Materials. One hundred and nine infertile males, as well as 50 control fertile males (proven fathers) have been tested for the presence of Y chromosome micro-deletions. Informed consent was obtained from all patients. On the basis of their semen analysis, the patients were categorized into four groups: 1) azoospermia (n = 36); severe oligo­asthenoteratozoospermia <5 x 10⁶/mL (n = 36); oligoasth­enoteratozoospermia >5 x 10⁶/mL (n = 14); and normo­asthenoteratozoospermia (n = 23). The control group con­sisted of 50 men who have fathered at least two children and whose paternity was proven by DNA analysis.

Methods. Semen analyses were performed according to the World Health Organization guidelines [12]. Serum FSH, LH and testosterone were measured by an elec-trochemi-luminescent method on Elecsys 1010 (Roche Diagnostics, F. Hoffmann-La Roche Ltd, Basel, Switzer­land). Testicular volume was evaluated using an orchido­meter. Testicular biopsy was performed by open surgery; the testicular tissue sample was fixed in Bouin's solution, and the histological examination of spermatogenesis was performed on hematoxylin and eosin-stained sections. DNA was isolated from leukocytes using the Proteinase K/SDS digestion-phenol/chloroform extraction-ethanol precipitation method.

The screening for Y micro-deletions was performed on genomic DNA by two multiplex PCR analyses (multiplex A and B), following the guidelines for molecular diagnosis of Y chromosomal micro-deletions [13]. A total of six STSs, two in each AZF region were analyzed (sY84 and sY86 in AZFa; sY127 and sY134 in AZFb; sY 254 and sY255 in the AZFc region). The ZFX/ZFY gene was analyzed in each multiplex PCR as an internal control. The SRY gene was also analyzed as a control for the testis determining factor and for the presence of Y-specific sequences. External positive (fertile male DNA) and nega­tive (female DNA and blank-no DNA) controls were run in parallel with each multiplex PCR.

The PCR reaction was performed in a total volume of 50 mL. The reaction mix contained 16.6 mM (NH₄)₂SO₄, 66.7 mM Tris-HCl pH 8.8, 4.0 mM MgCl₂, 6.8 mM EDTA, 5 mM b-mercaptoethanol, 160 ng/mL BSA, 10% DMSO, 200 mM each of the four dNTPs (dATP, dCTP, dGTP and dTTP), 10 pmol each of the primers, 0.5 mg DNA and 2.5 U Taq polymerase. The PCR was performed under the following conditions: initial denaturation step at 95°C for 5 min. (during which the Taq polymerase was added), followed by 30 cycles of 1 min. denaturation at 95°C, 1 min. annealing at 55°C and 4 min. elongation at 72°C; and final elongation at 72°C for 5 min. The PCR fragments were separated on a 1.8% agarose gel electro­phoresis, stained with ethidium bromide and visualized under UV light.