11_chapter 4

Chapter IV
Studies on microdeletion
of the Y-chromosome
in infertile men
Studies on microdeletion of the Y-chromosome in infertile men
4.1. Introduction
The genetic causes such as chromosomal aberrations, monogenic disorders, mutations in
mitochondrial DNA (mt DNA), Y-chromosome microdeletions and multifactorial disorders are
reported to be a contributing factor for male factor infertility; as about 10% genes in the genome
are related to spermatogenesis [1, 2]. One of the most commonly identified molecular genetic
causes of male infertility has been submicroscopic deletions on the Y chromosome which is
difficult to resolve in conventional chromosome analysis. The role of Y chromosome in male
infertility was first elucidated in 1976. Tiepolo and Zuffardi have proposed that a factor which
controls spermatogenesis, encoded by a gene that is localized within the euchromatic region of
the Y-chromosome long arm (Yq11), called as azoospermia factor (AZF) [3]. At present, three
different spermatogenetic loci on AZFa, AZFb and AZFc have been mapped to long arm of the
Y-chromosome. The Y-chromosome has been divided into seven deletion intervals that are
further divided into subintervals (like A, B, C D, E, F and G). Originally, three regions were
defined: AZFa, AZFb and AZFc (azoospermia factor), which maps on the long arm (Yq) from
centromere to telomere. A fourth region, named AZFd, located between AZFb and AZFc was
also reported, these regions may be associated with a particular testicular histology [4]. Vollrath
et al., (1992), constructed a 43-interval deletion map of human Y chromosome that contains
an array of sequence-tagged sites (STS) which spans entire length of the Y chromosome, and
reported to have involved in regulation of spermatogenesis [5].
The incidence of microdeletions within AZF regions varies from 1-55% of infertile men in
different studies; size and its position with infertile phenotype had shown a marked variation in
the deletion frequency; further wide variation was attributed due to selection of different patient
groups and use of different marker sets [6]. Microdeletions within the AZF region occur in
approximately 4% of men with oligozoospermia; 14% of men with severe oligozoospermia; and
18% in nonobstructive azoospermia men. In north European populations such as Scandinavian
countries, France, Germany, Netherlands for instance, the frequency of Y-chromosome
deletion in infertility cases is rather low (1–4%), while it is reported to be greater than 15% in
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Italy [7] and about 10% in Southeast Asian countries (China, Japan, Korea, Phillipines etc.,)
indicate the frequency to revolve around 10% [8, 9]. In the hitherto reported cases from Asian
populations, vast majority of deletions are confined to AZFc region with only rare cases of
AZFa/b deletions. Vogt et al., in 1996 correlated the position of AZF deletion with a phase in
which spermatogenesis was arrested and deletion representing that each AZF locus acts at a
different phase of spermatogenesis and causes spermatogenic arrest at a particular stage. Thus,
deletions within the AZF region can result in varying degrees of spermatogenic failure and
hence, the prevalence increases with severity of infertility [4].
The vast majority of microdeletions arise de novo and have been attributed to repetitive
palindromic DNA sequences termed amplicons, that are clustered along the Yq. The high
degree of homology between these palindromes promotes intrachromosomal recombination
and rearrangements with inevitable deletion patterns [18]. Microdeletions remove one or
more of these genes, and as a result cause varying defects in spermatogenesis; candidate genes
within the AZF regions are believed to play critical roles in germ cell cycle regulation and
meiosis. However, this has not yet led to the identification of molecular basis for defective
spermatogenesis in spite of genotype–phenotype correlations are emerging. Furthermore, as
only 3.42% of infertile men showed chromosomal abnormalities, the present study was extended
to examine microdeletions on the Y-chromosome. Hence, this study was aimed to correlate the
prevalence and frequency of microdeletions in AZFa, b and c sub-regions of azoospermic,
globozoospermic, SOAT and oligoasthenoteratozoospermic men.
4.2. Materials and methods
To study microdeletions on chromosome Y, genomic DNA was isolated as per the manufacturer’s
protocol (Bioserve-DNA Isolation Kit) as discussed in Section 2.4. The quantity and quality of
the DNA was checked by Spectrophotometer and 0.8% agarose gel electrophoresis as represented
under Section 2.4.2. and 2.4.3. Microdeletions were detected by performing STS PCR based
techniques on controls and case’s genomic DNA. All STS PCR primer sequences were obtained
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from published literature [6] and AZF regions studied were AZFa-sY 84 (326 bp), sY 86 (320
bp), AZFb-sY 127 (274 bp), sY134 (301 bp) and AZFc- sY 254 (400 bp), sY 255 (126 bp). The
PCR product was stained with ethidium bromide, resolved using 2% agarose gel electrophoresed
at 50V and observed under UV-transilluminator; presence or absence of a band for six loci were
investigated and documented as shown in Section 2.6. Fertile male and female samples were
used as positive and negative controls. The deletion frequency between cases and controls were
compared and the level of significance was calculated using student’s “t” test. 4.3. Results
A representative agarose gel electrophoresis of AZFa, AZFb and AZFc indicating the presence
or absence of the band of expected size is shown in Figure 4.1 and 4.6. Based on documented
information, the deletion frequency for six STS markers were calculated and given in the Table
4.1. The frequency of microdeletions was found to be 9.14% in the azoospermic and 3.42%
in the oligoasthenoteratozoospermic infertile men. An overall 12.56% of infertile subjects
showed microdeletions in one or more sequence-tagged sites (STS) with a percentage of 1.14,
2.28, 9.14 for AZFa (sY84, sY86), Figure 4.1 and 4.2; AZFb (sY127, sY134), Figure 4.3 and
4.4 and AZFc (sY254, sY255), Figure 4.5 and 4.6 respectively (Table 4.1). However, none of
the control subjects showed any deletion in all the six loci.
Table 4.1. Frequency of Yq microdeletions in infertile men
Microdeletions observed in AZF region (%)
Abnormal spermiogram
AZFa
AZFb
AZFc
Total
Azoospermia
1.14
2.28
5.72
9.14
OAT*
0.00
1.14
2.28
3.42
Total
1.14
3.42
8.00
12.56
*OAT-Oligoasthenoteratozoospermia
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Figure 4.1. Electrophoresis on a 2% agarose gel-AZFa (sY 84)
1
2
3
4
5
6
1000 bp
500 bp
300 bp
200 bp
326 bp
100 bp
Lane 1 Molecular weight DNA ladder (100 bp); Lane 2 absence of the band at sY 84 (326 bp);
Lane 3-6 presence of the band at sY 84 (326 bp).
Figure 4.2. Electrophoresis on a 2% agarose gel-AZFa (sY 86)
1
2
3
4
5
6
1000 bp
600 bp
400 bp
300 bp
320 bp
200 bp
100 bp
Lane 1 Molecular weight DNA ladder (100 bp); Lane 2-5 presence of the band at sY 86 (320
bp); Lane 6 absence of the band at sY 86 (320 bp).
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Figure 4.3. Electrophoresis on a 2% agarose gel-AZFb (sY 127)
1
2
3
4
5
6
1000 bp
500 bp
400 bp
300 bp
274 bp
200 bp
100 bp
Lane 1 Molecular weight DNA ladder (100 bp); Lane 2-4 and 6 presence of the band at sY 127
(274 bp); Lane 5 absence of the band at sY 127 (274 bp).
Figure 4.4. Electrophoresis on a 2% agarose gel-AZFb (sY 134)
1
2
3
4
5
1000 bp
500 bp
400 bp
300 bp
301 bp
200 bp
100 bp
Lane 1 Molecular weight DNA ladder (100 bp); Lane 2-3 and 5 presence of the band at sY 134
(301 bp); Lane 4 absence of the band at sY 134 (301 bp).
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Figure 4.5. Electrophoresis on a 2% agarose gel-AZFc (sY 254)
1
2
3
4
5
6
1000 bp
500 bp
300 bp
200 bp
400 bp
100 bp
Lane 1 Molecular weight DNA ladder (100 bp); Lane 2-4 and 6 presence of the band at sY 254
(400 bp); Lane 5 absence of the band at sY 254 (400 bp).
Figure 4.6. Electrophoresis on a 2% agarose gel-AZFc (sY 255)
1
2
3
1000 bp
600 bp
500 bp
400 bp
300 bp
200 bp
126 bp
Lane 1 absence of the band at sY 255 (126 bp); Lane 2 presence of the band at sY 255 (126 bp);
Lane 3 Molecular weight DNA ladder (100 bp).
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4.4. Discussion
Genetic factors contribute significantly to infertility and in about 50% of which are due to male
factor [10, 11]. Spermatogenesis is highly disorganized and that even healthy fertile men may
have certain percentage of sperm with morphological abnormality. It was also reported that
microdeletions in AZF region can lead to a variable phenotype with a significant reduction
in sperm count and secondarily to an increased loss of germ cells and progressive decline in
semen quality. The incidence of Yq microdeletions ranges from 7–21% in azoospermic men
and 0–14% in oligozoospermic patients respectively [8, 12, 13, 14]. Babu et al., (2002) found
15% of AZF deletions in azoospermic and severely oligozoospermic men in a population of 20
patients [15]. Thus, the incidence of microdeletion of chromosome Y is higher when patients
are selected by testicular histology [16, 17].
The deletion frequency in the current study was found to be 9.14% in azoospermia and 3.42%
in oligoasthenoteratozoospermia cases. However, Foresta et al., (1997) found a very high
percentage (55.5%) of Italian infertile men to carry these Y-chromosomal microdeletions in
azoospermia cases [7]. Kuroda-Kawaguchi et al., (2001) also have reported a higher deletion
frequency in azoospermia of 12% and a 6% in severe oligoasthenoteratozoospermia cases
[18]. Mohammed et al., (2007) reported 2.6% (n=7/266) microdeletions in the AZFb and
c regions [19]. Dada et al., in 2003 reported the frequency of Y microdeletions as 9.63%
among 83 infertile Indian men studied using six STS primers [20]. In the present study,
by using six STS markers in 175 infertile men, showed, 12.56% of microdeletions AZF
a, b and c loci on chromosome Y; the deletion frequency was calculated by resolving the
PCR product with 2% Agarose gel electrophoresis (AGE). Thangaraj et al (2003) studied
340 infertile men with 30 different STS markers by Southern hybridization technique
and sequencing and reported an 8.5% deletion [21]. Another study, had reported a 5%
deletion among 180 infertile men using 31 STS markers specific for AZF regions [39]. On the basis of testicular histology, the deletion of AZFa was associated with the complete absence
of germ cells, presence of sertoli cells in the seminiferous tubules, characteristic of sertoli cellonly syndrome (SCO) that is associated with azoospermia. The main candidate gene in the AZFb
region has a restricted expression in the testis [22] that is associated with developmental arrest of
germ cells at the pachytene stage and leads to meiotic maturation arrest [23, 24, 25]. Deletions in
AZFc regions are [26], associated with developmental arrest of germ cells at the spermatid stage,
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hypospermatogenesis, maturation arrest and low sperm counts. Thus, deletion of a particular AZF
locus results in a characteristic phenotype, and genes at each locus act at a particular stage of germ
cell differentiation.
Krausz et al., (2006) found that these microdeletions may cause deregulation of gene expression
by position effect and interfere with posttranscriptional modification of gene expression, or result
in the absence of genes critical for spermatogenesis [27]. The Y chromosome has the highest
spontaneous loss of genetic material in the human genome. Most of the ancestral genes are
functionally intact on the X chromosome, which undergoes crossing over; but because of the lack
of XY recombination, there is monotonic decline in gene function on the Y chromosome and thus
the accumulation of deleterious mutations. More than SNPs and point mutations in any specific
genes on the Y-chromosome, deletions of large regions within AZF have been found to be more
frequent in cases of idiopathic infertility.
In the present study, 12.56% of infertile men have shown Yq microdeletion in 175 cases, while
no microdeletion was observed in control samples. A 2% of the fertile men might harbor
microdeletions of chromosome Y that involving noncoding region [28]. The relative frequency of
individual microdeletions is reported to be 5%, 16% and 60% for AZFa, AZFb and AZFc regions
respectively. The first major gene identified in AZFa is sY84, but its role in spermatogenesis
remained to be confirmed. To date only three infertile patients have been reported carrying a
deletion of this gene, loss of these genes through AZFa have been seen to give rise to more severe
phenotypes such as SCO syndrome [29]. One of the hallmarks of the Y-chromosome is the high
frequency of amplified repeat sequences dispersed throughout the euchromatic and heterochromatic
regions. This genetic instability arises from the presence of highly repetitive segments in the long
and short interspersed repeats and from a large portion of Y chromosome (95%) that does not
undergo recombination during meiosis [30, 31]. Microdeletions restricted to AZFb or c, can result
in a range of phenotypes from sertoli cell-only syndrome to moderate oligozoospermia. The
DAZ gene cluster localized on the distal euchromatic region of the Y chromosome AZFc region
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is one of the most important candidate genes involved in infertility. Another study from South
India [32] reported 4/50 infertile men with AZFc deletions. The deletions in AZFc often include
all the copies of DAZ gene, and they are frequently associated with azoospermia, rarely with
oligozoospermia [33]. The presence of multiple copies is to create redundancy in this important
gene in case mutation damages one of those genes.
Estimations of the molecular extensions of AZF regions in numerous patients using PCR multiplex
interval mapping suggest similar breakpoints in Yq11 for AZFa [4, 34], AZFb [35, 36] and AZFc
patients [4, 33, 34, 36, 37]. This indicates breakage hotspots in Yq11 to be at their borderlines.
Such hotspots are frequently represented by homologous local chromosome-specific repetitive DNA
blocks [38]; these deletions probably occur due to unequal intrachromosomal crossing-over events
at meiosis during spermatogenesis in the father. This would explain for higher frequency of AZFc
deletions than AZFa and b deletions, as local repetitive DNA blocks are enriched in distal Yq11 in the
neighborhood of highly repetitive heterochromatic Yq12 region. However, to enable a more detailed
examination of AZF deletion’s origin, single cell deletion analyses in Yq11 of the spermatozoa of
sterile patients and fertile control men might be an essential prerequisite. Therefore, it should be of
critical interest to take geographical, environmental and ethnic axis into consideration on the genetic
basis of infertility. In a multifactorial disorder, such as idiopathic infertility, where environment and
the genetic components interact variously, data from more regions need to be generated to develop
further realistic picture.
4.5. Conclusion
This study has shown the prevalence of Y microdeletions as 12.6% in infertile men. Of three
AZF regions studied, AZFc region showed deletion in many infertile subjects, which is a strong
candidate for the azoospermia factor. The occurrence of Y microdeletions is reported to be de
novo, but there is a risk of transmission of the Yq microdeletions from father to son. Hence, Y
microdeletion analysis by STS-PCR is a simple, highly sensitive method and molecular analysis
can be a useful tool in identifying affected infertile men so that appropriate counseling can be
given before treatment by assisted reproductive technology.
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