regardless of the male’s age or the number of years since the last fatheredconception.

P-343

Clinical diagnosis in men undergoing infertility investigation. Fabio F.Pasqualotto, Antonio M. Lucon, Bernardo P. Sobreiro, Jorge Hallak,Eleonora B. Pasqualotto, Sami Arap. Univ de Sao Paulo, Sao Paulo, Brazil.

Purpose: We evaluated whether the changes in the diagnostic techniqueshave altered the epidemiology of male factor infertility in the 1990s. Anexamination of the patients evaluated and treated in a university hospital inBrazil, without charging for clinical appointment or treatment whatsoevermay yield a different epidemiologic data on the characterization of malefactor infertility services because costs is not a determining factor.

Design: Retrospective study.Setting: University-based center.Material and Methods: From September 1999 to November 2002, 822

patients were evaluated for infertility purposes. The mean age was 39.03 �7.34 (range 18 to 66 years old). We divided our infertility patients accordingto the clinical diagnosis.

Main Outcome Measure: Clinical diagnosis.Result(s): Most of the patients came for infertility diagnosis with vari-

cocele (n � 282; 34.3%), idiopathic infertility (n � 260; 31.6%) or had hadseminal tract obstruction (n � 85; 10.34%). Least commonly but equallyimportant causes found were mumps (n � 43; 5.23%), pyospermia (n �37;4.5%), systemic diaseases (n � 36; 4.37%), testicular failure (n � 34;4.13%), criptorchidism (� 14; 1.7%), ejaculatory dysfunction (n � 11;1.3%), genetics (n � 9; 1.1%), endocrinopathies (n � 4; 0.5%), testicularcancer (n � 4; 0.5%), and testicular torsion (n � 3; 0.36%). In 94 patients,a women was considered infertile.

Conclusion(s): Even with the changes in reproductive healthcare occurredin terms of availability of care because of managed care systems and thediagnostic testing and treatment modalities available, varicocele and semi-nal tract obstruction remain the main known causes of male infertility.However, a clinician should not forget other treatable causes of maleinfertility such as pyospermia, systemic diseases or testicular cancer. Westrongly believe that molecular biology techniques (microdelection of the Ychromosome, androgen receptor, CFTR testing for cystic fibrosis) willincrease even more the percentage of genetics causes diminishing thepercentage of idiopathic infertility.

P-344

Correlation of testis biopsy histology and fine needle aspiration innonobstructive azospermia. The Buenos Aires 3 years experience.Mariano Cohen, Jose Vazquez, Martin Del Sordo, Federico Muro, HernanNataskin, Osvaldo Mazza. Hosp de Clinicas, Buenos Aires, Argentina.

Correlation of testis biopsy histology and Fine Needle Aspiration Map-ping in nonobstructive azospermia. The Buenos Aires 3 yearsExperience

Objectives: To correlate the sperm finding from testicular biopsy to testisFine needle aspiration (FNA) mapping in infertile men with nonobstructiveazospermia (NOA).

Design: Retrospective study of infertile men with NOA.Methods: Testis FNA mapping was performed concurrently with open

“window” testis biopsies under local anesthesia in 34 men with NOA(sending to histology and criopreservation) over three year period at Hos-pital de Clinicas Jose de San Martin University Hospital .

Results: A total of 35 patients were studied by biopsy and FNA. A meannumber of 7 FNA sites and 1.0 biopsy per testes .Mature sperm with tailswere detected by FNA in 17 patients (48.5%) compared to 12 patients(34.2%) by biopsy histology alone. In addition 2/5 men with a complete latematuration arrest (n�5 patients) histologic pattern had successful spermretrieval by directed TESE after FNA suggested a focus of mature sperm.We consider late maturation arrest as a good predictor of finding sperm atthe retrieval .Other histologic patterns encountered include early maturationarrest (n � 3 patients) and pure Sertoli cell only (n�10 patients in one ofthese patients were found 5 sperm with criopreservation). No complicationswere encountered in any patient.

Conclusion: Our initial experience confirms the fact that a significantfraction of men with NOA can have sperm within the testis .In ours hands

,FNA mapping appears more sensitive than open testis biopsy in thedetection of mature sperm in these patients.

MALE REPRODUCTION AND UROLOGY: RESEARCH

P-345

Influence of Prenatal Diethylstilbestrol (DES) and dietary phytoestro-gens on sperm function and reproductive organ development in malemice. Ronald S. Suh, Xia Wang, Dana A. Ohl, Fred S. vom Saal, Gary D.Smith. Univ of Michigan, Ann Arbor, MI; Univ of Missouri, Columbia,MO.

Objective: The influence of exogenous estrogens on male reproductiveparameters remains controversial. Prenatal estrogen exposure may influencemale organ development and dietary phytoestrogens have been shown toexert physiologic estrogen activity, with the possible consequence of endo-crine disruption. We sought to determine the effects of both in uteroexogenous estrogen exposure and dietary phytoestrogen intake on malereproductive organ development and sperm physiology in mice.

Design: Experimental animal study.Materials and Methods: In utero exposure of males was performed on

pregnant mice fed corn oil with no DES (control), 0.1�g/kg/day DES(low-dose), and 50�g/kg/day DES (high-dose) on gestation days 11-17.Subsequent male pups (n�36) were then fed Purina 5001 (Diet A, 491mg/gdiet phytoestrogens) or Purina 5002 (Diet B, 159 mg/g diet phytoestrogens)until sacrifice on postnatal day 90. Testis and epididymal blotted weightswere obtained prior to sperm isolation from the cauda epididymis byswim-out after mincing. Morphology, sperm capacitation and acrosomereaction (AR) was evaluated. Cumulus masses from gonadotropin hyper-stimulated females were inseminated with sperm at a concentration of 1million motile sperm per ml for in vitro fertilization (IVF). Percent fertili-zation, defined as development to the 2-cell stage, and percent embryodevelopment to blastocyst was recorded for each male. Outcome meanswere analyzed with ANOVA, as well as 2-way ANOVA to detect interac-tive effects, with p�0.05 considered significant.

Results: In utero high-dose DES resulted in decreased testis and epidid-ymal weight, but was not statistically significant unless combined testis/epididymal weight was analyzed (p�0.023). No significant DES effect wasseen on sperm morphology, sperm capacitation or acrosome reaction (allp0.05). Fertilization (p�0.477) and embryo development (p�0.410) withIVF was also not adversely affected by in utero DES exposure. No dieteffect was seen on epididymal or testis weights or sperm morphology (allp0.05). A decrease in spontaneous capacitation (p�0.001) and AR(p�0.012) was found in the higher phytoestrogen diet. Higher dietaryphytoestrogen content also resulted in increased fertilization rates(p�0.014) but lower development to blastocyst (p�0.007). Two-wayANOVA analysis showed no interaction between DES exposure and dietaryphytoestrogen content among measured outcomes.

Conclusion: In utero high-dose DES exposure decreased combined epi-didymal/testis weight but had no influence on sperm function or in vitrofertilizing ability. Higher dietary phytoestrogen intake resulted in desirabledifferences in sperm capacitation and acrosome reaction, as well as im-proved rates of fertilization with IVF. No interaction between these twoexposures existed.

P-346

Sonic hedgehog pathway genes and protein in the adult mouse epidid-ymis. Terry T. Turner, Daniela Bomgardner, Jason P. Jacobs. Univ ofVirginia Sch of Medicine, Charlottesville, VA.

Objective: To determine if genes in the sonic hedgehog (shh) pathway areexpressed in the murine epididymis and, if present, to determine if theirexpression is affected by lumicrine factors from the testis.

Design: The shh pathway genes and proteins, shh, patched (ptc) and Gli-1were examined for in the murine epididymis and the effect of testicularfactors on their expression was determined.

Materials and Methods: Adult male mice were used in 4 groups of 9animals, each. The mice were subjected to unilateral efferent duct ligation(EDL) for 1, 3, 10, or 15 days. Their contralateral sides were subjected tosham operation. At the appropriate time all epididymides were harvested

FERTILITY & STERILITY� S235

and were used either for histology to examine lumen diameter, epithelialheight, and level of sperm clearance from lumen; for RNA extraction ofcaput, corpus, or cauda regions; or for obtaining protein extracts from thosesame regions. RNA and protein was also extracted from prostates andsham-side testes. Semi-quantitative RTPCR was used to assess reproductivetract expression of shh, ptc, and Gli-1 genes in each epididymal region ineach group after EDL. Western blot analysis was used to assess proteinpresence. Numerical data were analysed by analysis of variance followed byTukey’s range test(p�0.05).

Results: The murine epididymis is divided into 4-5 lobules or intrar-egional segments. Spermatozoa had cleared the caput segments by 24 hrsafter EDL and had cleared to the proximal cauda by 15 d after EDL. Shh,ptc, and Gli-1 genes are all three expressed in the adult mouse testis,epididymis, and the prostate. Epididymal expression of all three genes isgreater than in testis or prostate and shh gene expression increases fromproximal to distal epididymis. There was no significant change in ptc orGli-1 expression by region. Efferent duct ligation causes an increase in thesegene expressions in a region specific manner. Proteins evident from Westernblot analysis appear consistently with the gene expression data.

Conclusion: The shh pathway, important in pattern recognition duringdevelopment of the GU tract, continues to be expressed in the adultepididymis where testicular factors tend to suppress its expression. The shhpathway may play a heretofore unsuspected role in maintenance of adultepididymal function and resulting sperm fertility.

P-347

Cav2.3 (a1E) Ca2� channel participates in the control of sperm func-tion. Yu Sakata, Yasufumi Shimizu, Reiko Minaguchi, Takeshi Aso. To-kyo Medical and Dental Univ, Tokyo, Japan.

Objective: The voltage-dependent Ca2� channel (VDCC) is one of theimportant pathways of Ca2� influx into sperm. Ca2� channel containinga1 2.3 (a1E) subunit (Cav2.3 channel) is suggested to be present in sper-matozoa, though the function is not documented yet partly because of thelimited availability of its specific blocker. To know the function of Cav2.3channel in spermatozoa, we conducted the following experiments.

Design: Sperm function was compared between wild-type (Cav2.3�/�)and homozygous mutant (Cav2.3-/-) mice.

Materials and Methods: Epididymal sperm from wild-type (Cav2.3�/�)or homozygous mutant (Cav2.3-/-) mice (aged 8 to 30 weeks) were used inall the experiments in a blind manner. Computer-assisted sperm assay(CASA) system with a sperm motility analyzer (IVOS Ver 10.7; Hamilton-Thorne Research, Beverly, MA, USA) was used for the analysis of spermmotility. Ca2� influx was evoked by exposure of the sperm to solubilizedzona pellucida, mannose-BSA and high-K solution (K8.6). The fluorescenceintensities of Fura-2 were recorded and analyzed by Argus 50 Calciumimaging system (Hamamatsu Photonics, Japan). For statistical analyses, anunpaired t-test was used.

Results: A computer-assisted sperm motility assay (CASA) revealed thatstraight-line velocity and linearity were greater in Cav2.3-/- sperm thanthose in Cav2.3�/� sperm. ZP induced slightly slower tendency in Ca2�transient of Cav2.3-/- sperm than that of Cav2.3�/� sperm, whereasmannose-BSA induced significantly slower Ca2� transient in Cav2.3-/-sperm. On the contrary to these agonists, Ca2� transient caused by KCldepolarization tended to be higher in Cav2.3-/- sperm than Cav2.3�/�sperm.

Conclusion: The principal finding obtained in our study on the Cav2.3channel function in spermatozoa is that this channel participates in thecontrol of swimming behavior in sperm. Although the exact mechanismsunderlying these changes in the motility parameters remain elusive, ourresult is compatible with the idea that decreased [Ca2�]i in Cav2.3-/- spermlead to the decreased bending waveform asymmetry. In this study, we alsoinvestigated whether Cav2.3 channel is involved in Ca2� transient leadingto acrosome reaction. This suggests that Cav2.3 channel is involved in theCa2� transient induced by mannose-BSA, but this channel is not indis-pensable to the Ca2� transient induced by zona pellucida.

P-348

Sperm kinematics of normozoospermic specimens after stimulation byvarying concentrations of a specific inhibitor of CGMP phosphodies-

terase type-5 (Sildenafil). Sandro C. Esteves, S. Glina, R. Wonchockier,N. R. Correa. ANDROFERT-Centro de Referencia em Infertilidade Mas-culina, Campinas, Sao Paulo, Brazil; Unidade de Reproducao Humana,Hosp Israel Albert, Sao Paulo, Brazil.

Objectives: Cyclic nucleotides (cAMP and cGMP) are involved in spermmotility, capacitation, and in the acrosome reaction of many animal species.It is known that human sperm motility can be enhanced by cyclic adenosinemonophosphate (cAMP) phosphodiesterase (PDE) inhibitors. This studyinvestigated whether the direct addition of sildenafil, a specific inhibitor ofcGMP PDE type-5, to the ejaculates of normal men can enhance spermkinematics.

Design: Semen specimens were divided into four equal aliquots: the firstreceived no treatment (control) and the others were incubated with 0.001mi-croM, 0.01microM and 0.1microM of sildenafil (Pfizer, Sandwich, UK) for1 hour. After treatment, all aliquots were processed by the swim-up tech-nique and underwent further in vitro incubation for 3 hours.

Materials and Methods: IRB approval was obtained for this study. Semensamples were obtained from 21 healthy subjects with proven fertility. Eachspecimen was split and treated as described above. Each aliquot wasevaluated for percent motility, curvilinear velocity (VCL), straight-linevelocity (VSL), average path velocity (VAP), amplitude of lateral headdisplacement (ALH) and hyperactivation (HYPER) using a computer as-sisted semen analyzer (CASA). Sperm kinematics was assessed immedi-ately after sildenafil treatment and after swim-up and in vitro incubation.Analysis of variance (ANOVA) was used to test for statistical differences insperm motion characteristics.

Results: All motion parameters, except VSL and percent motility, weresignificantly increased after sperm treatment by sildenafil at the concentra-tion of 0.01microM (P�0.03). Curvilinear velocity, ALH and HYPER werealso significantly increased at the concentration of 0.1 microM (P�0.03).No effects were observed at the sildenafil concentration of 0.001microM. Adose-dependent effect of the type-5 PDE inhibitor on sperm motion char-acteristics was not observed. Sperm motion parameters were not statisticallydifferent between treated and control aliquots after swim-up and in vitroincubation.

Conclusions: This study suggests that direct addition of sildenafil, aspecific cGMP PDE type-5 inhibitor, to ejaculates of normal men enhancessperm motion characteristics. The concentration of 0.01microM seems to beoptimum to improve motility, which is lower than the one found in thehuman ejaculate after acute administration of 100mg oral sildenafil. Ourresults indicate that sildenafil was not able to maintain the initial motilityimprovement over time after removal of the seminal plasma by swim-up innormal sperm.

P-349

Semen quality and hormonal levels of smokers and nonsmokers normalfertile men. Jorge Hallak, Fabio F. Pasqualotto, Antonio M. Lucon, Ber-nardo P. Sobreiro, Eleonora B. Pasqualotto, Sami Arap. Univ de Sao Paulo,Sao Paulo, Brazil.

Purpose: Even though studies have showed that cigarette smoking isassociate with reduced semen quality, studies evaluating normal fertile menare lacking. The goal of our study was to evaluate semen quality andhormonal levels in mild, moderate, heavy and nonsmokers men undergoingvasectomy for sterilization purposes.

Settings: Prospective study.Material and Methods: The study was approved by our review board and

the patients involved granted their informed consent. �From January 1999to September 2002, 750 vasectomies for voluntary sterilization purposeswere performed. Computer-assisted semen analysis was performed on allspecimens, with a Motion Analysis VP 50 semen analyzer. We divided ourpatients into four groups: non-smokers (n � 232), mild smokers (� 10cigarettes/day; n � 156), moderate smokers (11-20 cigarettes/day; n �198), and heavy smokers (� 20 cigarettes/day; n � 164). We evaluatedsperm concentration, motility, motion parameters, and hormonal levels inthese men. Data was evaluated with ANOVA.

Results: No differences were seen in sperm concentration (p � 0.071),motility (0.679), follicle-stimulating hormone (p � 0.562), luteinizing hor-mone (p � 0.213), and serum total testosterone (p � 0.266). Also, spermmotion characteristics did not differed across the groups: linear velocity

S236 Abstracts Vol. 80, Suppl. 3, September 2003