Infertile men with varicoceles have fewer Leydig cells

Key Points

New Orleans-Infertile men with varicoceles demonstrate reductions in Leydig cell count, along with compensatory hypertrophy and signs of oxidative stress, according to findings presented at the American Society for Reproductive Medicine annual meeting here.

"We know that decreased androgen production results in decreased spermatogenesis and infertility, and there is a suspicion that androgen production is decreased with varicocele. But studies in humans and animals are contradictory as to whether Leydig cells are increased or decreased in number and whether they show atrophy or hyperplasia," Dr. Benoff said. "Our goals were to determine whether changes in Leydig cell number or size were observable in testis biopsies from infertile men with varicocele, and, if so, to determine how these changes participate in producing an infertile state."

Leydig cells were identified by location, morphology, and expression of SCC, and were counted per unit area using a microscope ocular grid. Apoptosis was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) in paraffin-embedded sections, and nNOS, iNOS, eNOS, SCC, and Fas expression were assessed by an indirect immunocytochemistry scale of 0 (absent) to 4 (maximum) by two blinded observers. Not all assays were done on all patients because of small biopsy size.

Leydig cell number per unit area was markedly decreased in patients with varicoceles, compared with controls. While control subjects demonstrated large numbers of Leydig cells, numbers were decreased with left varicocele and appeared to be even further diminished in patients with bilateral varicocele, Dr. Benoff reported.

She explained that an inverse relationship is seen between Leydig cell number and Fas immunostaining. A decrease in Leydig cell count increases Fas expression, ie, enhances the death cell pathway for apoptosis.

Nucleus shaped differently

The biopsies also showed the Leydig cell nucleus in normal controls was circular and relatively small. In infertile men with varicocele, however, the nucleus was oval-shaped and markedly enlarged (about one-third larger than normal). In controls, <10% of cells were hypertrophic, compared with >50% in men with varicoceles (p=.001).

An inverse relationship was shown between hypertrophy and Leydig cell count.

"When count goes down, abnormal morphology increases," Dr. Benoff said. "We also observed that these abnormal cells were trying to make more testosterone to make up for the fact that there were less of them."

nNOS was observed primarily in Leydig cells, while iNOS and eNOS were mostly observed within the seminiferous epithelium. In men with varicocele, the expression levels were highest for eNOS followed by nNOS, with very little expression of iNOS.

"In examining the relationship between the NOS isoforms, we found no association between iNOS and nNOS, but, interestingly, there was a positive correlation between nNOS and eNOS. eNOS also induces Fas and germ cell apoptosis," she noted.

"We put this together into a relatively simple model. As Leydig cell number decreases, Fas increases. As Leydig cell number decreases, nuclei become hyperplastic, and this is associated with increases in nNOS and Fas. nNOS will raise the levels of eNOS, which also brings up Fas. All this amplifies the death receptor pathway and results in germ cell apoptosis. The amount of steroid biosynthesis that is occurring is not enough to salvage the seminiferous epithelium."

These changes, which can be detected by minimal staining of a testis biopsy, provide evidence of oxidative stress in infertile men with varicocele and suggest that dietary antioxidants might be a beneficial adjunct to surgery, she concluded.

Commenting on the study, Marc Goldstein, MD, professor of urology, Weill Medical College of Cornell University, New York, noted that most previous research has focused on the effect of varicocele on spermatogenesis, but this study shows its effects on Leydig cell function.

"This is an area of great interest, and I think future investigations of varicocele will examine Leydig cell function and its effect on testosterone production," he said.