Subjects. A total of 225 male patients attending the Andrology Outpatient Unit at the Clinic for Endocrinology and Metabolic Disorders, Faculty of Medicine, Skopje, R. Macedonia, were enrolled in the study. All gave informed consent to participate in the study. Semen analysis was performed in accordance with World Health Organization (WHO) guidelines [12]. All patients were routinely screened for the presence of sex chromosomal aneuploi dies by QF polymerase chain reaction (PCR) analysis and for the presence of Y micro deletions by multiplex PCR analysis of several STS sites in the three AZF regions [13,14]. The group with known causes of infertility included 29 patients; 10 had Y micro deletions, eight were XXY patients, two were XX, one was XYY and eight had obstructive azoospermia. The other 196 patients were divided into four subgroups according to the severity of the spermatogenic defect as determined by semen analysis: azoospermia, n = 74; severe oligozoospermia (<5 × 10⁶/ mL), n = 56; mild oligozoospermia (>5 × 10⁶/mL), n = 27; and normozoospermia, n = 39. In addition, 123 men who have fathered at least one child by natural conception and whose paternity was proven by DNA analysis, served as controls.

Methods. Genomic DNA was isolated from leuko cytes using the Proteinase K/SDS digestionphenol/ chlo roform extraction-ethanol precipitation method [15]. The POLG CAG repeat number was determined by fluo rescent PCR amplification of exon 2 using the following primers: direct 5’-ROX-CCA GCT CCG TCC CCG CGT CCG ACC-3’ and reverse 5’-GCT GCC CGC CCT CCG AGG ATA GCA C-3’. The size of the PCR product was deter mined by capillary electrophoresis on an ABI PRISM™ 310 Genetic Analyzer (Applied BioSystems, Foster City, CA, USA) and the size of the PCR products was deter mined by GeneScan software (Figure 1). The number of CAG repeats predicted by the GenesScan soft ware was compared with the actual CAG repeats deter mined by direct dideoxy terminator cycle sequencing using a Big Dye Terminator Sequencing Kit v1.0 (Applied Bio Systems) in male DNA samples homozygous for 10 and 11 CAG repeats.

Statistical Analysis. Statistical analysis was perform ed using Chi square and Fisher’s exact test. A p value of <0.05 was considered statistically significant for each test.