligation was confirmed intra-operatively by observation of pulsatile twitching of the ligated vessel stump under 25⫻ magnification. Results: Accidental artery ligation was identified in 18 out of 2048 cases (0.9%). The mean age was 37.8 (30 – 49) yr. Infertility (89% primary), for an average duration of 2.2 (0.5– 6) yr, was the chief complaint in all cases, with 11% (2/18) with associated pain. The majority were grade II varicoceles (67% right and 56% left), while Grade III were found on the left side in 33% (6/18) of cases. Varicoceles were bilateral in 12/18 (67%) cases. Pre-operatively, average testis volume was 17 ml, testosterone was 178 (168-505) ng/dL and FSH 20.0 (3.1–72.6) U/L. Azoospermia was found in 28% (5/18) of cases. Median sperm counts was 12 (0 –78) million/ml with 43% (22–70%) motility and 41% (1–79%) normal morphology. More than one arterial structure was identified intra-operatively in all cases. A single major artery was ligated in 78% (14/18) of left- and 32% (4/12) of rightsided operation. The median follow-up period was 16.5 (1–120) months. No post-operative testicular atrophy occurred. Significant improvement in sperm count was noted in 83% (10/12) of men, including return of motile sperm to ejaculate in 40% (2/5) of azoospermic cases. Post-operative testosterone increased significantly in 80% of men by 262 ng/dL. In the 60% of men who were not on hormonal therapy, 83% (5/6) had a significant increase in serum testosterone. Pregnancies were achieved naturally in 17% (2/12) of cases, with IUI in 8% (1/12). Conclusions: Prior to the use of the operating microscope, the incidence of accidental testicular artery ligation during varicocelectomy was unknown. We report a low incidence (0.9%) of unilateral testicular artery ligation and no instances of bilateral ligation. Preservation of cremasteric and/or secondary testicular arteries likely contributed to a low incidence of adverse outcomes. The pregnancy rate in the ligated group (25%) however, was lower than that reported previously (43%) for the entire cohort. This may be due to the higher incidence of pre-operative azoospermia in the ligated cohort. It is possible that the smaller testes usually associated with azoospermia have smaller testicular arteries that are more prone to accidental ligation.

Tuesday, October 23, 2001 2:45 P.M. O-129 Characterization of the luxoid mouse: sterility due to spermatogonial stem cell defects. J. L. Morris, F. W. Buaas, R. E. Braun. Univ of Washington, Seattle, WA. Objective: The success of male reproduction depends on the ability of spermatogonial stem cells to balance two competing pathways: self-renewal versus differentiation. Disruptions in spermatogonial development lead to infertility. This study evaluated the sterility of a spontaneous mouse mutant, luxoid (lu), first described in 1955. Males homozygous for luxoid have skeletal abnormalities and are infertile. In this study, we characterized the histological phenotype of the luxoid mutant sterility and initiated gene identification experiments.

FERTILITY & STERILITY威

Design: A histological and immunohistochemical characterization of the male luxoid reproductive phenotype. Materials/Methods: Heterozygous mutant luxoid mice, obtained from the Jackson Laboratory, were intercrossed to generate homozygous mutant males. As the gene which is mutated in luxoid is unknown, micro-satellite markers were used to define the genetic interval that encompasses the mutated gene on chromosome 9. These markers were used for genotyping. Testicular histology was evaluated at several time points. Homozygous mutants and heterozygous controls were sacrificed at 4, 5, 8, 24, and 32 weeks. Heamatoxylin and eosin (H/E) sections of testes were evaluated for spermatogenesis. Immunohistochemistry was performed on testes sections to identify the presence of germ cell nuclear antigen (GCNA 1), an antigen present in spermatogonia and spermatocyte nuclei. Sections were analyzed for the presence or absence of staining in mutant and heterozygous males. Spontaneous recombinants and their backcross progeny were evaluated with micro-satellite markers. Chi-square statistical analysis was used when appropriate. Results: Homozygous luxoid mutants were found to have significantly more abnormal seminiferous tubules in the testes H/E cross sections than their controls (p ⬍ .001). Abnormalities noted were spermatogonial stem cell defects. Abnormal tubules were found to be missing spermatogenic waves while others displayed a sertoli cell only phenotype. Immunohistochemistry for GCNA 1 showed significantly decreased staining in luxoid mutants compared to controls (p ⬍ .001). 202 progeny analyzed from heterozygous intercrosses generated 28 homozygous mice, which deviated statistically from the expected amount of 51 (p ⬍ .005). Analysis of recombinant animals generated from the intercrosses enabled us to narrow the location of the luxoid gene to a 1.1 cM region on chromosome 9. Conclusions: These data indicate that the sterility of the luxoid male is due to a defect in spermatogonial stem cell maintenance and differentiation. The observation of decreased numbers of mutant progeny suggests that the luxoid gene is also involved in embryonic survival. With the region containing the mutated luxoid gene on chromosome 9 defined to 1.1 cM, we are closer to identifying the responsible gene. Identification of the gene causing this spermatogonial stem cell defect has implications for future stem cell therapies related to male infertility.

Tuesday, October 23, 2001 3:00 P.M. O-130 Separation of HIV-1 from the motile sperm fraction: comparison of gradient/swim-up and double tube techniques. J. A. Politch, C. Xu, L. Tucker, D. J. Anderson. Brigham and Women’s Hosp, Boston, MA. Objective: Semprini et al (Lancet 1992;340:1317) and Marina et al. (Fertil Steril 1998;70:35) have reported successful insemination of HIV seronegative women with processed semen from HIV seropositive partners without infection of the women or their resultant offspring. Both investigators utilized sperm processing protocols that combined density gradient with swim-up procedures. We recently have developed a new technique that utilizes a “double tube” design. Briefly, the method consists of an inner conical tube that has been inserted in an outer tube. A discontinuous gradient of sperm separation medium is layered inside the inner tube, which has an opening at its apical end. Following centrifugation, the motile sperm pass through this opening to the apical end of the outer tube. The inner tube and its contents are then removed and discarded, thus minimizing potential contamination of the motile sperm fraction. The present study sought to compare the double tube and the gradient/swim-up techniques in respect to HIV contamination of the motile sperm fraction and sperm yield. Design: Laboratory experiments utilizing semen samples from HIV seronegative normal donors spiked with HIV compared the gradient/swim-up and the double tube methods for exclusion of HIV and sperm yield. Materials/Methods: Semen samples (n ⫽ 13) from HIV seronegative normal donors were spiked with high concentrations (range: 10 -1 - 103 TCID50) of HIV-1 (MN HIV-1 strain) propagated in H9 cell cultures. Spiked samples were loaded on 15 ml tubes (gradient/swim-up) or double tubes containing gradients (47%/90%) of sperm separation medium (either Percoll or Isolate) and spun for 20 minutes at 400 ⫻ g. Upper layers and interfaces were carefully removed and pellets containing motile sperm were resuspended, placed in clean tubes and washed with 3 ml Ham’s F-10/HSA. For the gradient/swim-up condition, the motile sperm fraction was subjected

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