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

MATERIALS AND METHODS

Sample Population. Initially, 1234 men with primary infertility (the couples never conceived) were recruited from various diagnostic setups, hakeems (Muslim physicians) and private clinics in two cities of Rawalpindi and Islamabad, Pakistan, for a period of 2 years (2011 and 2012). A cohort of 348 proven fathers (had at least two children), residents of the same area, was also taken as a control group. Inclusion and Exclusion Criteria. After approval and permission of the concerned authorities, infertile men fulﬁlling the World Health Organization (WHO) criteria of failure to conceive during 1 year of unprotected intercourse with the same partner were recruited [1]. Only those men, whose partners were already screened and had normal reproductive functions, were included. The men with other physiological disorders, congenital and psychological disorders including diabetes, allergies, timing of the onset of puberty, cryptorchidism, testicular torsion, ectopic testis, single testis and testicular damage at birth and varicocele were excluded. The men with mental disorders, i.e., depression [12], anxiety [13] and stress [14], screened by using standard scales, respectively, were also excluded. The past and present infections (mumps, high fever, tuberculosis, lung infections, reproductive tract infections and antisperm antibodies) and testicular or abdominal injury/surgery or vasectomy and Y-chromosome micro-deletions, were also excluded. Moreover, men (both fertile and infertile) with hormonal disorders [tri-iodothyronine (T3), tetra-iodothyronine (T4), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T) and prolactin], and suspected lifestyle factors [smoking >5 cigarette (~5 gm tobacco)/day; tea/coffee/soft drinks (one or all) >3 times/ day or >600 mL (three cups)/day; wearing tight clothes >3 days a week and >12 hours/day for at least 1 year] were also excluded from the study. Consequently, a total 437 idiopathic infertile men including 57 azoospermic, 66 oligospermic, 44 asthenozoospermic, 29 teratozoospermic, 20 oligoasthenospermic and 221 infertile normospermic men were recruited, after ruling out nongenetic factors. Moreover, 218 normospermic fertile men who had two children (or more), were included as controls. The samples and controls screened for MTHFR C677T belonged to the Punjabi ethnic group as self-deﬁned by subjects. Semen Sampling. The semen samples were initially collected for analysis of a suspected fertility problem in men. The semen samples were also obtained from fertile men (control group). Each individual gave semen samples twice by masturbation after 3-5 days of abstinence, their written consent was obtained for the sample to be used further in molecular research. Blood Sampling. At least 10 mL of a single blood sample was collected from the antecubital vein of each subject and stored in vacutainers with EDTA as anticoagulant. Blood samples were immediately transported to the Genetics Laboratory, Institute of Biomedical Sciences and Genetic Engineering (IBGE), Islamabad, Pakistan, where the plasma was separated from all samples by cen-trifugation and stored at ‒25 °C until further analysis. The cellular portion was used for DNA extraction for genetic analysis.

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