Indian Journal of Urology Users online:1136  
IJU
Home Current Issue Ahead of print Editorial Board Archives Symposia Guidelines Subscriptions Login 
Print this page  Email this page Small font sizeDefault font sizeIncrease font size


 
ORIGINAL ARTICLE
Year : 2008  |  Volume : 24  |  Issue : 4  |  Page : 480-485
 

Y chromosome microdeletions in sperm DNA of infertile patients from Tamil Nadu, south India


1 Centre for Computational Engineering and Networking, Amrita Vishwa Vidyapeetham, Kettukarar Thottam, Coimbatore-641105, Tamil Nadu, India
2 Emerald Heights College for Women, Ooty, Tamil Nadu, India

Correspondence Address:
Poongothai J Sakthivel
Research Associate, Centre for Computational Engineering and Networking, Amrita Vishwa Vidyapeetham, Coimbatore-641105, Tamil Nadu
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-1591.44252

Rights and Permissions

 
   Abstract 

Context: Y chromosome microdeletions in infertile men of Tamil Nadu, South India.
Aim: The paper assesses the association of Y chromosome microdeletions among infertile patients using several STS markers from each AZF (AZoospermic Factor) region and also aspires to determine whether the blood DNA microdeletion picture matches the semen DNA Yq microdeletion map.
Materials and Methods: A total of 287 men, including 147 infertile men and 140 normozoospermic fertile controls were included for the study.
Results: Screening 72 semen samples with the STS markers specific to AZF (a,b,c) regions showed Y chromosome microdeletions in 19 (12.9%) individuals. No deletion was observed in all the three AZF regions by screening 45 blood and 30 paired samples. None of the control men showed deletion for the 28 STS markers, which were used for the primary screening of the deletion of AZF a,b,c regions.
Conclusion: Germ cell DNA can be analyzed for Yq microdeletions rather than blood DNA.


Keywords: AZoospermic factor, microdeletions, Y chromosome


How to cite this article:
Sakthivel PJ, Swaminathan M. Y chromosome microdeletions in sperm DNA of infertile patients from Tamil Nadu, south India. Indian J Urol 2008;24:480-5

How to cite this URL:
Sakthivel PJ, Swaminathan M. Y chromosome microdeletions in sperm DNA of infertile patients from Tamil Nadu, south India. Indian J Urol [serial online] 2008 [cited 2019 Sep 16];24:480-5. Available from: http://www.indianjurol.com/text.asp?2008/24/4/480/44252



   Introduction Top


Infertility is a major health problem today affecting 10-15% of the couples [1] and in these couples, male factor infertility accounts for ~50% of causes. The known causes of male infertility are quite numerous but can be grouped into a moderate number of major categories. It has been associated with several genetic and non-genetic conditions, such as hypogonadotrophic hypogonadism, testicular maldescence, structural abnormalities of the male genital tract, genital infections, impotency, previous scrotal or inguinal surgery, varicoceles, chronic illness, medication and exposure to chemicals. [2]

Genetic causes of infertility are an important etiological factor leading to irreversible partial or complete spermatogenetic arrest. Y chromosome microdeletions are a common molecular cause of spermatogenic failure. It has been identified in 9% of azoospermic, 7-10% of idiopathic severe oligozoospermic and 11.6% of severe oligoasthenozoospermic infertile patients. [3] The microdeletions that occur in the AZF [AZoospermic Factor] region of the long arm of Y chromosome affect genes that are involved in spermatogenesis. There are three recurrently deleted nonoverlapping subregions in the proximal, middle and distal Yq11 regions in the deleted interval designated as AZFa, AZFb and AZFc. Recently, a fourth region AZFd has been identified which lies between AZFb and AZFc. [4]

To date, most studies from India and the majority of studies worldwide have analyzed Yq microdeletions from DNA isolated from blood. [1],[5],[6],[7],[8] However, blood DNA might not be representative of sperm DNA, which is of different embryological origin. Sperm DNA might have a higher rate of deletions and DNA damage as a result of oxidative stress. Only few studies have analyzed sperm DNA for Y chromosome microdeletions. [9],[10] Further, few markers confining to each AZF region were used in most of STS-based Y chromosome microdeletion studies. We, for the first time made an attempt to assess the association of Y chromosome microdeletions among Tamilian men of South India with azoospermia, oligozoospermia and oligoasthenozoospermia using several STS markers from each AZF region and also aimed to determine whether the blood DNA microdeletion picture matches the semen DNA Yq microdeletion map.


   Materials and Methods Top


Patients

Samples of blood were collected from 45 infertile men; semen samples from 72 infertile men; paired samples (blood and semen of same patient) from 30 infertile men at infertility clinics from Erode and Nilgiri districts of Tamil Nadu, South India. The patients were classified according to alterations detected in spermograms, based on the WHO technique, [11] into groups [Table 1] with oilgozoospermia (1-20 X 10 6 spermatozoa /ml), asthenozoospermia (>60% of non-motile sperms), oligoasthenozoospermia, varicocele (normal count), azoospermia (no sperms in the ejaculate), teratozoospermia (>40% of abnormal sperms), and necrozoospermia (100% dead sperms). One hundred and forty normozoospermic (> 20 million sperms /ml of semen) males [90 blood, 30 semen, 10 paired samples] of proven fertility served as controls. Appropriate written informed consent according to protocols approved by the ethical committee of the center for Cellular and Molecular Biology, Hyderabad, India was obtained from all participants in this study. Only patients with an apparently normal 46,XY karyotype were included in this study. One patient's scrotal Doppler study revealed bilateral significant varicocele with reflux on the left side.

DNA extraction and deletion mapping by polymerase chain reaction

DNA was extracted from 10 ml of peripheral blood [12] and from semen [13] using standard procedures. Polymerase chain reaction (PCR)-based studies for Y chromosome microdeletions on both infertile and control men were carried out using STS markers on the long arm of the Y chromosome. Initially the screening for AZF regions was done using 28 STS markers [Table 2]. To detect the heterochromatic region, sY160 was used in all cases. Individuals who had deletions in any of these STS markers were further analyzed by using the flanking STS markers [3],[14] in order to map the deletion breakpoints.

Primers were synthesized using ABI392 Oligosynthesizer (Perkin Elmer, Foster City, Calif). Each marker was amplified separately in a 0.2 mL thin wall tube using an MJ Research Thermal Cycler (Waltham, MA, USA) with a female negative control sample. The PCR conditions used for STS markers were as follows: initial denaturation (94C for 5 min) and subsequent denaturations (94C for 45 sec) were the same for all the samples. Different annealing temperatures that were used for different STS markers were as follows: 53C for 30 sec for sY84 and sY158; 55C for 30 sec for sY86; 60C for 1 min for sY746, sY740, sY741, sY745, DFFRY, DBY, sY615, sY743 sY709 and sY744; 57.4C for 1 min for sY99, sY100, sY129 and sY133; 55C for 1 min for sY109, sY138, sY143, sY146, sY152, and sY156; 50C for 1 min for sY113; 60C for 30 sec for sY254; 58C for 30 sec for sY127,sY134 and sY255. Extension was 65C for 1 min for sY127, sY134, and sY255, whereas for the other STS markers extension was 72C for 2 min. Amplified PCR products were electrophoresed at 120V in 2% agarose gel impregnated with ethidium bromide at 5 g/mL and visualized under UV light.


   Results Top


Primary screening

Screening 72 semen samples with the STS markers specific to AZF(a,b,c) regions showed Y chromosome microdeletions in 19 (five oligozoospermic, seven asthenozoospermic, seven oligoasthenozoospermic) individuals [Figure 1], which accounted for 12.9% of the total infertile men analyzed. The deletions that were detected in sperm DNA were confirmed thrice to rule out amplification failure. Surprisingly, no deletion was observed in all the three AZF regions by screening 45 blood and 30 paired (blood and semen) samples with STS markers. None of the control men showed deletion for the 28 STS markers, which were used for the primary screening of the deletion of AZFa, AZFb, and AZFc regions.

Deletion mapping of Y chromosome

Micro and macrodeletions were noticed by further scrutinizing the deletions in these infertile men [Figure 2]. Deletion of the AZFa region alone was detected in two oligoasthenozoospermic men (SS59, SS78), which accounted for 10.5% of the total deletion [Figure 3]. One mild oligozoospermic man (SS84) showed deletion of the AZFb region whereas AZFab deletion was exhibited by SS129 severe oligospermic patient both of which accounted for 21.1% of the total deletions [Figure 3]. Deletion of AZFc alone was detected in 13 individuals (SS16, SS29, SS42, SS58, SS72, SS80, SS83, SS92, SS124, SS133, SS143, SS145, SS146), which accounted for 68.4% [Figure 3] of the total deletion. As a whole, deletion of AZFc region accounts for 78.9% [Figure 3] inclusive of AZFc region alone and AZFbc regions.


   Discussion Top


For the first time, an attempt was made to evaluate the role of Y chromosome microdeletion in male infertility among Tamilians using copious STS markers from AZFa,b,c regions and also to determine whether the blood DNA microdeletion picture correlates with the semen DNA Yq microdeletion map. PCR-based STS analysis of 147 infertile men revealed micro- and macrodeletions on the Y chromosome in 19 individuals [Figure 1] and [Figure 2], accounting for 12.9% of the total infertile men analyzed. To our astonishment deletions were noticed only in the semen samples of infertile patients whereas no deletion was observed in blood and paired samples which correlates with the results of Mantas et al., 2007. [9] It was interesting to note that none of the azoospermic, necrozoospermic, teratozoospermic and varicocele sample showed deletion in any of the AZF regions. Previous studies revealed that the Y chromosome microdeletions were responsible for 3-13% of the infertile men. [7],[8],[15],[16]

In this study, approximately one-sixth (15.8%) of the total Y chromosome deletion was in the AZFa region [Figure 3]. Of these 19 patients, deletion of the AZFa region alone was detected in two infertile men who were oligoasthenozoospermic (SS59, SS78), which accounted for 10.5% of the total deletion [Figure 3]. The size of the deletion in the AZFa region was very narrow (microdeletion) compared with the AZFb and AZFc regions. Neither of the individuals showed deletion of same STS marker. It was interesting that SS59 patient showed sY84 deletion and SS78 patient showed sY86 deletion [Figure 1], as these two markers were recommended for the diagnosis of the AZFa region by Simoni et al., 1999. [17] The further characterization of the microdeletions by extension analysis revealed that mild oligoasthenozoospermic (SS59) men showed deletion of middle region of AZFa and that of severe oligoasthenozoospermic men (SS78) confirmed proximal AZFa deletion [Figure 2]. Larger deletion of AZFa region in SS78 patient compared to SS59 might correlate with his severe phenotype. In general, frequency of deletion in the AZFa region was less compared with the AZFb and AZFc regions. [18],[19] In most of the studies, deletion of the complete AZFa was found to be associated with SCOS. [18],[20],[21],[22] This study demonstrated that even deletion of a narrow region on the AZFa region can cause SCOS as indicated by Thangaraj et al., 2003. [7]

Altogether, deletion of the AZFb region was involved in 21.1% of the total deletions, of which 5.3% was due to AZFb alone; another 5.3% was in association with AZFa and remaining 10.5% with AZFc [Figure 3]. Two different patterns of partial deletions [Figure 2] within the AZFb region were observed in one mild oligozoospermic (SS84) man in the patient group, which accounted for 1.4% (1/69) of the total oligozoospermic men included in this study. Arruda et al., 2007 [23] reported 5% of AZFb deletion in oligozoospermic patients of the Brazilian population. Complete screening of the above patient for AZFb region revealed interesting results. Mild oligozoospermic patient (SS84) showed deletion of complete stretch of sY100, sY2522, sY108, sY109 and sY127, sY129, sY1211, sY133, sy134 STS markers. AZFab deletion was observed in severe oligozoospermic patient (SS129), but this individual showed deletion of one STS marker (sY151) in the AZFa region, whereas microdeletions of four STS markers (sY134, sY138, sy139, sY141) were observed in the AZFb region [Figure 2].

As a whole, deletion of the AZFc region accounts for 78.9% inclusive of AZFc region alone and AZFbc regions. Deletion of AZFc alone was detected in 13 individuals (SS16, SS29, SS42, SS58, SS72, SS80, SS83, SS92, SS124, SS133, SS143, SS145, SS146), which accounted for 68.4% [Figure 3] of the total deletion. In this study, minimum of total Y chromosome deletion was observed in AZFa and AZFb region whereas maximum deletion was seen in AZFc region. This is in agreement with the earlier studies which showed that the incidence of deletion in the AZFc region was high compared with the AZFa and AZFb regions. [24],[25],[26],[27] Deletion of almost the same STS markers were found in SS143 and SS145 oligoasthenozoospermic patients; microdeletion of the same AZFc region in five asthenozoospermic men correlated with their phenotype [Figure 2]. Interestingly, two infertile men of different sperm anomalies showed deletion in both AZFb and AZFc regions [Figure 2], accounting for 10.5% of total deletion [Figure 3]. AZFbc deletion was observed by Fernando et al., 2006 [28] with a frequency of 28.6% in the Sri Lankan population. Spermiograms of these infertile men showed that deletion of the AZFb and AZFc interval was associated with either asthenozoospermia, or severe oligozoospermia and insufficient production of mature sperm to enable reproduction.

Surprisingly, none of the infertile men from Nilgiri district showed deletion of AZF region which was evident from the results. This may be due to small sample size. None of the infertile and normal controls showed the deletion of the heterochromatic [Figure 2] region (DYZ). Although the Yq heterochromatic region predominantly consists of DYZ1 and DYZ2 classes of repeat sequences, [29] a few studies have shown its association with infertility and reproductive failure.


   Conclusion Top


The results of this work on the Tamilian population of South India highlights the need for larger more extensive studies to determine the frequency of deletions in blood and sperm DNA. Even though the percentage of Y chromosome microdeletions is lower in the selected population and it is noticed only in sperm DNA, in all cases opting for ART it is more applicable and necessary to analyze germ cell DNA than DNA isolated from blood for Yq microdeletions, since detection of these deletions has a prognostic value in predicting potential success of testicular sperm retrieval for assisted reproduction.

 
   References Top

1.Dada R, Gupta NP, Kucheria K. AZF microdeletions associated with idiopathic and non idiopathic cases with cryptorchidism and varicocele. Asian J Androl 2002;4:259-63.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Crosignani PG, Collins J, Cooke ID, Diczfalusy E, Rubin B. Recommendations of the ESHRE workshop on 'Unexplained Infertility'. Anacapri, August 28-9,1992. Hum Reprod 1993;8:977-80.  Back to cited text no. 2    
3.Chiang HS, Wei HJ, Chen YT. Genetic screening for patients with azoospermia and severe oligo- asthenospermia. Int J Androl 2000;2:20-5.   Back to cited text no. 3    
4.Kent-First M, Muallem A, Shultz J, Pryor J, Roberts K, Nolten W, et al . Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection. Mol Reprod Dev 1999;53:27-41.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Mitra A, Dada R, Kumar R, Gupta NP, Kucheria K, Gupta SK. Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome. Asian J Androl 2006;8:81-8.   Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Chiang HS, Yeh SD, Wu CC, Huang BC, Tsai HJ, Fang CL. Clinical and pathological correlation of the microdeletion of Y chromosome for the 30 patients with azoospermia and severe oligoasthenospermia. Asian J Androl 2004;6:369-75.   Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Thangaraj K, Gupta NJ, Pavani K, Reddy AG, Subramainan S, Rani DS, et al . Y chromosome deletions in azoospermic men in India. J Androl 2003;24:588-97.   Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Viswambharan N, Suganthi R, Simon AM, Manonayaki S. Male infertility: Polymerase chain reaction-based deletion mapping of genes on the human chromosome. Singapore Med J 2007;48:1140 2.   Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Mantas D, Angelopoulou R, Msaouel P, Plastira K. Evaluation of sperm chromatin quality and screening of Y chromosome microdeletions in Greek males with severe oligozoospermia. Arch Androl 2007;53:5-8.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Dada R, Kumar R, Shamsi MB, Kumar R, Kucheria K, Sharma RK, et al . Higher frequency of Yq microdeletions in sperm DNA as compared to DNA isolated from blood. Asian J Androl 2007;9:720-2.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 3rd ed. 1999. p. 44-5.   Back to cited text no. 11    
12.Thangaraj K, Joshi MB, Reddy AG, Gupta NJ, Chakravarthy B, Singh L. CAG repeat expansion in androgen receptor gene is not associated with male infertility in Indian populations. J Androl 2002;23:815-8.   Back to cited text no. 12    
13.Thangaraj K, Joshi MB, Reddy AG, Rasalkar AA, Singh L. Sperm mitochondrial mutations as a cause of low sperm motility. J Androl 2003;24:388-92.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Ma K, Inglis JD, Sharkey A, Bickmore WA, Hill RE, Prosser EJ, et al . A Y chromosome gene family with RNA-binding protein homology: Candidates for the azoospermia factor AZF controlling human spermatogenesis. Cell 1993;75:1287-95.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]
15.Rejeb I, M'rad R, Maazoul F, Trabelsi M, Ben Jemaa L, et al . Y chromosome microdeletions in Tunisian infertile males. Pathol Biol (Paris), 2008 Epub ahead of print.  Back to cited text no. 15    
16.Simoni M, Tuttelmann F, Gromoll J, Nieschlag E. Clinical consequences of microdeletions of the Y chromosome: The extended Munster experience. Reprod Biomed Online 2008;16:289-303.  Back to cited text no. 16    
17.Simoni M, Bakker E, Eurlings MC, Matthijs G, Moro E, Muller R, et al . Laboratory guidance for molecular diagnosis of Y-chromosomal microdeletions. Int J Androl 1999;22:292-9.  Back to cited text no. 17    
18.Fujisawa M, Shirakawa T, Kanzaki M, Okada H, Arakawa S, Kamidono S. Y-chromosome microdeletion and phenotype in cytogenetically normal men with idiopathic azoospermia. Fertil Steril 2001;76:491-5.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]
19.Layman LC. Human gene mutations causing infertility. J Med Genet 2002;39:153-61.  Back to cited text no. 19  [PUBMED]  [FULLTEXT]
20.Kamp C, Huellen K, Fernandes S, Sousa M, Schlegel PN, Mielnik A, et al . High deletion frequency of the complete AZFa sequence in men with Sertoli cell only syndrome. Mol Hum Reprod 2001;7:987-94.  Back to cited text no. 20  [PUBMED]  [FULLTEXT]
21.Dohle GR, Halley DJ, Van Hemel JO, van den Ouwel AM, Pieters MH, Weber RF, et al . Genetic risk factors in infertile men with severe oligozoospermia and azoospermia. Hum Reprod 2002; 17:13-6.  Back to cited text no. 21  [PUBMED]  [FULLTEXT]
22.Sun C, Skaletsky H, Birren B, Devon K, Tang Z, Silber S, et al . An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y. Nat Genet 1999;23:429-32.  Back to cited text no. 22  [PUBMED]  [FULLTEXT]
23.Arruda JT, Bordin BM, Santos PR, Mesquita WE, Silva RC, Maia MC, et al . Y chromosome microdeletions in Brazilian fertility clinic patients. Genet Mol Res 2007;6:461-9.  Back to cited text no. 23  [PUBMED]  [FULLTEXT]
24.Vutyavanich T, Piromlertamorn W, Sirirungsi W, Sirisukkasem S. Frequency of Y chromosome microdeletions and chromosomal abnormalities in infertile Thai men with oligozoospermia and azoospermia. Asian J Androl 2007;9:68-75.  Back to cited text no. 24  [PUBMED]  [FULLTEXT]
25.Omrani MD, Samadzadae S, Bagheri M, Attar K. Y chromosome microdeletions in idiopathic infertile men from west azarbaijan. Urol J 2006;3:38-43.  Back to cited text no. 25  [PUBMED]  
26.Hadj-Kacem L, Hadj-Kacem H, Ayadi H, Ammar-Keskes L, Chakroun-Fki N, Rebai T, et al . Screening of Y chromosome microdeletions in Tunisian infertile men. Arch Androl 2006; 52:169-74.  Back to cited text no. 26  [PUBMED]  [FULLTEXT]
27.Dinic J, Kusic J, Nikolic A, Divac A, Ristanovic M, Radojkovic D. Analysis of Y chromosome microdeletions and CFTR gene mutations as genetic markers of infertility in Serbian men. Vojnosanit Pregl 2007;64:253-6.  Back to cited text no. 27    
28.Fernando L, Gromoll J, Weerasooriya TR, Nieschlag E, Simoni M. Y-chromosomal microdeletions and partial deletions of the Azoospermia Factor c (AZFc) region in normozoospermic, severe oligozoospermic and azoospermic men in Sri Lanka. Asian J Androl 2006;8:39-44.  Back to cited text no. 28  [PUBMED]  [FULLTEXT]
29.Manz E, Alkan M, Buhler E, Schmidtke J. Arrangement of DYZ1 and DYZ2 repeats on the human Y-chromosome: A case with presence of DYZ1 and absence of DYZ2. Mol Cell Probes 1992;6:257-9.  Back to cited text no. 29    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]

This article has been cited by
1 Genomic integrity of the Y chromosome sequence-tagged-sites in infertile and Down syndrome Jordanian males
S. R. Yasin,L. H. Tahtamouni,N. S. Najeeb,N. M. Issa,Z. A. Al-Mazaydeh,A. A. Alfaouri
Andrologia. 2013; : n/a
[Pubmed] | [DOI]
2 Y chromosome microdeletions in infertile men: prevalence, phenotypes and screening markers for the Indian population
S. Sen, A. R. Pasi, R. Dada, M. B. Shamsi, D. Modi
Journal of Assisted Reproduction and Genetics. 2013; 30(3): 413
[VIEW] | [DOI]
3 Efficient combined FISH and PRINS technique for detection of DAZ microdeletion in human sperm
Hossein Mozdarani,Pegah Ghoraeian
Journal of Assisted Reproduction and Genetics. 2012; 29(9): 979
[Pubmed] | [DOI]
4 Efficient combined FISH and PRINS technique for detection of DAZ microdeletion in human sperm
Mozdarani, H. and Ghoraeian, P.
Journal of Assisted Reproduction and Genetics. 2012; 29(9): 979-984
[Pubmed]



 

Top
Print this article  Email this article
Previous article Next article

    

 
   Search
 
   Next article
   Previous article 
   Table of Contents
  
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Article in PDF (175 KB)
    Citation Manager
    Access Statistics
    Reader Comments
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
    Introduction
    Materials and Me...
    Results
    Discussion
    Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed10580    
    Printed91    
    Emailed1    
    PDF Downloaded228    
    Comments [Add]    
    Cited by others 4    

Recommend this journal

HEALTHWARE INDIA