Abstract
The azoospermia factor (AZF) region of the human Y chromosome contains essential genes for spermatogenesis. Microdeletions in AZF region has been shown to cause male infertility. The aim of this investigation was to determine the frequency of AZF microdeletions in Jordanian infertile males. A sample of 100 infertile males (36 with azoospermia and 64 with oligozoospermia) was screened for microdeletions using 16 AZF markers and polymerase chain reaction (PCR) technique. Two subjects were found to have microdeletions in AZFc region and one subject has microdeletion that includes AZFb and part of AZFc and AZFa. The three deletions were found in azoospermic subjects (8.3%). No microdeletions were found in oligozoospermic group. The frequency of AZF microdeletions in Jordanian azoospermic infertile males is comparable to that observed in other populations (1%-15%). The results suggest the importance of AZF microdeletion analysis for genetic counseling prior to providing assisted reproduction technique.
Keywords: AZF, microdeletion, Jordan, infertility, male
Introduction
Infertility is defined as failure to conceive after one year of regular unprotected sexual intercourse [1]. Infertility affects 15% of couples worldwide, and in roughly half of these cases, the defect can be traced to the male factors [1]. Several factors have been implicated in male infertility such as hormonal abnormalities, erectile dysfunction, infections, antisperm antibodies, exposure to chemical agents and radiations, testicular cancer, varicose, genetic factors and others [2-5]. Thus, male infertility can be considered as multifactorial syndrome that has a collection of different conditions with a variety of etiologies. However, in about 40% of male cases, causing factors of infertility are unknown or called idiopathic [6].
The main genetic cause of male infertility is chromosomal abnormalities, which account for almost 5% of infertile males, and the prevalence increases to 15% in the azoospermic males [7]. Men with non-obstructive azoospermia have high prevalence of aneuploidy, particularly in their sex chromosomes [8]. The second most common genetic cause of male infertility is microdeletion in the azoospermia factor (AZF) region of the Y chromosome [9]. Micro deletions in this region cause defect in spermatogenesis that leads to development of azoospermia and oligozoospermia [10]. Three major loci have been identified in the AZF and named AZFa, AZFb and AZFc regions. The three loci contain 16 coding genes that play a role in the process of spermatogenesis such as regulation of gene expression, RNA processing and trafficking [11].
In this study, we examined the frequency of microdeletions in the AZF in a sample of infertile men with idiopathic azoospermia and oligozoospermia using polymerase chain reaction (PCR) technique. In addition, we compared the detected microdeletion frequencies with numbers identified in other countries and populations.
Methods
Subjects
Unrelated 100 infertile men with oligozoospermia (n = 64, sperm count; < 15 million/ml) and azoospermia (n = 36) were participated in the study. The subjects were recruited from infertility clinics in Middle of Jordan. Subjects with chromosomal abnormalities or known causes of infertility were excluded from study. A structured questionnaire was used to collect information about subject’s medical history and demographic characteristics. Informed consent was taken from all subjects according to Institutional Review Board at Jordan University of Science and Technology.
DNA extraction
Genomic DNA was extracted from blood specimens collected from subjects in EDTA tubes using commercially available kit (Promega, Madison, USA). The concentration of DNA in the samples was measured spectrophotometrically and stored at -35°C until used.
Detection of AZF microdeletions
Microdeletions in AZF region were detected using polymerase chain reaction (PCR) technique as previously described [12,13]. Sixteen sequence tagged sites (STS) in the AZF region were used as markers for analysis of microdeletion. The STS markers were ZFY and SRY for internal control (IC) region; SY 81, SY 82, SY 84 and SY 86 for AZFa; RBM1, SY 127, SY 134, SY 142 and SY 164 for AZFb; DAZ, CDY, SY 152, SY 255 and SY 277 for AZFc. STS PCR multiplexing groups and their amplified fragments are shown in Table 1. PCR was carried out in 30 μL reaction mixture containing 10 ng of DNA, 1 μM of each primer and ready to use PCR master mix (Promega, Madison, USA). The PCR conditions were: denaturation at 95°C for 6 minutes followed by 35 cycles of 95°C for 60 s, 62°C for 60 s and extension at 72°C for 90 s, and a final extension at 72°C for 8 minutes.
Table 1.
Sequence tagged sites (STS)- PCR multiplexing groups, locations and their amplified sizes
| Multiplex set | Name of STS | Region | Amplified fragments (Bp) |
|---|---|---|---|
| 1 | ZFY | IC | 495 |
| SY 86 | AZFa | 320 | |
| SY 127 | AZFb | 274 | |
| SY 254 | AZFc | 400 | |
| 2 | SRY | IC | 472 |
| SY 84 | AZFa | 362 | |
| SY 134 | AZFb | 301 | |
| SY 255 | AZFc | 126 | |
| 3 | SY 152 | AZFc | 125 |
| DAZ | AZFc | 1300 | |
| 4 | SY 142 | AZFb | 180 |
| SY 164 | AZFb | 590 | |
| SY 277 | AZFc | 275 | |
| 5 | SY 82 | AZFa | 280 |
| 6 | SY 81 | AZFa | 200 |
| 7 | CDY | AZFc | 132 |
Results
In this study 100 infertile males with azoospermia (n = 36) and oligozoospermia (n = 64) were screened for AZF microdeletions in the Y chromosome. The mean age of subjects was 32.9 ± 7 years (range 23-43).
None of the patients showed complete microdeletion of all AZF regions. However 3 patients with azoospermia out of 36 had partial microdeletions (8.3%). Distribution of deleted markers in the three patients is shown in Table 2. Two subjects had microdeletion in AZFc region while one had a microdeletion that involved AZFb region and part of AZFa and AZFc regions.
Table 2.
Pattern of microdeletions in the 3 azoospermic infertile males
| Region | Phenotype STS | Patient 1 | Patient 2 | Patient 3 |
|---|---|---|---|---|
| IC | ZFY | |||
| SRY | ||||
| AZFa | SY 81 | |||
| SY 82 | ||||
| SY 86 | ||||
| SY 84 | DeL | |||
| AZFb | SY 127 | DeL | ||
| SY 134 | DeL | |||
| SY 142 | DeL | |||
| SY 164 | DeL | |||
| RBMI | DeL | |||
| AZFc | SY 152 | DEL | DeL | DeL |
| DAZ | DeL | DeL | DeL | |
| SY 255 | Del | DeL | DeL | |
| SY 277 | DeL | DeL | ||
| CDY | Del |
Discussion
In this study, we examined microdeletions in AZF region of Y chromosome in 100 male with unknown cause of infertility. The results showed that 3 out of 36 azoospermic infertile males had microdeletions in AZF region, whereas no microdeletions were detected in oligozoospermic infertile males.
The frequency of AZF microdeletion observed in this study was about 8.3% among azoospermic males. This frequency is similar (Table 3) to what reported in patients from China (8.6%), India (7.6%) and Netherland (8.1%) [14-16] while it is slightly lower (Table 3) than that detected in patients from USA (11%), Japan (11.7%) and Tunisia (11.8%) [17-19]. However, very low frequency of AZF microdeletions was reported in studies from Algeria (2%), Slovakia (4.6%) and Turkey (1.3%) [12,20,21]. The variation in the detected frequencies of AZF microdeletions among azoospermic infertile males could be due to method of detection of deletions, inclusion criteria and sample sizes. In our sample, no microdeletions were detected among 64 oligozoospermic infertile males. Similar findings were reported in studies from India, Algeria and Turkey [12,15,21]. The result is also consistent with the majority of the studies that observed higher frequency of microdeletions in infertile males with azoospermia than those with oligozoospermia [12,13,15,18,19,21-24].
Table 3.
The frequency of AZF microdeletions among azoospermic infertile males in selected populations
| Population | Number of azoospermic males | Frequency of AZF microdeletion | Reference |
|---|---|---|---|
| Jordanian | 34 | 8.3% | This study |
| Indian | 119 | 7.6% | [15] |
| Spanish | 57 | 14% | [24] |
| Chinese | 137 | 8.7% | [16] |
| 945 | 11.5% | [13] | |
| Algerian | 49 | 2.0% | [21] |
| Japanese | 60 | 11.7% | [18] |
| Tunisian | 76 | 11.8% | [19] |
| Turkish | 52 | 1.3% | [12] |
| USA | 385 | 10.4% | [23] |
| Netherlander | 37 | 8.1% | [14] |
| Brazillian | 60 | 6.6% | [22] |
| Mexican | 50 | 12% | [11] |
In the current study, the distribution of microdeletions detected in AZFc region was higher than the other AZF regions. This result is consistent with most of the literature that indicate that the prevalence of microdeletions in AZFc is high compared to AZFb and AZFa regions [12,13,15,18,19,21-23]. AZFc region contains the DAZ family genes that encode proteins with RNA-binding motive and involved in the regulation of RNA metabolism during testicular development [25,26]. Deletions in DAZ gene cluster have been shown to be associated with a variety of spermatogenic alterations [25,26]. The reasons behind high frequency of microdeletions in AZFc could be due to the presence of repetitive sequences of the genes in this region that predispose it to intrachromosomal recombination.
In conclusion, AZFc Y chromosome microdeletion among infertile azoospermic males is common. This highlights the importance of examination of AZF microdeletions and genetic counseling prior to providing IVF service in Jordan.
Acknowledgements
This work was supported from fund provided by the Deanship of Research at Jordan University of Science and Technology.
Disclosure of conflict of interest
Nothing to declare.
References
- 1.Poongothai J, Gopenath TS, Manonayaki S. Genetics of human male infertility. Singapore Med J. 2009;50:336–347. [PubMed] [Google Scholar]
- 2.Bonde JP. Occupational causes of male infertility. Curr Opin Endocrinol Diabetes Obes. 2013;20:234–239. doi: 10.1097/MED.0b013e32835f3d4b. [DOI] [PubMed] [Google Scholar]
- 3.Check JH. Treatment of male infertility. Clin Exp Obstet Gynecol. 2007;34:201–206. [PubMed] [Google Scholar]
- 4.Jarow JP, Troiani J, McNellis D, Wiederhorn R, Fang G, Handelsman H. Use of biomarkers to assess tissue specific androgen adequacy: defining male hypogonadism. J Urol. 2013;189:633–637. doi: 10.1016/j.juro.2012.09.025. [DOI] [PubMed] [Google Scholar]
- 5.O’Flynn O’Brien KL, Varghese AC, Agarwal A. The genetic causes of male factor infertility: a review. Fertil Steril. 2010;93:1–12. doi: 10.1016/j.fertnstert.2009.10.045. [DOI] [PubMed] [Google Scholar]
- 6.Hamada AJ, Esteves SC, Agarwal A. A comprehensive review of genetics and genetic testing in azoospermia. Clinics (Sao Paulo) 2013;68(Suppl 1):39–60. doi: 10.6061/clinics/2013(Sup01)06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Emery BR, Carrell DT. The effect of epigenetic sperm abnormalities on early embryogenesis. Asian J Androl. 2006;8:131–142. doi: 10.1111/j.1745-7262.2006.00127.x. [DOI] [PubMed] [Google Scholar]
- 8.Palermo GD, Colombero LT, Hariprashad JJ, Schlegel PN, Rosenwaks Z. Chromosome analysis of epididymal and testicular sperm in azoospermic patients undergoing ICSI. Hum Reprod. 2002;17:570–575. doi: 10.1093/humrep/17.3.570. [DOI] [PubMed] [Google Scholar]
- 9.Slezak R, Sasiadek M. [Chromosome Y microdeletions in the pathogenesis of male infertility] . Pol Merkur Lekarski. 2002;13:229–233. [PubMed] [Google Scholar]
- 10.Briton-Jones C, Haines CJ. Microdeletions on the long arm of the Y chromosome and their association with male-factor infertility. Hong Kong Med J. 2000;6:184–189. [PubMed] [Google Scholar]
- 11.Shinka T, Nakahori Y. The azoospermic factor on the Y chromosome. Acta Paediatr Jpn. 1996;38:399–404. doi: 10.1111/j.1442-200x.1996.tb03514.x. [DOI] [PubMed] [Google Scholar]
- 12.Balkan M, Tekes S, Gedik A. Cytogenetic and Y chromosome microdeletion screening studies in infertile males with Oligozoospermia and Azoospermia in Southeast Turkey. J Assist Reprod Genet. 2008;25:559–565. doi: 10.1007/s10815-008-9272-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Li D, Zhang H, Wang R, Zhu H, Li L, Liu R. Chromosomal abnormalities in men with pregestational and gestational infertility in northeast China. J Assist Reprod Genet. 2012;29:829–836. doi: 10.1007/s10815-012-9783-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Dohle GR, Halley DJ, Van Hemel JO, van den Ouwel AM, Pieters MH, Weber RF, Govaerts LC. Genetic risk factors in infertile men with severe oligozoospermia and azoospermia. Hum Reprod. 2002;17:13–16. doi: 10.1093/humrep/17.1.13. [DOI] [PubMed] [Google Scholar]
- 15.Mitra A, Dada R, Kumar R, Gupta NP, Kucheria K, Gupta SK. Screening for Y-chromosome microdeletions in infertile Indian males: utility of simplified multiplex PCR. Indian J Med Res. 2008;127:124–132. [PubMed] [Google Scholar]
- 16.Zhang YS, Dai RL, Wang RX, Zhang HG, Chen S, Liu RZ. Analysis of Y chromosome microdeletion in 1738 infertile men from northeastern China. Urology. 2013;82:584–588. doi: 10.1016/j.urology.2013.04.017. [DOI] [PubMed] [Google Scholar]
- 17.Brandell RA, Mielnik A, Liotta D, Ye Z, Veeck LL, Palermo GD, Schlegel PN. AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum Reprod. 1998;13:2812–2815. doi: 10.1093/humrep/13.10.2812. [DOI] [PubMed] [Google Scholar]
- 18.Nakashima M, Koh E, Namiki M, Yoshida A. Multiplex sequence-tagged site PCR for efficient screening of microdeletions in Y chromosome in infertile males with azoospermia or severe oligozoospermia. Arch Androl. 2002;48:351–358. doi: 10.1080/01485010290099309. [DOI] [PubMed] [Google Scholar]
- 19.Rejeb I, M’Rad R, Maazoul F, Trabelsi M, Ben Jemaa L, Chaabouni M, Zhioua F, Chaabouni H. Y chromosome microdeletions in Tunisian infertile males. Pathol Biol (Paris) 2008;56:111–115. doi: 10.1016/j.patbio.2007.05.002. [DOI] [PubMed] [Google Scholar]
- 20.Behulova R, Varga I, Strhakova L, Bozikova A, Gabrikova D, Boronova I, Repiska V. Incidence of microdeletions in the AZF region of the Y chromosome in Slovak patients with azoospermia. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2011;155:33–38. doi: 10.5507/bp.2011.006. [DOI] [PubMed] [Google Scholar]
- 21.Chellat D, Rezgoune ML, McElreavey K, Kherouatou N, Benbouhadja S, Douadi H, Cherifa B, Abadi N, Satta D. First study of microdeletions in the Y chromosome of Algerian infertile men with idiopathic oligo- or azoospermia. Urol Int. 2013;90:455–459. doi: 10.1159/000347046. [DOI] [PubMed] [Google Scholar]
- 22.Pina-Neto JM, Carrara RC, Bisinella R, Mazzucatto LF, Martins MD, Sartoratto E, Yamasaki R. Somatic cytogenetic and azoospermia factor gene microdeletion studies in infertile men. Braz J Med Biol Res. 2006;39:555–561. doi: 10.1590/s0100-879x2006000400017. [DOI] [PubMed] [Google Scholar]
- 23.Stahl PJ, Masson P, Mielnik A, Marean MB, Schlegel PN, Paduch DA. A decade of experience emphasizes that testing for Y microdeletions is essential in American men with azoospermia and severe oligozoospermia. Fertil Steril. 2010;94:1753–1756. doi: 10.1016/j.fertnstert.2009.09.006. [DOI] [PubMed] [Google Scholar]
- 24.Martinez C, Mar C, Azcarate M, Pascual P, Aritzeta JM, Lopez-Urrutia A. Sperm motility index: a quick screening parameter from sperm quality analyser-IIB to rule out oligo- and asthenozoospermia in male fertility study. Hum Reprod. 2000;15:1727–1733. doi: 10.1093/humrep/15.8.1727. [DOI] [PubMed] [Google Scholar]
- 25.Ferlin A, Moro E, Garolla A, Foresta C. Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY. Hum Reprod. 1999;14:1710–1716. doi: 10.1093/humrep/14.7.1710. [DOI] [PubMed] [Google Scholar]
- 26.Kleiman SE, Yogev L, Gamzu R, Hauser R, Botchan A, Paz G, Lessing JB, Yaron Y, Yavetz H. Three-generation evaluation of Y-chromosome microdeletion. J Androl. 1999;20:394–398. [PubMed] [Google Scholar]