Original Article

Cytogenet Genome Res Accepted: May 18, 2018

by M. Schmid
DOI: 10.1159/000494464
Published online: November 23, 2018

Localization of the SRY Gene on Chromosome

3 in a Patient with Azoospermia and a Complex
Karyotype 45,X/46,X,i(Y)(q10)/46,XX/
47,XX,i(Y)(q10)
Linda C. Barnabas a Anbu Sumathy a Muthuswamy A. Indumathi b
Thankam R. Varma b Swathi Shetty c Jayarama S. Kadandale c Bibhas Kar a
a
Center for Genetic Studies and Research, b Institute of Reproductive Medicine and Women’s Health,
The Madras Medical Mission, Chennai, India; c Centre for Human Genetics, Bangalore, India

Keywords can be attributed to this abnormal genotype. The impor-

Azoospermia · Isochromosome · Male infertility · Mosaicism tance of genetic testing in infertile patients and the need for
genetic counselling to prevent the transmission of the de-
fect are emphasized. © 2018 S. Karger AG, Basel
Abstract
This study aimed to identify the cause of azoospermia in a
38-year-old infertile man who was referred for genetic test-
ing. Cytogenetic evaluation was performed by G-banding, Infertility is the inability to conceive after a year of
C-banding, and FISH using centromeric probes for chromo- unprotected sex and has been observed in about 15% of
somes X and Y and showed the presence of a monocentric couples worldwide. About 50% of the etiology of infer-
isochromosome Y with a complex, mosaic karyotype tility can be attributed to male factors, of which 15–30%
45,X/46,X,i(Y)(q10)/46,XX/47,XX,i(Y)(q10). Multiplex PCR for contribute to genetic abnormalities like chromosomal
the commonly deleted genes in the AZFa, AZFb, and AZFc defects, Y chromosome microdeletion, single gene mu-
regions of the Y chromosome was performed and indicated tations, and epigenetic disorders [Neto et al., 2016]. The
the presence of all 3 regions. Further, PCR amplification fol- frequency of chromosomal abnormalities is higher in
lowed by DNA sequencing of the SRY gene was done, which patients with abnormal spermatozoa, with the number
ruled out mutations in that gene. To identify the position of of sex chromosome abnormalities being greater in azo-
the SRY gene, FISH using a locus-specific probe was used and ospermia patients and autosomal chromosome abnor-
showed that the gene had been translocated to chromo- malities being greater in patients with oligozoospermia
some 3. Subtelomere FISH for 3q and Yp evidenced that the [Vijayalakshmi et al., 2011]. A total of 35% of the azo-
subtelomeric region of the Y chromosome was found on the ospermia and 12.7% of oligozoospermia patients have
terminal region of 3q. The clinical symptoms of the patient chromosomal abnormalities which are found to be sig-
130.235.66.10 - 11/24/2018 8:36:10 AM

© 2018 S. Karger AG, Basel Dr. Bibhas Kar

Center for Genetic Studies and Research
Lund University Libraries

The Madras Medical Mission

E-Mail karger@karger.com
Chennai 600037 (India)
www.karger.com/cgr
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E-Mail drbibhas_kar @ yahoo.co.in
 1 2 3 4 5 1 2 3 4 5

6 7 8 9 10 11 12 6 7 8 9 10 11 12

13 14 15 16 17 18 13 14 15 16 17 18

19 20 21 22 X Y 19 20 21 22 X Y

Fig. 1. GTG analysis showing the 45,X and 46,X,i(Yq) karyotype of the patient.

nificantly higher than in patients with normal sperma- Materials and Methods
tozoa [Pylyp et al., 2013]. Y chromosome microdele-
tions have been observed in 10–15% of patients with Clinical Data
A 38-year-old man, weighing 61 kg with a height of 165 cm,
azoospermia. Azoospermia factor (AZF) is located on visited the Institute of Reproductive Medicine & Women’s Health
Yq11.23 and has 4 sub-regions: AZFa, AZFb, AZFc, and on account of primary infertility of 7 years after a failed ayurvedic
a possible AZFd region, the presence of which is contro- and homeopathic treatment for 1.5 years and 7 months, respec-
versial. A deletion in one or more of these regions can tively.
cause different degrees of spermatogenetic failure [Yu et Semen analysis performed at 2 different times showed the ab-
sence of spermatozoa indicating azoospermia. His hormone levels
al., 2015]. Another important gene is the sex-determin- of FSH (9.48 mIU/mL), testosterone (3.680 ng/mL), and LH (7.09
ing region on Y chromosome (SRY) gene which is lo- mIU/mL) were within the normal range, whereas there was an in-
cated on the short arm of the Y chromosome. This re- crease in the level of prolactin (21.67 ng/mL). The Doppler study
gion may be deleted or translocated to the X chromo- of the scrotum revealed bilateral small testes, the right one being
some or to any of the autosomes. The translocation of smaller than the left. A calcification of 4 mm was observed in the
epididymal tail which could be a result of an old infection. Bilat-
the SRY gene has been observed in 46,XX males who are eral minimal hydrocele was also noted. He had a normal penis and
genotypically female but have a male-like appearance no clinical varicocele was observed. He experienced decreased li-
and may or may not exhibit genital ambiguity. The SRY bido and erectile dysfunction.
gene encodes the testis-determining factor, and an ab-
sence of this region can affect masculinization [Akinsal Analysis of GTG- and CBG-Banded Metaphases
Phytohaemagglutinin-stimulated peripheral blood lympho-
et al., 2017]. cytes were cultured for 72 h and were further treated with colcemid
In association with infertility, 46,XXY, 46,XYY, 46,XX using the standard procedure. The cells were harvested, and GTG-
males, 45,X/46,XY [Neto et al., 2016], 45,X/46,X,i(Yq) banding was performed on 25 metaphases. C-banding (CBG) was
[Ferguson et al., 1969], 45,X/46,X,i(Yq)/46,XY [Taylor et done using barium hydroxide and stained with Giemsa to identify
al., 1978], 45,X/46,X,i(Yq)/46,X,idic(Yq) [Jiang et al., the number of centromeres present in the Y chromosome. No-
menclature was according to ISCN [2016].
2017], 45,X/46,X,idic(Y)/46,XY,idic(Y) [Caglayan et al.,
2009] karyotypes and a male with 8 different cell lines, FISH Analysis
45,X/46,X,i(Y)/46,XY/46,X,i(Y),+mar/46,X,trc(Y)/47,X,i Centromere enumeration probes for X (DXZ1, Xp11.1-q11.1,
(Y),i(Y)/47,XY,i(Y)/47,XYY [Haaf and Schmid, 1990] Spectrum Green, Cytocell) and Y chromosomes CEPY (DYZ3,
have been reported. Here, an azoospermic male from Yp11.1-q11.1, Spectrum Orange, Cytocell) were used to study the
percentage of mosaicism, and the locus-specific probe LSI SRY
India was found to have 4 different cell lines, 45,X/46,X,i (Yp11.32, Spectrum Orange, Abbott) was used to identify the posi-
(Yq)/46,XX/47,XX,i(Yq), which is reported for the first tion of the SRY gene. Subtelomeric FISH was performed using sub-
time to the best of our knowledge. telomere-specific probes for 3q (LPT 03QR, Cytocell) and Yp (LPT
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DOI: 10.1159/000494464 Shetty/Kadandale/Kar
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 A B

Fig. 2. FISH analysis with CEP probes

showing a green signal for chromosome X
and red signal for chromosome Y. A One X
chromosome. B One X and one Y chromo-
some. C Two X chromosomes. D Two X
and a Y chromosome. C D

YPG, Cytocell). The instruction given by the manufacturers were

followed, and the analysis was performed using CytoVision 7.4
software.

Molecular Genetic Analysis

Blood sample was collected in EDTA tubes, and genomic DNA
was extracted. Multiplex PCR was performed to check for the
presence of 9 sequence-tagged sites in the AZFa (sY84, sY86),
AZFb (sY127, sY134, sY143), and AZFc (sY254, sY255, sY156,
sY158) regions of the Y chromosome. The isolated DNA was also
used to amplify the SRY gene by PCR using 2 different sets of
primers (5′-cggagaagctcttccttcct-3′, 3′-ccctgaactttgagaaacatga-5′
and 5′-tcagcagggcaagtagtca-3′, 3′-ccctgaactttgagaaacatga-5′), am-
plifying 500 bp upstream and downstream of the SRY gene and
yielding a 1.6-kb and a 1.9-kb DNA fragment. A fertile male and Fig. 3. C-banding showing the presence of one centromere on the
female sample was used as positive and negative controls, respec- Y isochromosome.
tively. To check for DNA contamination, water was used as a
blank and was run in parallel with each set of primers. For further
verification, the PCR product was sequenced using an ABI 3130
genetic analyzer. mosome to check for the presence of other cell lines. Two
additional cell lines, a female cell line with 2 signals for
the X chromosome and a Klinefelter cell line with 2 green
Results and 1 red signal for chromosomes X and Y were ob-
served: 69% 45,X; 25% 46,X,i(Y); 3% 47,XX,i(Y); and 3%
Two different cells lines with the karyotype 46,XX (Fig. 2). The final karyotype can be summarized
45,X/46,X,i(Y)(q10) were observed by GTG banding as 45,X/46,X,i(Y)(q10)/46,XX/47,XX,i(Y)(q10). C-band-
(Fig. 1). Of the 25 cells scored, 16 cells (64%) had a 45,X ing was performed to detect the number of centromeres
and 9 cells (36%) a 46,X,i(Y) karyotype. FISH was per- present in the abnormal Y chromosome and revealed a
formed using centromeric probes for the X and Y chro- monocentric isochromosome Y (Fig. 3). Due to his azo-
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SRY on Chromosome 3 in a Patient with Cytogenet Genome Res 3

Azoospermia DOI: 10.1159/000494464
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Fig. 4. FISH with SRY(R)/CEP X(G) probe showing 1 green signal on the X chromosome
and 1 red signal on a large metacentric chromosome. Inverted DAPI image proved the
large metacentric chromosome to be chromosome 3. Fig. 5. FISH for the subtelomere region of
3q (red) and Yp (green) showing the loca-
tion of Ypter in the terminal region of the
q arm of chromosome 3.

ospermic condition, the patient was referred for Y chro- Turner syndrome, male pseudohermaphroditism, or
mosome microdeletion study to determine the presence mixed gonadal dysgenesis [Kilic et al., 2010]. A male
of the commonly deleted genes in AZFa, AZFb, and with azoospermia and small testes having a mosaic
AZFc using multiplex PCR, which revealed the presence karyotype with 8 different cell populations has been re-
of all 3 regions. Since the karyotype indicated an absence ported. The patient had 45,X, 46,XY, and 47,XYY cell
of the Yp region, PCR was performed to test for the pres- lines, with the Y chromosome being an isochromosome
ence of the SRY gene, which is usually located on Yp11.32. or a marker and the isochromosome Y having 1–3 cen-
As a result, SRY was shown to be present, and the DNA tromeres [Haaf and Schmid, 1990]. Our patient also ex-
sequence of the gene, when further analyzed, showed no hibited small testes and azoospermia but had only 4 cell
mutation. To detect the location of the SRY gene, a locus- lines, with the Y chromosome being an isochromosome
specific probe was used, and by reverse DAPI staining, with only 1 centromere. CBG-banding detected the pres-
the gene was found to be located on the long arm of chro- ence of 1 centromere on the Y isochromosome. Further-
mosome 3 (Fig. 4). Subtelomere FISH using probes for more, FISH performed using a centromere-specific
3q and Yq was performed, and the Yp signal was ob- probe for the Y chromosome confirmed the presence of
served on the 3q region of the derivative chromosome 3 1 centromere. Karyotype analysis picked up 2 different
(Fig. 5). Due to the risk of transmission of the abnormal- cell lines in the patient, and FISH analysis detected 2
ity, after comprehensive counselling the couple opted for more cell lines. To check for the presence of these
in vitro fertilization (IVF) using donor sperm. Four em- 47,XX,i(Y) and 46,XX cell lines, the GTG-banded slides
bryos were transferred but the implantation was not suc- were re-examined, but as the frequency of these cell lines
cessful. A second IVF was attempted with a different do- was low, they were not detectable by the standard cyto-
nor’s sperm. One embryo was transferred, but pregnancy genetic technique. This highlights the importance of
was not achieved. FISH in studying mosaicism. The necessity to perform
FISH after karyotyping for detecting low level mosa-
icism has also been emphasized in a study where 64% of
Discussion the mosaic karyotypes revealed an additional cell line
[Abulhasan et al., 1999].
Azoospermia has been observed in phenotypically Mosaicism is the occurrence of 2 or more cell lines de-
normal men with a 45,X/46,XY karyotype. Men with this rived from the same stem cell in an individual [Caglayan
karyotype can appear normal or be associated with et al., 2009]. In GTG-banding, the presence of extra chro-
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DOI: 10.1159/000494464 Shetty/Kadandale/Kar
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mosomes or structural abnormalities in a minimum of 2 was continued, and a phenotypically normal male was
cells or missing chromosomes in 3 cells is considered as a born [Chien et al., 2009]. In our patient, an asynaptic type
mosaic, and in FISH, a mosaic is considered by the pres- of meiosis may occur, hampering the pairing of homolo-
ence of a minimum of 2 cells with an extra signal or 3 cells gous chromosomes especially on chromosome 3 where
with a missing signal [Okada et al., 2001]. the SRY gene is translocated. Karyotyping the patient’s
A study was conducted in which 2 patients with a pseu- father would have helped us to understand the origin of
dodicentric Y chromosome were reported, who exhibited the translocation, but this was not possible as he did not
relatively small testes and showed an absence of sperm in give his consent.
their semen, but the reason for their azoospermia could Men with an abnormal karyotype have the same pro-
be attributed to the absence of the AZFb and AZFc re- portion of sperm aneuploidy when compared to infertile
gions [Li et al., 2013; Nailwal and Chauhan, 2017]. Micro- men with a normal karyotype. The sperm aneuploidy
deletions in the AZF region are commonly observed in rate in a male with an XO/XYY karyotype was almost
cases of severe oligozoospermia and azoospermia. These similar to that seen in controls, whereas in another study
microdeletions have mostly a de novo origin, but there is of a Klinefelter male with an XXY/XXXY/XY chromo-
a high chance that they can get transmitted to their off- some composition, the number of hyperhaploid sperm
spring through artificial reproductive techniques (ART) was greater than in a normal male [Kruse et al., 1998;
or through natural pregnancy [Yu et al., 2015]. The pres- Dale et al., 2002; Ren et al., 2015]. Thus, if sperm is avail-
ence of the AZFa, AZFb, and AZFc regions was con- able in the testes of the patient, ART using surgical
firmed in our patient using 9 STS markers, indicating that sperm retrieval, like percutaneous epididymal or testic-
the cause for the azoospermia may not be linked to these ular sperm aspiration (PESA or TESA), followed by in-
regions. A set of primers for AZFa (sY84, sY86), AZFb tracytoplasmic sperm injection (ICSI) can help men
(sY127, sY134), and AZFc (sY254, sY255) is capable of with a mosaic karyotype. However, prenatal diagnosis
detecting over 90% of deletions in the 3 AZF regions. becomes very crucial to prevent the genetic abnormality
However, recent advancement in research has shown that from getting transmitted to the offspring [Francisco et
these deletions can be ethnicity specific and there might al., 2010; Kilic et al., 2010]. In our patient, TESE was not
be additional regions deleted in the Indian population performed due to the presence of many abnormal cell
[Sachdeva et al., 2011]. Hence based on the literature lines which indicated a high risk of transmission to the
available, 3 additional STS primers, AZFa (sY84), AZFb offspring. The couple hence opted for IVF using donor
(sY134), and AZFc (sY255), showing the highest deletion sperm. Five oocytes were retrieved and 4 of them were
frequency in the Indian population, were selected and fertilized by artificial insemination. Four embryos, 1
added to the deletion testing procedure. each at 6- and 8-cell and 2 at 10-cell stages, were trans-
A translocation between the Y chromosome and an ferred to the uterus after laser-assisted hatching, but im-
autosome has been observed in 1: 2,000 of the general plantation was not successful. A second cycle of IVF was
population. A 38-year-old male with oligozoospermia attempted using sperm from a different donor. Two oo-
has been reported with a translocation between the acro- cytes were retrieved and fertilized, but only 1 embryo
centric chromosome 13 and the Y [Alves et al., 2002], and cleaved and embryo transfer was done. However, the
2 females, aged 30 and 26 years, had a portion of the Y βhCG level was low, indicating an unsuccessful preg-
chromosome translocated to the short arm of chromo- nancy. The wife was reluctant to have her karyotype
some 1 which they had inherited from their fathers. In analysis done as the cause of infertility seemed to be one-
these women, the SRY gene was absent, and hence they sided. However, genetic testing of an infertile couple is
were phenotypically female [Morel et al., 2002]. About very crucial for planning the management. Performing
130 cases of Y and autosome translocations have been re- ART without genetic testing can transmit the abnormal-
ported, and 21 of them were unbalanced translocations of ity directly to the offspring, and hence, at least karyotyp-
which 13 had a male phenotype [Hsu, 1994]. In our pa- ing has to be made mandatory for couples undergoing
tient, the locus-specific probe for the SRY gene showed its IVF. Preimplantation genetic screening (PGS) can help
translocation to the q arm of chromosome 3, but this in improving the chances of pregnancy by choosing an
could not be visualized by GTG-banding. A similar trans- embryo with a better chance for developing, and for pa-
location between chromosomes 3 and Y was identified tients who have terminated their pregnancy following
during prenatal testing in a fetus with a 46,XX karyotype prenatal testing, PGS seems to be a better option [Soini
and apparently normal male genitalia. The pregnancy et al., 2006].
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SRY on Chromosome 3 in a Patient with Cytogenet Genome Res 5

Azoospermia DOI: 10.1159/000494464
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 In conclusion, the clinical symptoms of our patient can Statement of Ethics
be attributed to his abnormal genotype. FISH as a diag- Informed written consent was given by the couple prior to their
nostic technique has been shown to have a great potential participation in this study.
in identifying mosaicism. This report emphasizes the im-
portance of genetic testing in infertility patients and the
need for genetic counselling to prevent the recurrence of Disclosure Statement
an abnormality in the offspring.
The authors have no conflicts of interest to declare.

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