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Volume 11, Issue 4 (6-2013)
IJRM 2013, 11(4): 335-0 |
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Sheikhha M H, Zaimy M A, Soleimanian S, Kalantar S M, Rasti A, Golzade M et al . Multiplex PCR Screening of Y-chromosome microdeletions in azoospermic ICSI candidate men. IJRM 2013; 11 (4) :335-0
URL: http://ijrm.ir/article-1-406-en.html
URL: http://ijrm.ir/article-1-406-en.html
Mohammad Hasan Sheikhha1 
, Mohammad Ali Zaimy1 , Saeede Soleimanian * 2, Seyed Mehdi Kalantar1 , Azam Rasti1 , Maryam Golzade1 , Hamid Hoseini Fahraji3
, Mohammad Ali Zaimy1 , Saeede Soleimanian * 2, Seyed Mehdi Kalantar1 , Azam Rasti1 , Maryam Golzade1 , Hamid Hoseini Fahraji3
1- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran ,saeedesoleimanian@gmail.com
3- Department of Animal Sciences, Agriculture Faculty, Urumia University, Urumia, Iran
2- Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran ,
3- Department of Animal Sciences, Agriculture Faculty, Urumia University, Urumia, Iran
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Introduction
It is estimated that around 15% of the couple at reproductive age are infertile, and approximately half of the infertilities are caused by male factor (1). Defective spermatogenesis is the result of several different disorders, such as endocrinological disorders, malnutrition, genetic defects and maybe environmental condition (2). Around 10% of males with azoospermia and oligozoospermia, have interstitial microdeletion on the Y-chromosome (3-5).
Male-related genes including sex-determining region of Y-chromosome (SRY) and several spermatogenesis-related genes are accumulated in Y chromosome (6). The AZFa, AZFb and AZFc are three major candidate regions of azoospermic factor on long arm of Y-chromosome. These regions have several candidate for the factor: AZFa; USP9Y and DBY that encode an ubiquitin specific protease and a RNA-helicase respectively; AZFb contains several candidate genes such as RBMY, encode an RAN-binding protein; and AZFc contains DAZ and CDY family (5, 7-11).
Current analysis of human genome has showed that the Y-chromosome long arm has many palindromes or inverted repeats. This typical structure is presumed to cause the microdeletion in AZF regions found in some of infertile men (11-13). There are 300 sequence tagged sites (STS) in the Y-chromosome that mapped for the above three AZF regions (14). This study aim was to calculate the frequency of AZF microdeletions among azoospermic intracytoplasmic sperm injection (ICSI) candidate men that attended to Yazd Research and Clinical Center for Infertility.
Materials and methods
In this case-control study, 25 infertile men attending to Yazd Research and Clinical Center for Infertility who were candidate for intracytoplasmic sperm injection (ICSI) were selected randomly in 2011. All of the selected patients were azoospermic with normal 46XY karyotype and had a positive history of male factor infertility. The control group consists of 25 normozoospermic men with normal karyotype. The Ethical Committee of Yazd Research and Clinical Center for Infertility approved the study and all of the participants consented to enter this study verbally. Inclusion criteria were 25-40 year-old men with azoospermia and exclusion criteria was azoospermic men with obstructive tract or any chromosomal abnormality. DNA was extracted from leukocytes of peripheral blood samples by a salting out method (15).
A series of 6 STS markers on the long arm of Y-chromosome were used for detection of interstitial microdeletions according to the European Academy of Andrology (EAA), the European Molecular Genetic Quality network (EMQN) and other protocols. The markers consisted of sY84 and Sy86 for AZFa, sY127 and sY134 for AZFb, sY254 and sY255 for AZFc regions. The sequence and size of all of primers are shown in table I.
Multiplex-PCR
DNA amplified by multiplex PCR method. Two sets of amplification reactions were used. In each PCR 3 STS primers including two internal controls were used. Primers of each reactions had similar melting temperature (Tm) (Table II). PCR amplification condition had a thermocycling procedure consisted of 4min in 94oC for initial denaturation. The procedure followed by 32 cycles of 30s at 94oC, 30s at 59oC and 4min in 65oC with a final extension at 65oC for 5min.
Gel electrophoresis
The products of PCR were run by electrophoresis on a 2% agarose gel.
Statistical analysis
X2 test was carried out to compare difference between the cases and controls. The statistical analyses were performed with SPSS 16 statistical software when p-value was under 0.05 the difference was considered significant.
Results
A total of 25 infertile men who were candidate for ICSI wereselected. The average age in case group was 30.5 years (26-36.5 years) and in control group was 28 years (25-34 years). Of these patients, 5 cases have deletions in more than one region of AZF loci on Y-chromosome but no microdeletion was detected among control group. Totally 17 microdeletions was observed. Among the regions, AZFc had the most microdeletions 42% (7/17) followed by AZF a 35% (6/17) and the AZFb microdeletions have the less frequency 23% (4/17). In this study, among patients with microdeletion one case had deletions in all three AZF regions. Four cases had deletions in two regions and two cases had deletion just in one region. In total, 16% of all cases have deletions in AZFa, 16% in AZFc and 12% in AZFb regions.
Table I. Primers sequence and size
90-107-6/Table_1.jpg)
Table II. Primer mix of each Multiplex-PCR reaction
90-107-6/Table_2.jpg)
Table III. Results of different studies
90-107-6/Table_3.jpg)
Discussion
Deletions of AZF regions are deletions of the euchromatine part of the Y chromosome long arm. It is assumed that deletions of this part of Y chromosome can damage genes in this region that is responsible for the proper course of spermatogenesis. Many factors including somatic and sex chromosome genes interaction candicate to the normal spermatogenesis and the AZF deletions are the most frequent cause of spermatogenetic failure (6).
After Klinefelter’s syndrome, Y-chromosomal deletions are the second most frequent spermatogenesis disorder in infertile men. In the last few years some of research and clinical institutions have described screening of Y chromosome microdeletions in infertile men and molecular diagnostics of this type of Y-chromosomal disorders has become an important diagnostic test within laboratories worldwide dealing with these disorders (16-18).
Y chromosome AZF regions microdeletions are frequently found in azoospermic patients. Deletions incidence has been found from 3-55% (19, 20). In table III we compared our results whit other similar studies. EAA and EMQN published the guidelines for molecular screening of Y-chromosome microdeletions (21). In this study we used six sequences according to European guidelines in which all three sub-regions are represented by Y sequences: sY84, sY86, sY127, sY134, sY254 and sY255. Whit use of these STSs we reported 16% of microdeletions in AZFa, 12% in AZFb and 16% in AZFc regions.
The deletions in an infertile man could provide a proper understanding of the disease allows the medical stuff to avoid unnecessary expensive treatment to fertility improvement. Microdeletions of azoospermia factors are characteristic for spermatogenic failures and lead to oligozoospermia or azoospermia. PCR analysis of these deletions helps to determine site and the frequency of gene deletion and presents a defined prognosis and valuable counseling for couple whit fertility disorders. In the patients' with microdeletions that want to undertake ICSI procedure the deletions may be passed to their son. Because of this probability ethical consequences should be important dimensions of this technique.
Acknowledgements
The grant for this study was given by Yazd Research and Clinical Center for Infertility.
Conflict of interest
There is no conflict of interest in this study.
It is estimated that around 15% of the couple at reproductive age are infertile, and approximately half of the infertilities are caused by male factor (1). Defective spermatogenesis is the result of several different disorders, such as endocrinological disorders, malnutrition, genetic defects and maybe environmental condition (2). Around 10% of males with azoospermia and oligozoospermia, have interstitial microdeletion on the Y-chromosome (3-5).
Male-related genes including sex-determining region of Y-chromosome (SRY) and several spermatogenesis-related genes are accumulated in Y chromosome (6). The AZFa, AZFb and AZFc are three major candidate regions of azoospermic factor on long arm of Y-chromosome. These regions have several candidate for the factor: AZFa; USP9Y and DBY that encode an ubiquitin specific protease and a RNA-helicase respectively; AZFb contains several candidate genes such as RBMY, encode an RAN-binding protein; and AZFc contains DAZ and CDY family (5, 7-11).
Current analysis of human genome has showed that the Y-chromosome long arm has many palindromes or inverted repeats. This typical structure is presumed to cause the microdeletion in AZF regions found in some of infertile men (11-13). There are 300 sequence tagged sites (STS) in the Y-chromosome that mapped for the above three AZF regions (14). This study aim was to calculate the frequency of AZF microdeletions among azoospermic intracytoplasmic sperm injection (ICSI) candidate men that attended to Yazd Research and Clinical Center for Infertility.
Materials and methods
In this case-control study, 25 infertile men attending to Yazd Research and Clinical Center for Infertility who were candidate for intracytoplasmic sperm injection (ICSI) were selected randomly in 2011. All of the selected patients were azoospermic with normal 46XY karyotype and had a positive history of male factor infertility. The control group consists of 25 normozoospermic men with normal karyotype. The Ethical Committee of Yazd Research and Clinical Center for Infertility approved the study and all of the participants consented to enter this study verbally. Inclusion criteria were 25-40 year-old men with azoospermia and exclusion criteria was azoospermic men with obstructive tract or any chromosomal abnormality. DNA was extracted from leukocytes of peripheral blood samples by a salting out method (15).
A series of 6 STS markers on the long arm of Y-chromosome were used for detection of interstitial microdeletions according to the European Academy of Andrology (EAA), the European Molecular Genetic Quality network (EMQN) and other protocols. The markers consisted of sY84 and Sy86 for AZFa, sY127 and sY134 for AZFb, sY254 and sY255 for AZFc regions. The sequence and size of all of primers are shown in table I.
Multiplex-PCR
DNA amplified by multiplex PCR method. Two sets of amplification reactions were used. In each PCR 3 STS primers including two internal controls were used. Primers of each reactions had similar melting temperature (Tm) (Table II). PCR amplification condition had a thermocycling procedure consisted of 4min in 94oC for initial denaturation. The procedure followed by 32 cycles of 30s at 94oC, 30s at 59oC and 4min in 65oC with a final extension at 65oC for 5min.
Gel electrophoresis
The products of PCR were run by electrophoresis on a 2% agarose gel.
Statistical analysis
X2 test was carried out to compare difference between the cases and controls. The statistical analyses were performed with SPSS 16 statistical software when p-value was under 0.05 the difference was considered significant.
Results
A total of 25 infertile men who were candidate for ICSI wereselected. The average age in case group was 30.5 years (26-36.5 years) and in control group was 28 years (25-34 years). Of these patients, 5 cases have deletions in more than one region of AZF loci on Y-chromosome but no microdeletion was detected among control group. Totally 17 microdeletions was observed. Among the regions, AZFc had the most microdeletions 42% (7/17) followed by AZF a 35% (6/17) and the AZFb microdeletions have the less frequency 23% (4/17). In this study, among patients with microdeletion one case had deletions in all three AZF regions. Four cases had deletions in two regions and two cases had deletion just in one region. In total, 16% of all cases have deletions in AZFa, 16% in AZFc and 12% in AZFb regions.
Table I. Primers sequence and size
Table II. Primer mix of each Multiplex-PCR reaction
Table III. Results of different studies
Discussion
Deletions of AZF regions are deletions of the euchromatine part of the Y chromosome long arm. It is assumed that deletions of this part of Y chromosome can damage genes in this region that is responsible for the proper course of spermatogenesis. Many factors including somatic and sex chromosome genes interaction candicate to the normal spermatogenesis and the AZF deletions are the most frequent cause of spermatogenetic failure (6).
After Klinefelter’s syndrome, Y-chromosomal deletions are the second most frequent spermatogenesis disorder in infertile men. In the last few years some of research and clinical institutions have described screening of Y chromosome microdeletions in infertile men and molecular diagnostics of this type of Y-chromosomal disorders has become an important diagnostic test within laboratories worldwide dealing with these disorders (16-18).
Y chromosome AZF regions microdeletions are frequently found in azoospermic patients. Deletions incidence has been found from 3-55% (19, 20). In table III we compared our results whit other similar studies. EAA and EMQN published the guidelines for molecular screening of Y-chromosome microdeletions (21). In this study we used six sequences according to European guidelines in which all three sub-regions are represented by Y sequences: sY84, sY86, sY127, sY134, sY254 and sY255. Whit use of these STSs we reported 16% of microdeletions in AZFa, 12% in AZFb and 16% in AZFc regions.
The deletions in an infertile man could provide a proper understanding of the disease allows the medical stuff to avoid unnecessary expensive treatment to fertility improvement. Microdeletions of azoospermia factors are characteristic for spermatogenic failures and lead to oligozoospermia or azoospermia. PCR analysis of these deletions helps to determine site and the frequency of gene deletion and presents a defined prognosis and valuable counseling for couple whit fertility disorders. In the patients' with microdeletions that want to undertake ICSI procedure the deletions may be passed to their son. Because of this probability ethical consequences should be important dimensions of this technique.
Acknowledgements
The grant for this study was given by Yazd Research and Clinical Center for Infertility.
Conflict of interest
There is no conflict of interest in this study.
Type of Study: Original Article |
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