Methods: M469I mutation occurrance that so far has not been studied in Iran, was studied by using RFLP-PCR in 100 azoospermia's men.
Results: One hundered azoospermia's men and 60 controls have been studied by NdeI restriction enzyme.
Conclusion: This study showed there was one M469I mutation heterozygote in these patients.
Key words: Infertility, congenital bilateral absence of vase deferens, cystic fibrosis, obstructive azoospermia, non-obstructive azoospermia.
Infertility is the most important human problemes that causes couples interrupting and lots of later issues in social life. Fertility ascribe to ability of a couple in reproduction and infertility is non-reproduction in common living after one year and copulation without using of antipregnancy way but sterility is complete and innate disabiling in reproduction(1). Infertility can be primitive or secondary. Primitive infertility is about individuals who didn't become pregnant at all and secondary infertility is about who had pregnancy background according datas represented in America 15% of people are infertile in reproduction age which including 10 million couples. Each of couples (single or both) participate in infertility (1,2). Consequently, causes detecting and treatment of infertility in many cases effectuate continuation a common living and families immaterial quiescence, investigation of infertility causations can lead to tremendous programming in treatmentable cases.
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Congenital bilateral absence of the vas deferens (CBAVD) is a genital form of cystic fibrosis (CF) that is responsible for 2%-6% of male infertility. The incidence of CF varies in different populations; therefore, the incidence of CBAVD will also vary in different populations. The spectrum and distribution of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations vary between CBAVD and CF patients and are comparable to control individuals. Combinations of particular alleles at several polymorphic loci yield insufficient functional CFTR protein (3,4,5).
The CFTR gene contains 27 exons encompassing 180 kb of DNA on chromosome 7q31.2. The CFTR protein is a glycosylated transmembrane protein, which functions as a chloride channel. CFTR is expressed in epithelial cells of exocrine tissues, such as the lungs, sweat glands, and vas deferens. The CFTR molecule is made up of 2 homologous repeats, each containing 6 transmembrane (TM) regions followed by an intracellular nucleotide-binding domain (NBD). These 2 halves are joined by an intracellular regulatory (R) domain (3,6,7,8).
Mammalian sperm for fertilizing need one process called capacitation that is related with increasing in intracellular pH and hyperpolarization of sperm's membrane. These changes are depended on extracellular HCO3Ö¿. CFTR is channel that conduct ClÖ¿ and HCO3Ö¿ transportation and mutation in this gene cause non-capacitation of sperm. This situation ultimately causes male infertility (7). Most of CFTR mutations were located in exons 2-5, 7, 9-13, 17b and 19-21, which encoding transmembrane domain (TMD) and nucleotide domain (NBD)(8,9,10).
A few CFTR mutations that cause male infertility have already been detected like ΔF508,M470V,ΔI507,N1303K (11,12,13). The M469I mutation is studied in IranianÛ¥s infertile men.
This study is as a fundamental survey. Samples were infertile men who approached in Isfahan Infertility Center and Sari Saint Mary Infertility Center. By surveying profile of these men, obstructive and non-obstructive azoospermia's men were selected. Samples were 23-48 years old (mean age 31.5). One hundered azoospermia's men and 60 controls were studied. Two ml blood sample was collected from patients and some normal men as controls. For preventing blood clotting, Tubes contain EDTA was used. The collected blood samples were gently shaked promptly and kept on ice until extraction. Genomic DNA was extracted from blood by using salting-out procedure.
Sequence of exon 10 CFTR gene was selected by Ensemble site and was detected M469I mutation location in this exon. Because of this polymorphism don't create or don't delete particular restriction enzyme site, primer was designed. (by using oligo6 and CLC software). That cutting site for NdeI restriction enzyme was contrived in forward primer, therefore by amplifying sequence of exon 10 this cutting site is recognized by NdeI if don't exist M469I mutation but if exist this mutation the cutting site isnot recognized by NdeI. PCR product size is 240bp. If this fragment is cut with NdeI, two fragments are created (25bp, 215bp) (Fig 1).
(We should mention that this enzyme was bought from Fermentase corporation).
Always on Time
Marked to Standard
By designed primer exon10 was amplified by PCR technique, then these products were influenced by NdeI and products were observed in 2% agarose gel. The sequence of primers was shown in table1.
The diagnosis of CBAVD or CUAVD patients was initially suggested by impalpable scrotal vas on physical examination and transabdominal/rectal ultrasonography , subsequently confirmed by cytobiochemical characteristics â€•azoospermia with low semen volume (<1.5 mL) and decrease of fructose (vesicular marker) and carnitine (epididymal marker) concentrationsâ€•followed by hormonal analysis(8,14).
By using RFLP-PCR in this study 60 controls were cut by NdeI and all of azoospermia's smples were cut except one of them that was heterozygote. it means this sample contains eighter mutant and normal sequences (Fig 2).
M469I polymorphism was studied by RFLP technique. The GG, GT, TT genotypes of this polymorphism were observed in 100%, 0%, 0% of the control group and in 99%, 1%, 0% patients(table2). Compared with GG wild genotype, a significant correlation wasn't found between GT nad TT genotypes and infertility (OR= 0.990, 95% confidence interval CI= 0.971-1.010 , P= 0.437 ).
M469I mutation that occurs in NBD1 was observed for the first time in Taiwanese patients in 2005. Of 36 CBAVD patients one of them had M469I mutation, who was heterozygote for this mutation (15,16). The technique which was used in Taiwane was TTGE. TTGE analysis reveals homozygous change as band shift and heterozygous change as multiple bands. The DNA fragments that showed abnormal banding patterns on TTGE analysis were sequenced using the Big Dye terminator cycle sequencing kit and analysed on an ABI Prism 377 DNA Sequencer according to the manufacturer's protocols(17).
Because of this technique is time-consuming and expensive RFLP-PCR technique was opted that is easier and profitable and its conclusion is documentable.
Purpose of M469I mutation investigation was detecting frequence of one in this population and its association with male infertility. If there is association between them, this mutation can use in panel of mutations for screening in couples that use assisted reproductive techniques for treatment of infertility such as intra Cytoplasmic sperm injection (ICSI) and IVF is recommended (18) .