Balkan Journal of Medical Genetics

ASSOCIATION OF THE MTHFR C677T (rs1801133) POLYMORPHISM WITH IDIOPATHIC MALE INFERTILITY IN A LOCAL PAKISTANI POPULATION
Irfan M, Ismail M, Azhar Beg M, Shabbir A, Rashid Kayani A, Kaukab Raja G
*Corresponding Author: Muhammad Irfan, M.Phil., Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, Pakistan. Tel: +92-344-551-8382. Email: muhammadirfan11@gmail.com
page: 51

SAMPLE ANALYSES

Semen Analysis. Semen parameters (semen volume, sperm concentration, sperm motility, sperm morphology, liquefaction time, pH and colour) were determined in a private laboratory by an expert using standard methods. The semen samples were categorized into one of the semen group deﬁned by WHO [1]. (Table 3.1). The volume was measured using a graduated glass pipette. The sperm concentration was counted using a sperm counting chamber (Jiansu Sanwe Medical Science and Technology Co. Ltd., Xuzhou, China). The concentration, motility and morphology of sperm cells were observed using a binocular microscope at x100 magniﬁcation (Olympus Optical Co. Ltd., Tokyo, Japan). The semen pH was determined with a digital pH meter. Genetic Analysis. Total genomic DNA was extracted from blood samples using a standard phenol/ chloroform extraction method. MTHFR C677T Genotyping. The MTHFR C677T polymorphism was analyzed by polymerase chain reaction (PCR) of the genomic DNA with primers 5’-ACC CAC AGA AAA TAC CCA G-3’ (forward) and 5’-TGC CCC ATT ATT TA-3’ (reverse) (Alpha DNA, Montreal, Quebec, Canada) with an initial step consisting of denaturation for 4 min. at 94 °C, annealing for 45 seconds at 60 °C, extension for 45 seconds at 72 °C, followed by 35 cycles of denaturation at 94 °C for 45 seconds, annealing at 60 °C for 45 seconds, extension at 72 °C for 45 seconds, and a ﬁnal extension at 72 °C for 10 min. The ampliﬁed PCR products were digested with HinfI restriction endonuclease (Fermentas Life Sciences, Burlington, ON, Canada) as the C677T polymorphism creates a restriction site for it. Gel Electrophoresis. The digested product was elec-trophoresed on a 3.0% agarose gel with ethidium bromide staining which was then visualized through ultraviolet transillumination. The normal allele with cytosine at position 677 (C677) formed an undigested fragment of 198 bp, while the mutant allele with thymine in position 677 (T677) formed fragments of 175 and 23 bp. Statistical Analysis. The allele frequency of the MTHFR 677C>T polymorphism was determined by counting alleles through electrophoresis gel analysis. The χ2 analysis was used to determine the Hardy-Weinberg equilibrium of the alleles in the population. The association of the MTHFR 677C>T polymorphism with subgroups of male infertility was determined by logistic regression analysis adjusting the effects of age, body mass index (BMI), occupation, working hours, and working shift. A p value of <0.05 was considered statistically signiﬁcant. All statistical analyses were performed using the International Business Machines (IBM) Statistical Package for the Social Sciences (SPSS) v20.0 for Windows (IBM SPSS; http:// www.ibm.com).

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