Materials. Two hundred and five infertile/subfertile men and a control group of 145 proven fathers were included in this study. Informed consent was obtained from all patients. On the basis of their semen analysis, the subjects were categorized into six groups: azoospermia (n = 77); severe oligozoospermia, sperm counts £1.0 x 10⁶/mL (n = 34); moderate oligozoospermia, sperm counts 1-5 x 10⁶/m (n = 16); mild oligozoospermia, sperm counts 5-20 x 10⁶/m (n = 22); normozoospermia (n = 40) and patients with a known cause of infertility – azoospermia factor (AZF) deletions and chromosome abnormalities (XXY and XX males) (n = 16). The control group consisted of 145 men who have fathered at least one child and whose paternity was proven by DNA analysis.
Methods. Semen analyses were performed according to the World Health Organization (WHO) guidelines [17]. DNA was isolated from leukocytes using a Proteinase K/SDS digestion-phenol/chloroform extraction-ethanol precipitation method [18]. Exons 2 and 4 of the HFE gene containing the H63D and C282 mutations were amplified by polymerase chain reaction (PCR), using oligonucleo­tide primer sequences, as reported previously [6]. Aliquots of amplified products were digested with RsaI and BclI restriction enzymes to detect the C282 and H63D mutations, respectively. The detection of the HFE H63D mutation by PCR and BclI digestion, followed by agarose gel electrophoresis, is shown in Figure 1.
Chi square and Fisher’s exact test were used to determine the significance of the differences in the frequencies of the HFE H63D mutation between the various groups of subjects studied. The associations with a value of p <0.05 were considered to be significant.