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Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia
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ARTICLE
Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia Sanjukta Sen a, Rupesh Agarwal a, Prafulla Ambulkar b, Indira Hinduja c, Kusum Zaveri c, Jyotsna Gokral d, Asoke Pal b, Deepak Modi a,* a
Molecular and Cellular Biology Laboratory, National Institute for Research in Reproductive Health (ICMR), JM Street, Parel, Mumbai, 400 012, India; b Human Genetic Division, Department of Anatomy, Mahatma Gandhi Institute of Medical Sciences, Sevagram, 442102, Wardha, India; c Hinduja IVF Centre, PD Hinduja Hospital and Medical Research Center, Veer Savarkar Marg, Mahim, Mumbai, 400016, India; d Department of Reproductive Endocrinology and Infertility, National Institute for Research in Reproductive Health (ICMR), JM Street, Parel, Mumbai, 400012, India * Corresponding author.
E-mail address: [email protected] (D Modi). Sanjukta Sen has completed her masters degree in biochemistry, and is presently pursuing a doctorate degree in applied biology. Her area of interest is genetics of male infertility, with a focus on Y chromosomal genes and male infertility. She is also interested in the reproductive outcome of patients with male infertility opting for ICSI for biological parenthood.
The AZFc locus on the human Y chromosome harbours several multicopy genes, some of which are required for spermatogenesis. It is believed that deletion of one or more copies of these genes is a cause of infertility in some men. GOLGA2LY is one of the genes in the AZFc locus and it exists in two copies, GOLGA2P2Y and GOLGA2P3Y. The involvement of GOLGA2LY gene copy deletions in male infertility, however, is unknown. This study aimed to investigate the association of deletions of GOLGA2P2Y and GOLGA2P3Y gene copies with male infertility and with sperm concentration and motility. The frequency of GOLGA2P3Y deletion was significantly higher in oligozoospermic men compared with normozoospermic men (7.7% versus 1.2%; P = 0.0001), whereas the frequency of GOLGA2P2Y deletion was comparable between oligozoospermic and normozoospermic men (10.3% versus 11.3%). The deletion of GOLGA2P3Y but not GOLGA2P2Y was significantly higher (P = 0.03) in men with gr/gr rearrangements, indicating that GOLGA2P3Y deletions increase the susceptibility of men with gr/gr rearrangements to oligozoospermia. Furthermore, men with GOLGA2P3Y deletion had reduced sperm concentration and motility compared with men without deletion or with deletion of GOLGA2P2Y. These findings indicate GOLGA2P3Y gene copy may be candidate AZFc gene for male infertility. Abstract
© 2015 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. KEYWORDS: AZFc, GOLGA2LY, gr/gr rearrangements, oligozoospermia, sperm count, sperm motility
http://dx.doi.org/10.1016/j.rbmo.2015.11.001 1472-6483/© 2015 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001
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Introduction Cytogenetic and molecular analyses of men with defective spermatogenesis have identified microdeletions in the q arm of the Y chromosome. Between 2 and 10% of men with oligozoospermia or azoospermia are estimated to harbour Yq microdeletion, and it is currently the leading genetic cause of male infertility (Abid et al., 2008a; Ambulkar et al., 2014; Krausz et al., 2014; Massart et al., 2012; Sen et al., 2013a). Yq microdeletions involve loss of one (or more) of the three AZF loci namely, AZFa, AZFb and AZFc. Among these three, the deletion of AZFc locus is most commonly observed (Ambulkar et al., 2014; Foresta et al., 2001; Massart et al., 2012; Sen et al., 2013a; Simoni et al., 2008). The human AZFc is palindromic in nature, and has several repeat regions making it susceptible to self recombination during meiosis, which makes it most susceptible to deletions (Kuroda-Kawaguchi et al., 2001; Skaletsky et al., 2003). Withstanding this fact, along with complete AZFc deletions, genetic analysis of infertile men has led to identification of partial deletions within the AZFc locus. Referred to as the gr/ gr rearrangements or AZFc subdeletions, these rearrangements remove 1.6–1.8 Mb, which is about 50% of the AZFc region (Ferlin et al., 2005; Repping et al., 2003; Stouffs et al., 2008). The gr/gr rearrangements are more frequently observed in azoospermic and oligozoospermic men compared with normozoospermic counterparts (Ferlin et al., 2005; Giachini et al., 2005; Lo Giacco et al., 2014; Sen et al., 2015; Stouffs et al., 2011). Unlike the Yq microdeletions, however, the gr/gr rearrangements are also observed in 2–5% of fertile and normozoospermic men (Lo Giacco et al., 2014; Sen et al., 2015; Stouffs et al., 2011; Tüttelmann et al., 2007). What makes some men harbouring gr/gr rearrangements susceptible to male infertility is presently unclear. Although ethnicity and genetic background of Y chromosome have been demonstrated to be a cause; variation in the genes deleted within the AZFc locus in individuals with gr/gr rearrangements has been suspected to be the causative factor (Lo Giacco et al., 2014; Sen et al., 2015). The AZFc locus is structurally unique, and is composed of three palindromes housing 11 families of transcription units (Kuroda-Kawaguchi et al., 2001; Skaletsky et al., 2003). All the genes in AZFc locus exist in multiple copies and are expressed in the testis (Kuroda-Kawaguchi et al., 2001; Skaletsky et al., 2003; Tse et al., 2003). It is believed that gr/gr rearrangements remove copies of some key AZFc genes like Deleted in Azoospermia (DAZ) and Chromodomain on the Y chromosome,1 (CDY1) causing male infertility (Ferlin et al., 2005; Krausz et al., 2009; Sen et al., 2015; Stouffs et al., 2008). In addition to these genes, however, the gr/gr rearrangements also remove copies of three additional genes, namely Golgi autoantigen, golgin subfamily a, 2-like, Y linked (GOLGA2LY), Basic protein on Y chromosome, 2 (BPY2) and Testis specific transcript, Y linked 4 (TTY4) (Noordam et al., 2011; Stouffs et al., 2008). Whether the deletion of one or more copies of these genes may also be a cause of infertility is presently unclear. As not all men harbouring gr/gr rearrangements are infertile, it is possible that there might be heterogeneity in the deletion of these genes, which may be the determinant of the susceptibility of some men to infertility. Although variation in DAZ and CDY1 copy deletions in
men with gr/gr rearrangements has been reported to increase the susceptibility of gr/gr rearrangements to male infertility (Choi et al., 2012; Krausz et al., 2009; Lu et al., 2009; Sen et al., 2015), the involvement of other genes is still unknown. GOLGA2LY is a 4.8Kb gene present in two copies on the AZFc locus, referred to as GOLGA2P2Y and GOLGA2P3Y for the 5′ and the 3′ copy, respectively. The gr/gr deletions generally remove either the GOLGA2P2Y copy or GOLGA2P3Y copy depending on the site of recombination (Krausz et al., 2009; Noordam et al., 2011). Controversies exist on the association of GOLGA2LY copy deletions and male infertility. Noordam et al. (2011) showed that infertile males harbouring the AZFc subdeletion encompassing the GOLGA2LY had lower total motile sperm count compared with controls. Lu et al. (2014) failed to report any significant association of GOLGA2LY gene copy number variations with male infertility. Therefore, it is unclear if deletions of GOLGA2P2Y/3Y may be a causative factor for male infertility. As GOLGA2LY is transcribed in the testis (Kuroda-Kawaguchi et al., 2001), and is located within the region deleted in gr/gr rearrangements, we hypothesize that loss of GOLGA2LY gene copies may be also associated with male infertility. In the present study, we aimed to determine the association of the deletions of the GOLGA2LY gene copies GOLGA2P2Y and GOLGA2P3Y with oligozoospermia. The effect of these gene copy deletions were also evaluated on sperm concentration and motility.
Materials and methods Ethics statement The study was conducted independently at the National Institute for Research in Reproductive Health, Mumbai, and Mahatma Gandhi Institute of Medical Sciences, Sevagram, India. The Mahatma Gandhi Institute of Medical Sciences Ethical Committee approved the study on 30 December 2006 and the Ethics Committee for Clinical Studies of National Institute for Research in Reproductive Health approved the study on 20 December 2005. Written informed consent was obtained from all study participants.
Study participants The details of inclusion and exclusion criteria for the participants have been described previously (Ambulkar et al., 2014; Sen et al., 2013b). Participants were classified on the basis of sperm concentration, and were grouped into normozoospermic men (sperm concentration ≥15 million/ ml) and oligozoospermic men (sperm concentration <15 million/ml) as per the World Health Organization 2010 guidelines (WHO, 2010). Participants with karyotypic abnormalities, obstructive azoospermia, hypogonadism, hypoandrogenism, chronic diseases, history of mumps, history of fever, varicocele and history of pelvic/spinal injuries were excluded. None of the patients reported heavy smoking, alcohol intake, or both. All the men were screened for Yq microdeletion
Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001
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according to the protocol previously published (Abid et al., 2008a, 2008b; Ambulkar et al., 2014; Sen et al., 2013a, 2013b).
Detection of GOLGA2P2Y and GOLGA2P3Y deletions Genomic DNA was isolated from the whole blood using a commercial kit (Sigma-Aldrich Co, St Louis., USA) as described previously (Sen et al., 2013b). The GOLGA2LY gene copies were detected by Sequence Family Variant (SFV) analysis, which is based on the fact that the GOLGA2P2Y differs from GOLGA2P3Y by a SNP that can be detected by HhaI restriction site (Fernandes et al., 2004). The fragment of GOLGA2P2Y/3Y that encompasses the SNP was amplified by polymerase chain reaction (PCR) using the Red Amp master mix (Sigma-Aldrich Co, USA) for 35 cycles. The PCR products were digested overnight using HhaI (Fermentas International Inc., Ontario, Canada) and resolved on 15% PAGE. The primer sequences and the annealing temperatures are given in Table 1.
the frequency of GOLGA2P2Y and GOLGA2P3Y gene deletions were evaluated using the chi-squared test. Differences of sperm concentration and motility were analysed by analysis of variance. The GOLGA2LY copy deletion and the risk of male infertility were estimated by computing the odds ratio, 95% confidence interval and P-values. P < 0.05 were regarded as statistically significant.
Results A single band of expected size (531 bp) was detected in all the samples amplified for GOLGA2LY. No bands were detected when female genomic DNA and water were used as template indicating specificity of the product. Direct sequencing of the PCR products revealed its 100% identity to GOLGA2LY. Restriction digestion of the PCR amplified product gave three bands of 289 and 242 bp for GOLGA2P2Y and 531 bp for GOLGA2P3Y. Absence of the 289 and 242 bp products indicate a deletion of GOLGA2P2Y, whereas absence of 531 bp product indicates deletion of GOLGA2P3Y (Figure 1).
Detection of gr/gr rearrangements gr/gr rearrangement typing was carried by PCR amplification of sequence tagged site markers sY1291/sY1191 and SRY gene as internal control. As described above, PCR was carried out and run on 2% agarose gel. The AZFc subdeletions were classified as follows: the absence of sY1291 as gr/gr, the absence of sY1191 as b2/b3 and the absence of both the markers sY1291/sY1191 as b1/b3 deletions (Giachini et al., 2005). The optimized annealing temperature and primer sequences are given in Table 1.
Statistical analysis All the statistical analyses were carried out using the SPSS software (SPSS Inc., Chicago, IL, USA, version 16). Differences in Table 1
Primer sequences and the annealing temperatures.
Primers
GOLGA2LY
sY1191
sY1291
sY14/SRY
Sequence
Expected size (bp)
Annealing temperature (°C)
F 5′ TTGGCCTGTT GCTTCTAGGGTT 3′ R 5′ ACAGGGAGGG TGCTGTCACA 3′ F 5′ CCAGACGTTCT ACCCTTTCG 3′ R 5′ GAGCCGAGATC CAGTTACCA 3′ F 5′ TAAAAGGCAGAA CTGCCAGG 3′ R 5′ GGGAGAAAAGTT CTGCAACG 3′ F 5′ GAATATTCCCG CTCTCCGGA 3′ R 5′ GCTGGTGCTCC ATTCTTGAG 3′
531
68
385
62
527
62
472
62
Frequency of deletion of GOLGA2P2Y and GOLGA2P3Y in fertile and infertile men A total of 609 individuals who met the inclusion and exclusion criteria were included in the study: 336 men had normozoospermia and 273 had oligozoospermia. Of this, 12.5% (42/336) of the normozoospermic men and 17.9% (49/273) of the oligozoospermic men had any one of the two GOLGA2LY copies deleted. This difference was not statistically significant. Deletion of GOLGA2P2Y was observed in 11.3% of normozoospermic men and 10.3% of oligozoospermic men. This difference was not statistically significant. Deletion of GOLGA2P3Y was observed in 1.2% of normozoospermic men and 7.7% of oligozoospermic men. The frequency of the GOLGA2P3Y deletion was significantly higher in oligozoospermic men compared with normozoospermic men (P = 0.0001) (Table 2).
Figure 1 Association of deletions of GOLGA2LY copies with oligozoospermia. (A) Polymerase chain reaction restriction fragment length polymorphism for the detection of GOLGA2LY copy deletion. Lane 1: 100 bp ladder, Lane 2, 3: GOLGA2P3Y deletion, Lane 4: GOLGA2P2Y deletion, Lane 5: female control, Lane 6: water control, Lane 7: no deletion, Lane 8: undigested DNA.
Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001
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Table 2
Frequency of men with deletions of gene copies GOLGA2P2Y and GOLGA2P3Y.
Gene copy deleted
GOLGA2P2Y GOLGA2P3Y
Oligozoospermic (n = 273)
Normozoospermic (n = 336)
Number with deletion (%)
Number with deletion (%)
28 (10.3) 21 (7.7)
38 (11.3) 4 (1.2)
P-value
Odds ratio
95% CI
NS 0.0001
0.9 6.9
0.5 to 1.5 2.3 to 20.4
CI, confidence interval, NS, not statistically significant.
Frequency of deletion of GOLGA2P2Y and GOLGA2P3Y in men with gr/gr rearrangements Forty-eight out of 609 individuals included in this study had gr/gr rearrangements. Of these, 70.8% had gr/gr, 6.3% had b1/b3 and 22.9% had b2/b3 deletion (Supplementary Table S1). GOLGA2LY copy deletions were observed in 50% (8/16) of men with normozoospermia and 72% (23/32) of men with oligozoospermia who had gr/gr rearrangements. A total of 37.5% of normozoospermic men and 28.1% of oligozoospermic men had GOLGA2P2Y deletion. This difference was not statistically significant. A total of 12.5% of normozoospermic men and 43.8% of oligozoospermic men had the GOLGA2P3Y copy deleted; this difference was statistically significant (P = 0.03) (Table 3).
Association of deletions of GOLGA2P2Y and GOLGA2P3Y with sperm concentration and motility To analyse the effect of GOLGA2LY copy deletions on spermatogenesis, the sperm concentration and motility were evaluated in men with GOLGA2LY copy deletions, with and without gr/gr rearrangements. Irrespective of the gr/gr rearrangements, the sperm concentration and motility in men with GOLGA2P3Y deletion were significantly lower compared with men without any deletion (without GOLGA2LY copy deletions and gr/gr rearrangements; P = 0.001 and P = 0.003, respectively (Supplementary Table S2 and Figure 2A and 2B). The sperm concentration and motility of men with and without GOLGA2P2Y deletion were similar (Supplementary Table S2 and Figure 2A and 2B). To further determine the effect of GOLGA2LY copy deletions on phenotypic manifestations of gr/gr rearrangements, we evaluated the sperm concentration and motility of men with GOLGA2LY deletions also harbouring gr/gr rearrangements. In the group with gr/gr rearrangements, compared with non-deleted controls (without GOLGA2LY copy deletions and gr/gr rearrangements), the sperm concentration and motility were significantly lower in men with
Table 3
GOLGA2P3Y deletions (P = 0.006 and P = 0.009, respectively) (Supplementary Table S3 and Figure 2C and 2D). The sperm concentration and motility in men with GOLGA2P2Y deletions were similar to those observed in men with intact GOLGA2LY (Supplementary Table S3 and Figure 2C and 2D). Irrespective of the gr/gr status, the sperm concentration and motility in men with GOLGA2P3Y deletions were significantly lower compared with those in men with GOLGA2P2Y deletions (Sperm concentration: P = 0.002; percentage motile sperm: P = 0.006).
Odds ratio of GOLGA2P2Y and GOLGA2P3Y gene deletion on sperm concentration To determine if deletions of GOLGA2P2Y or GOLGA2P3Y gene copies are independent risk factor for oligozoospermia, we calculated the odds ratio for GOLGA2P2Y and GOLGA2P3Y gene deletions based on sperm concentration. In men having sperm concentration 0.1–5 million/ml, the odds ratio for GOLGA2P3Y deletion was 9 and in men with sperm concentration greater than 5 but less than 15 million/ml the odds ratio for GOLGA2P3Y deletion was 4.7 (Table 4). The odds ratio for GOLGA2P2Y deletion in men with sperm concentration 0.1–5 million/ml was 0.9 and in men with sperm concentration greater than 5 but less than 15 million/ml was 0.8 (Table 4).
Discussion The results of the present study demonstrate that the frequency of GOLGA2P3Y but not GOLGA2P2Y deletion is significantly higher in men with oligozoospermia compared with men with normozoospermia. The deletion of GOLGA2P3Y gene copy leads to significant lowering of sperm concentration and motility, and is a risk factor for oligozoospermia. GOLGA2LY gene copies were first identified electronically by sequence analysis of AZFc locus of human Y chromosome (Kuroda-Kawaguchi et al., 2001). In the AZFc locus, the
Frequency of deletion of gene copies GOLGA2P2Y and GOLGA2P3Y in men with gr/gr rearrangements only.
Gene copy deleted
GOLGA2P2Y GOLGA2P3Y
Oligozoospermic (n = 32)
Normozoospermic (n = 16)
Number with deletion (%)
Number with deletion (%)
9 (28.1) 14 (43.8)
6 (37.5) 2 (12.5)
P-value
Odds ratio
95% CI
NS 0.03
0.65 5.4
0.2, 2.3 1.1, 28.0
CI, confidence interval, NS, not statistically significant.
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Figure 2 Effect of GOLGA2LY gene copy deletions on sperm concentration and motility. (A) sperm concentration (million/ml); and (B) motility (percentage motile sperm) in men with and without GOLGA2P2Y and GOLGA2P3Y irrespective of gr/gr rearrangements; (C) and (D) are sperm concentration and motility in men with gr/gr rearrangements. No deletion = without GOLGA2LY copy deletions and gr/gr rearrangements, n are the number of men assessed in each group. Data are expressed as mean and 95% CI. *, values statistically significant (P < 0.05) compared with no deletion group. Table 4
Frequency of GOLGA2P2Y and GOLGA2P3Y deletion in men with different sperm concentration. GOLGA2P2Y
Sperm concentration (million/ml) (number of men) Number of men with deletion (%) Odds ratio (CI) P-value
GOLGA2P3Y
0.1–5 (n = 143) >5 <15 (n = 130) ≥15 (n = 336) 0.1–5 (n = 143) >5 <15 (n = 130)
≥15 (n = 336)
15 (10.5)
4 (1.2)
13 (10)
0.9 (0.5 to 1.8) 0.8 (0.5 to 1.6) NS NS
38 (11.3)
14 (9.8)
7 (5.4)
– –
9 (2.9 to 27.8) 0.00001
4.7 (1.4 to 16.4) – 0.02 –
CI, confidence interval, NS, not statistically significant.
GOLGA2LY gene is present in two copies named GOLGA2P2Y and GOLGA2P3Y. The copies are 99% identical and are predicted to transcribe and translate a protein of 108 amino acids. Although the functions of GOLGA2LY are unknown; the fact that AZFc subdeletions (gr/gr, b1/b3 and b2/b3) that remove either GOLGA2P2Y or GOLGA2P3Y are associated with male
infertility and low total motile sperm count (Noordam et al., 2011) led us to postulate that loss of one of these two copies may be a cause of infertility in a subset of men. In the present study, we observed that deletions of GOLGA2P3Y but not GOLGA2P2Y are frequently observed in men with oligozoospermia compared with men with normozoospermia (OR 6.9).
Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001
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These observations are in contrast with those reported by Lu et al. (2014) where they did not detect any association of GOLGA2LY copy number variations with male infertility. Herein, it is important to note that Lu et al. (2014) have used quantitaive PCR to detect the copy number variations of GOLGA2LY, which cannot differentiate between GOLGA2P2Y and GOLGA2P3Y, so they did not consider the individual GOLGA2LY gene copies specifically. In our study, when we specifically looked at individual deletions of GOLGA2LY copies, we observed that the deletion of GOLGA2P3Y but not GOLGA2P2Y is associated with oligozoospermia. Therefore, it is likely that the failure to find an association of GOLGA2LY copy number variation with male infertility by Lu et al. (2014) may be a result of methodological differences. Indeed, when the data from this study were analysed based on GOLGA2LY copy deletions without considering individual copies (as with Lu et al.), we failed to find any significant difference in the frequency of GOLGA2LY deletions and male infertility (data not shown). To the best of our knowledge, this is the first report showing the association of GOLGA2LY copy deletions with oligozoospermia. It will be of interest to study more populations to determine the association of GOLGA2LY copy deletions with male infertility. Beyond Yq microdeletions, the gr/gr rearrangements are the next leading cause of male infertility (Sen et al., 2015; Stouffs et al., 2011). These gr/gr rearrangements are expected to remove about 1.6–1.8 Mb of the AZFc locus spanning the GOLGA2LY gene (Ferlin et al., 2005; Repping et al., 2003; Stouffs et al., 2008). A wide heterogeneity, however, is observed in copy number variations of AZFc genes in men with gr/gr rearrangements, and it is believed that the susceptibility of gr/gr rearrangements to male infertility may depend on the types of gene copies deleted in these men (Krausz et al., 2009; Lu et al., 2009; Sen et al., 2015). In the present study, we observed that not all men with gr/gr rearrangements had deletions of GOLGA2LY copies. Only 31 out of 48 men (64.6%) with gr/gr rearrangements had either GOLGA2P2Y or GOLGA2P3Y gene copy deleted. Interestingly, the frequency of GOLGA2P3Y deletion was higher in oligozoospermic men compared with normozoospermic men with gr/gr rearrangements. These observations imply that loss of GOLGA2P3Y gene copy increases the susceptibility of men with gr/gr rearrangements to oligozoospermia. Indeed low total motile sperm count has been reported in men with gr/gr rearrangements having GOLGA2LY copy deletions compared with non-deleted men, i.e. those without any GOLGA2LY deletions (Noordam et al., 2011). To test the functional significance of GOLGA2LY gene copy deletions, we next compared the sperm concentration and motility in men with and without GOLGA2LY deletions. The results reveal that men with GOLGA2P3Y had significantly lower sperm concentration and motility compared with men with GOLGA2P2Y gene deletions or non-deleted controls (without GOLGA2LY copy deletions and gr/gr rearrangements). Interestingly in the group with gr/gr rearrangements, the sperm concentration and motility were significantly lower in men that had GOLGA2P3Y deletions but not GOLGA2P2Y deletions. Our results are also supported by a previous observation where deletion of GOLGA2LY gene copies in men with gr/gr rearrangements has lower total motile sperm count than those without GOLGA2LY gene copy deletion (Noordam et al., 2011). This reduction is not as a consequence
of gr/gr rearrangements but is specifically related to GOLGA2P3Y deletion, as the sperm concentration and motility was lowest in men with GOLGA2P3Y deletion compared with men that had gr/gr rearrangements but not GOLGA2LY copy deletions. Together, these observations imply that GOLGA2P3Y deletion is a locus of oligozoospermia. To determine if the deletion of GOLGA2P3Y is a risk factor for oligozoospermia, we next tested the occurrence of GOLGA2P3Y deletion based on sperm concentration in men irrespective of gr/gr rearrangements. The results demonstrated that, the risk of harbouring a GOLGA2P3Y (but not GOLGA2P2Y copy deletion) is higher in men with severe oligozoospermia (OR 9) and oligozoospermia (OR 4.7) compared with men with normozoospermia. These observations confirm that loss of GOLGA2P3Y is an independent risk factor for oligozoospermia. We propose that GOLGA2P3Y deletion screening may be offered as a part of genetic evaluation to oligozoospermic men in a clinical setup. More populationbased studies, however, will be needed to determine the usefulness of this screening in a clinical setting. In conclusion, the results of the present study indicate that GOLGA2P3Y gene is an essential factor for the maintenance of spermatogenesis, and its deletion is a cause of oligozoospermia. It will be of interest to determine functionally how GOLGA2P2Y vs GOLGA2P3Y differ and determine the molecular mechanism by which loss of GOLGA2P3Y may lead to male infertility.
Acknowledgement SS and PA are thankful to Indian Council of Medical Research (ICMR) for Senior Research Fellowship. SS is also thankful to Lady Tata Memorial Trust (LTMT) for Junior Research Fellowship. RA is thankful to Indian Academy of Sciences (IAS) for Summer Research Fellowship. We are also thankful to the help extended by staff of Hinduja IVF Centre and INKUS IVF centre. The work included in this publication (RA/271/062015) has been supported financially by grants from the Indian Council of Medical Research (ICMR), New Delhi, India to JG, DM and AP.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.rbmo.2015.11.001.
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Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001
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7 Repping, S., Skaletsky, H., Brown, L., van Daalen, S.K., Korver, C.M., Pyntikova, T., Kuroda-Kawaguchi, T., de Vries, J.W., Oates, R.D., Silber, S., van der Veen, F., Page, D.C., Rozen, S., 2003. Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat. Genet. 35, 247–251. Sen, S., Pasi, A.R., Dada, R., Shamsi, M.B., Modi, D., 2013a. Y chromosome microdeletions in infertile men: prevalence, phenotypes and screening markers for the Indian population. J. Assist. Reprod. Genet. 30, 413–422. Sen, S., Dixit, A., Thakur, C., Gokral, J., Hinduja, I., Zaveri, K., Thangaraj, K., Modi, D., 2013b. Association of progesterone receptor gene polymorphism with male infertility and clinical outcome of ICSI. J. Assist. Reprod. Genet. 30, 1133–1139. Sen, S., Ambulkar, P., Hinduja, I., Zaveri, K., Gokral, J., Pal, A., Modi, D., 2015. Susceptibility of gr/gr rearrangements to azoospermia or oligozoospermia is dependent on DAZ and CDY1 gene copy deletions. J. Assist. Reprod. Genet. 32, 1333–1341. PMID: 26149076. Epub ahead of print. Simoni, M., Tüttelmann, F., Gromoll, J., Nieschlag, E., 2008. Clinical consequences of microdeletions of the Y chromosome: the extended Münster experience. Reprod. Biomed. Online 16, 289–303. Skaletsky, H., Kuroda-Kawaguchi, T., Minx, P.J., Cordum, H.S., Hillier, L., Brown, L.G., Repping, S., Pyntikova, T., Ali, J., Bieri, T., Chinwalla, A., Delehaunty, A., Delehaunty, K., Du, H., Fewell, G., Fulton, L., Fulton, R., Graves, T., Hou, S.F., Latrielle, P., Leonard, S., Mardis, E., Maupin, R., McPherson, J., Miner, T., Nash, W., Nguyen, C., Ozersky, P., Pepin, K., Rock, S., Rohlfing, T., Scott, K., Schultz, B., Strong, C., Tin-Wollam, A., Yang, S.P., Waterston, R.H., Wilson, R.K., Rozen, S., Page, D.C., 2003. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423, 825–837. Stouffs, K., Tournaye, H., Van der Elst, J., Haentjens, P., Liebaers, I., Lissens, W., 2008. Do we need to search for gr/gr deletions in infertile men in a clinical setting? Hum. Reprod. 23, 1193– 1199. Stouffs, K., Lissens, W., Tournaye, H., Haentjens, P., 2011. What about gr/gr deletions and male infertility? Systematic review and metaanalysis. Hum. Reprod. Update 17, 197–209. Tse, J.Y., Wong, E.Y., Cheung, A.N., O, W.S., Tam, P.C., Yeung, W.S., 2003. Specific expression of VCY2 in human male germ cells and its involvement in the pathogenesis of male infertility. Biol. Reprod. 69, 746–751. Tüttelmann, F., Rajpert-De Meyts, E., Nieschlag, E., Simoni, M., 2007. Gene polymorphisms and male infertility – a meta-analysis and literature review. Reprod. Biomed. Online 15, 643–658. World Health Organization, 2010. WHO Laboratory Manual for the Examination and Processing of Human Semen, fifth ed. World Health Organization, Geneva.
Declaration: The study was funded by the Indian Council of Medical Research (ICMR), New Delhi, India. The authors have no competing interests to declare.
Received 23 June 2015; refereed 16 October 2015; accepted 3 November 2015.
Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001
w w w. s c i e n c e d i r e c t . c o m w w w. r b m o n l i n e . c o m
ARTICLE
Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia Sanjukta Sen a, Rupesh Agarwal a, Prafulla Ambulkar b, Indira Hinduja c, Kusum Zaveri c, Jyotsna Gokral d, Asoke Pal b, Deepak Modi a,* a
Molecular and Cellular Biology Laboratory, National Institute for Research in Reproductive Health (ICMR), JM Street, Parel, Mumbai, 400 012, India; b Human Genetic Division, Department of Anatomy, Mahatma Gandhi Institute of Medical Sciences, Sevagram, 442102, Wardha, India; c Hinduja IVF Centre, PD Hinduja Hospital and Medical Research Center, Veer Savarkar Marg, Mahim, Mumbai, 400016, India; d Department of Reproductive Endocrinology and Infertility, National Institute for Research in Reproductive Health (ICMR), JM Street, Parel, Mumbai, 400012, India * Corresponding author.
E-mail address: [email protected] (D Modi). Sanjukta Sen has completed her masters degree in biochemistry, and is presently pursuing a doctorate degree in applied biology. Her area of interest is genetics of male infertility, with a focus on Y chromosomal genes and male infertility. She is also interested in the reproductive outcome of patients with male infertility opting for ICSI for biological parenthood.
The AZFc locus on the human Y chromosome harbours several multicopy genes, some of which are required for spermatogenesis. It is believed that deletion of one or more copies of these genes is a cause of infertility in some men. GOLGA2LY is one of the genes in the AZFc locus and it exists in two copies, GOLGA2P2Y and GOLGA2P3Y. The involvement of GOLGA2LY gene copy deletions in male infertility, however, is unknown. This study aimed to investigate the association of deletions of GOLGA2P2Y and GOLGA2P3Y gene copies with male infertility and with sperm concentration and motility. The frequency of GOLGA2P3Y deletion was significantly higher in oligozoospermic men compared with normozoospermic men (7.7% versus 1.2%; P = 0.0001), whereas the frequency of GOLGA2P2Y deletion was comparable between oligozoospermic and normozoospermic men (10.3% versus 11.3%). The deletion of GOLGA2P3Y but not GOLGA2P2Y was significantly higher (P = 0.03) in men with gr/gr rearrangements, indicating that GOLGA2P3Y deletions increase the susceptibility of men with gr/gr rearrangements to oligozoospermia. Furthermore, men with GOLGA2P3Y deletion had reduced sperm concentration and motility compared with men without deletion or with deletion of GOLGA2P2Y. These findings indicate GOLGA2P3Y gene copy may be candidate AZFc gene for male infertility. Abstract
© 2015 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd. KEYWORDS: AZFc, GOLGA2LY, gr/gr rearrangements, oligozoospermia, sperm count, sperm motility
http://dx.doi.org/10.1016/j.rbmo.2015.11.001 1472-6483/© 2015 Published by Elsevier Ltd on behalf of Reproductive Healthcare Ltd.
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Introduction Cytogenetic and molecular analyses of men with defective spermatogenesis have identified microdeletions in the q arm of the Y chromosome. Between 2 and 10% of men with oligozoospermia or azoospermia are estimated to harbour Yq microdeletion, and it is currently the leading genetic cause of male infertility (Abid et al., 2008a; Ambulkar et al., 2014; Krausz et al., 2014; Massart et al., 2012; Sen et al., 2013a). Yq microdeletions involve loss of one (or more) of the three AZF loci namely, AZFa, AZFb and AZFc. Among these three, the deletion of AZFc locus is most commonly observed (Ambulkar et al., 2014; Foresta et al., 2001; Massart et al., 2012; Sen et al., 2013a; Simoni et al., 2008). The human AZFc is palindromic in nature, and has several repeat regions making it susceptible to self recombination during meiosis, which makes it most susceptible to deletions (Kuroda-Kawaguchi et al., 2001; Skaletsky et al., 2003). Withstanding this fact, along with complete AZFc deletions, genetic analysis of infertile men has led to identification of partial deletions within the AZFc locus. Referred to as the gr/ gr rearrangements or AZFc subdeletions, these rearrangements remove 1.6–1.8 Mb, which is about 50% of the AZFc region (Ferlin et al., 2005; Repping et al., 2003; Stouffs et al., 2008). The gr/gr rearrangements are more frequently observed in azoospermic and oligozoospermic men compared with normozoospermic counterparts (Ferlin et al., 2005; Giachini et al., 2005; Lo Giacco et al., 2014; Sen et al., 2015; Stouffs et al., 2011). Unlike the Yq microdeletions, however, the gr/gr rearrangements are also observed in 2–5% of fertile and normozoospermic men (Lo Giacco et al., 2014; Sen et al., 2015; Stouffs et al., 2011; Tüttelmann et al., 2007). What makes some men harbouring gr/gr rearrangements susceptible to male infertility is presently unclear. Although ethnicity and genetic background of Y chromosome have been demonstrated to be a cause; variation in the genes deleted within the AZFc locus in individuals with gr/gr rearrangements has been suspected to be the causative factor (Lo Giacco et al., 2014; Sen et al., 2015). The AZFc locus is structurally unique, and is composed of three palindromes housing 11 families of transcription units (Kuroda-Kawaguchi et al., 2001; Skaletsky et al., 2003). All the genes in AZFc locus exist in multiple copies and are expressed in the testis (Kuroda-Kawaguchi et al., 2001; Skaletsky et al., 2003; Tse et al., 2003). It is believed that gr/gr rearrangements remove copies of some key AZFc genes like Deleted in Azoospermia (DAZ) and Chromodomain on the Y chromosome,1 (CDY1) causing male infertility (Ferlin et al., 2005; Krausz et al., 2009; Sen et al., 2015; Stouffs et al., 2008). In addition to these genes, however, the gr/gr rearrangements also remove copies of three additional genes, namely Golgi autoantigen, golgin subfamily a, 2-like, Y linked (GOLGA2LY), Basic protein on Y chromosome, 2 (BPY2) and Testis specific transcript, Y linked 4 (TTY4) (Noordam et al., 2011; Stouffs et al., 2008). Whether the deletion of one or more copies of these genes may also be a cause of infertility is presently unclear. As not all men harbouring gr/gr rearrangements are infertile, it is possible that there might be heterogeneity in the deletion of these genes, which may be the determinant of the susceptibility of some men to infertility. Although variation in DAZ and CDY1 copy deletions in
men with gr/gr rearrangements has been reported to increase the susceptibility of gr/gr rearrangements to male infertility (Choi et al., 2012; Krausz et al., 2009; Lu et al., 2009; Sen et al., 2015), the involvement of other genes is still unknown. GOLGA2LY is a 4.8Kb gene present in two copies on the AZFc locus, referred to as GOLGA2P2Y and GOLGA2P3Y for the 5′ and the 3′ copy, respectively. The gr/gr deletions generally remove either the GOLGA2P2Y copy or GOLGA2P3Y copy depending on the site of recombination (Krausz et al., 2009; Noordam et al., 2011). Controversies exist on the association of GOLGA2LY copy deletions and male infertility. Noordam et al. (2011) showed that infertile males harbouring the AZFc subdeletion encompassing the GOLGA2LY had lower total motile sperm count compared with controls. Lu et al. (2014) failed to report any significant association of GOLGA2LY gene copy number variations with male infertility. Therefore, it is unclear if deletions of GOLGA2P2Y/3Y may be a causative factor for male infertility. As GOLGA2LY is transcribed in the testis (Kuroda-Kawaguchi et al., 2001), and is located within the region deleted in gr/gr rearrangements, we hypothesize that loss of GOLGA2LY gene copies may be also associated with male infertility. In the present study, we aimed to determine the association of the deletions of the GOLGA2LY gene copies GOLGA2P2Y and GOLGA2P3Y with oligozoospermia. The effect of these gene copy deletions were also evaluated on sperm concentration and motility.
Materials and methods Ethics statement The study was conducted independently at the National Institute for Research in Reproductive Health, Mumbai, and Mahatma Gandhi Institute of Medical Sciences, Sevagram, India. The Mahatma Gandhi Institute of Medical Sciences Ethical Committee approved the study on 30 December 2006 and the Ethics Committee for Clinical Studies of National Institute for Research in Reproductive Health approved the study on 20 December 2005. Written informed consent was obtained from all study participants.
Study participants The details of inclusion and exclusion criteria for the participants have been described previously (Ambulkar et al., 2014; Sen et al., 2013b). Participants were classified on the basis of sperm concentration, and were grouped into normozoospermic men (sperm concentration ≥15 million/ ml) and oligozoospermic men (sperm concentration <15 million/ml) as per the World Health Organization 2010 guidelines (WHO, 2010). Participants with karyotypic abnormalities, obstructive azoospermia, hypogonadism, hypoandrogenism, chronic diseases, history of mumps, history of fever, varicocele and history of pelvic/spinal injuries were excluded. None of the patients reported heavy smoking, alcohol intake, or both. All the men were screened for Yq microdeletion
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according to the protocol previously published (Abid et al., 2008a, 2008b; Ambulkar et al., 2014; Sen et al., 2013a, 2013b).
Detection of GOLGA2P2Y and GOLGA2P3Y deletions Genomic DNA was isolated from the whole blood using a commercial kit (Sigma-Aldrich Co, St Louis., USA) as described previously (Sen et al., 2013b). The GOLGA2LY gene copies were detected by Sequence Family Variant (SFV) analysis, which is based on the fact that the GOLGA2P2Y differs from GOLGA2P3Y by a SNP that can be detected by HhaI restriction site (Fernandes et al., 2004). The fragment of GOLGA2P2Y/3Y that encompasses the SNP was amplified by polymerase chain reaction (PCR) using the Red Amp master mix (Sigma-Aldrich Co, USA) for 35 cycles. The PCR products were digested overnight using HhaI (Fermentas International Inc., Ontario, Canada) and resolved on 15% PAGE. The primer sequences and the annealing temperatures are given in Table 1.
the frequency of GOLGA2P2Y and GOLGA2P3Y gene deletions were evaluated using the chi-squared test. Differences of sperm concentration and motility were analysed by analysis of variance. The GOLGA2LY copy deletion and the risk of male infertility were estimated by computing the odds ratio, 95% confidence interval and P-values. P < 0.05 were regarded as statistically significant.
Results A single band of expected size (531 bp) was detected in all the samples amplified for GOLGA2LY. No bands were detected when female genomic DNA and water were used as template indicating specificity of the product. Direct sequencing of the PCR products revealed its 100% identity to GOLGA2LY. Restriction digestion of the PCR amplified product gave three bands of 289 and 242 bp for GOLGA2P2Y and 531 bp for GOLGA2P3Y. Absence of the 289 and 242 bp products indicate a deletion of GOLGA2P2Y, whereas absence of 531 bp product indicates deletion of GOLGA2P3Y (Figure 1).
Detection of gr/gr rearrangements gr/gr rearrangement typing was carried by PCR amplification of sequence tagged site markers sY1291/sY1191 and SRY gene as internal control. As described above, PCR was carried out and run on 2% agarose gel. The AZFc subdeletions were classified as follows: the absence of sY1291 as gr/gr, the absence of sY1191 as b2/b3 and the absence of both the markers sY1291/sY1191 as b1/b3 deletions (Giachini et al., 2005). The optimized annealing temperature and primer sequences are given in Table 1.
Statistical analysis All the statistical analyses were carried out using the SPSS software (SPSS Inc., Chicago, IL, USA, version 16). Differences in Table 1
Primer sequences and the annealing temperatures.
Primers
GOLGA2LY
sY1191
sY1291
sY14/SRY
Sequence
Expected size (bp)
Annealing temperature (°C)
F 5′ TTGGCCTGTT GCTTCTAGGGTT 3′ R 5′ ACAGGGAGGG TGCTGTCACA 3′ F 5′ CCAGACGTTCT ACCCTTTCG 3′ R 5′ GAGCCGAGATC CAGTTACCA 3′ F 5′ TAAAAGGCAGAA CTGCCAGG 3′ R 5′ GGGAGAAAAGTT CTGCAACG 3′ F 5′ GAATATTCCCG CTCTCCGGA 3′ R 5′ GCTGGTGCTCC ATTCTTGAG 3′
531
68
385
62
527
62
472
62
Frequency of deletion of GOLGA2P2Y and GOLGA2P3Y in fertile and infertile men A total of 609 individuals who met the inclusion and exclusion criteria were included in the study: 336 men had normozoospermia and 273 had oligozoospermia. Of this, 12.5% (42/336) of the normozoospermic men and 17.9% (49/273) of the oligozoospermic men had any one of the two GOLGA2LY copies deleted. This difference was not statistically significant. Deletion of GOLGA2P2Y was observed in 11.3% of normozoospermic men and 10.3% of oligozoospermic men. This difference was not statistically significant. Deletion of GOLGA2P3Y was observed in 1.2% of normozoospermic men and 7.7% of oligozoospermic men. The frequency of the GOLGA2P3Y deletion was significantly higher in oligozoospermic men compared with normozoospermic men (P = 0.0001) (Table 2).
Figure 1 Association of deletions of GOLGA2LY copies with oligozoospermia. (A) Polymerase chain reaction restriction fragment length polymorphism for the detection of GOLGA2LY copy deletion. Lane 1: 100 bp ladder, Lane 2, 3: GOLGA2P3Y deletion, Lane 4: GOLGA2P2Y deletion, Lane 5: female control, Lane 6: water control, Lane 7: no deletion, Lane 8: undigested DNA.
Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001
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Table 2
Frequency of men with deletions of gene copies GOLGA2P2Y and GOLGA2P3Y.
Gene copy deleted
GOLGA2P2Y GOLGA2P3Y
Oligozoospermic (n = 273)
Normozoospermic (n = 336)
Number with deletion (%)
Number with deletion (%)
28 (10.3) 21 (7.7)
38 (11.3) 4 (1.2)
P-value
Odds ratio
95% CI
NS 0.0001
0.9 6.9
0.5 to 1.5 2.3 to 20.4
CI, confidence interval, NS, not statistically significant.
Frequency of deletion of GOLGA2P2Y and GOLGA2P3Y in men with gr/gr rearrangements Forty-eight out of 609 individuals included in this study had gr/gr rearrangements. Of these, 70.8% had gr/gr, 6.3% had b1/b3 and 22.9% had b2/b3 deletion (Supplementary Table S1). GOLGA2LY copy deletions were observed in 50% (8/16) of men with normozoospermia and 72% (23/32) of men with oligozoospermia who had gr/gr rearrangements. A total of 37.5% of normozoospermic men and 28.1% of oligozoospermic men had GOLGA2P2Y deletion. This difference was not statistically significant. A total of 12.5% of normozoospermic men and 43.8% of oligozoospermic men had the GOLGA2P3Y copy deleted; this difference was statistically significant (P = 0.03) (Table 3).
Association of deletions of GOLGA2P2Y and GOLGA2P3Y with sperm concentration and motility To analyse the effect of GOLGA2LY copy deletions on spermatogenesis, the sperm concentration and motility were evaluated in men with GOLGA2LY copy deletions, with and without gr/gr rearrangements. Irrespective of the gr/gr rearrangements, the sperm concentration and motility in men with GOLGA2P3Y deletion were significantly lower compared with men without any deletion (without GOLGA2LY copy deletions and gr/gr rearrangements; P = 0.001 and P = 0.003, respectively (Supplementary Table S2 and Figure 2A and 2B). The sperm concentration and motility of men with and without GOLGA2P2Y deletion were similar (Supplementary Table S2 and Figure 2A and 2B). To further determine the effect of GOLGA2LY copy deletions on phenotypic manifestations of gr/gr rearrangements, we evaluated the sperm concentration and motility of men with GOLGA2LY deletions also harbouring gr/gr rearrangements. In the group with gr/gr rearrangements, compared with non-deleted controls (without GOLGA2LY copy deletions and gr/gr rearrangements), the sperm concentration and motility were significantly lower in men with
Table 3
GOLGA2P3Y deletions (P = 0.006 and P = 0.009, respectively) (Supplementary Table S3 and Figure 2C and 2D). The sperm concentration and motility in men with GOLGA2P2Y deletions were similar to those observed in men with intact GOLGA2LY (Supplementary Table S3 and Figure 2C and 2D). Irrespective of the gr/gr status, the sperm concentration and motility in men with GOLGA2P3Y deletions were significantly lower compared with those in men with GOLGA2P2Y deletions (Sperm concentration: P = 0.002; percentage motile sperm: P = 0.006).
Odds ratio of GOLGA2P2Y and GOLGA2P3Y gene deletion on sperm concentration To determine if deletions of GOLGA2P2Y or GOLGA2P3Y gene copies are independent risk factor for oligozoospermia, we calculated the odds ratio for GOLGA2P2Y and GOLGA2P3Y gene deletions based on sperm concentration. In men having sperm concentration 0.1–5 million/ml, the odds ratio for GOLGA2P3Y deletion was 9 and in men with sperm concentration greater than 5 but less than 15 million/ml the odds ratio for GOLGA2P3Y deletion was 4.7 (Table 4). The odds ratio for GOLGA2P2Y deletion in men with sperm concentration 0.1–5 million/ml was 0.9 and in men with sperm concentration greater than 5 but less than 15 million/ml was 0.8 (Table 4).
Discussion The results of the present study demonstrate that the frequency of GOLGA2P3Y but not GOLGA2P2Y deletion is significantly higher in men with oligozoospermia compared with men with normozoospermia. The deletion of GOLGA2P3Y gene copy leads to significant lowering of sperm concentration and motility, and is a risk factor for oligozoospermia. GOLGA2LY gene copies were first identified electronically by sequence analysis of AZFc locus of human Y chromosome (Kuroda-Kawaguchi et al., 2001). In the AZFc locus, the
Frequency of deletion of gene copies GOLGA2P2Y and GOLGA2P3Y in men with gr/gr rearrangements only.
Gene copy deleted
GOLGA2P2Y GOLGA2P3Y
Oligozoospermic (n = 32)
Normozoospermic (n = 16)
Number with deletion (%)
Number with deletion (%)
9 (28.1) 14 (43.8)
6 (37.5) 2 (12.5)
P-value
Odds ratio
95% CI
NS 0.03
0.65 5.4
0.2, 2.3 1.1, 28.0
CI, confidence interval, NS, not statistically significant.
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Figure 2 Effect of GOLGA2LY gene copy deletions on sperm concentration and motility. (A) sperm concentration (million/ml); and (B) motility (percentage motile sperm) in men with and without GOLGA2P2Y and GOLGA2P3Y irrespective of gr/gr rearrangements; (C) and (D) are sperm concentration and motility in men with gr/gr rearrangements. No deletion = without GOLGA2LY copy deletions and gr/gr rearrangements, n are the number of men assessed in each group. Data are expressed as mean and 95% CI. *, values statistically significant (P < 0.05) compared with no deletion group. Table 4
Frequency of GOLGA2P2Y and GOLGA2P3Y deletion in men with different sperm concentration. GOLGA2P2Y
Sperm concentration (million/ml) (number of men) Number of men with deletion (%) Odds ratio (CI) P-value
GOLGA2P3Y
0.1–5 (n = 143) >5 <15 (n = 130) ≥15 (n = 336) 0.1–5 (n = 143) >5 <15 (n = 130)
≥15 (n = 336)
15 (10.5)
4 (1.2)
13 (10)
0.9 (0.5 to 1.8) 0.8 (0.5 to 1.6) NS NS
38 (11.3)
14 (9.8)
7 (5.4)
– –
9 (2.9 to 27.8) 0.00001
4.7 (1.4 to 16.4) – 0.02 –
CI, confidence interval, NS, not statistically significant.
GOLGA2LY gene is present in two copies named GOLGA2P2Y and GOLGA2P3Y. The copies are 99% identical and are predicted to transcribe and translate a protein of 108 amino acids. Although the functions of GOLGA2LY are unknown; the fact that AZFc subdeletions (gr/gr, b1/b3 and b2/b3) that remove either GOLGA2P2Y or GOLGA2P3Y are associated with male
infertility and low total motile sperm count (Noordam et al., 2011) led us to postulate that loss of one of these two copies may be a cause of infertility in a subset of men. In the present study, we observed that deletions of GOLGA2P3Y but not GOLGA2P2Y are frequently observed in men with oligozoospermia compared with men with normozoospermia (OR 6.9).
Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001
ARTICLE IN PRESS 6
S Sen et al.
These observations are in contrast with those reported by Lu et al. (2014) where they did not detect any association of GOLGA2LY copy number variations with male infertility. Herein, it is important to note that Lu et al. (2014) have used quantitaive PCR to detect the copy number variations of GOLGA2LY, which cannot differentiate between GOLGA2P2Y and GOLGA2P3Y, so they did not consider the individual GOLGA2LY gene copies specifically. In our study, when we specifically looked at individual deletions of GOLGA2LY copies, we observed that the deletion of GOLGA2P3Y but not GOLGA2P2Y is associated with oligozoospermia. Therefore, it is likely that the failure to find an association of GOLGA2LY copy number variation with male infertility by Lu et al. (2014) may be a result of methodological differences. Indeed, when the data from this study were analysed based on GOLGA2LY copy deletions without considering individual copies (as with Lu et al.), we failed to find any significant difference in the frequency of GOLGA2LY deletions and male infertility (data not shown). To the best of our knowledge, this is the first report showing the association of GOLGA2LY copy deletions with oligozoospermia. It will be of interest to study more populations to determine the association of GOLGA2LY copy deletions with male infertility. Beyond Yq microdeletions, the gr/gr rearrangements are the next leading cause of male infertility (Sen et al., 2015; Stouffs et al., 2011). These gr/gr rearrangements are expected to remove about 1.6–1.8 Mb of the AZFc locus spanning the GOLGA2LY gene (Ferlin et al., 2005; Repping et al., 2003; Stouffs et al., 2008). A wide heterogeneity, however, is observed in copy number variations of AZFc genes in men with gr/gr rearrangements, and it is believed that the susceptibility of gr/gr rearrangements to male infertility may depend on the types of gene copies deleted in these men (Krausz et al., 2009; Lu et al., 2009; Sen et al., 2015). In the present study, we observed that not all men with gr/gr rearrangements had deletions of GOLGA2LY copies. Only 31 out of 48 men (64.6%) with gr/gr rearrangements had either GOLGA2P2Y or GOLGA2P3Y gene copy deleted. Interestingly, the frequency of GOLGA2P3Y deletion was higher in oligozoospermic men compared with normozoospermic men with gr/gr rearrangements. These observations imply that loss of GOLGA2P3Y gene copy increases the susceptibility of men with gr/gr rearrangements to oligozoospermia. Indeed low total motile sperm count has been reported in men with gr/gr rearrangements having GOLGA2LY copy deletions compared with non-deleted men, i.e. those without any GOLGA2LY deletions (Noordam et al., 2011). To test the functional significance of GOLGA2LY gene copy deletions, we next compared the sperm concentration and motility in men with and without GOLGA2LY deletions. The results reveal that men with GOLGA2P3Y had significantly lower sperm concentration and motility compared with men with GOLGA2P2Y gene deletions or non-deleted controls (without GOLGA2LY copy deletions and gr/gr rearrangements). Interestingly in the group with gr/gr rearrangements, the sperm concentration and motility were significantly lower in men that had GOLGA2P3Y deletions but not GOLGA2P2Y deletions. Our results are also supported by a previous observation where deletion of GOLGA2LY gene copies in men with gr/gr rearrangements has lower total motile sperm count than those without GOLGA2LY gene copy deletion (Noordam et al., 2011). This reduction is not as a consequence
of gr/gr rearrangements but is specifically related to GOLGA2P3Y deletion, as the sperm concentration and motility was lowest in men with GOLGA2P3Y deletion compared with men that had gr/gr rearrangements but not GOLGA2LY copy deletions. Together, these observations imply that GOLGA2P3Y deletion is a locus of oligozoospermia. To determine if the deletion of GOLGA2P3Y is a risk factor for oligozoospermia, we next tested the occurrence of GOLGA2P3Y deletion based on sperm concentration in men irrespective of gr/gr rearrangements. The results demonstrated that, the risk of harbouring a GOLGA2P3Y (but not GOLGA2P2Y copy deletion) is higher in men with severe oligozoospermia (OR 9) and oligozoospermia (OR 4.7) compared with men with normozoospermia. These observations confirm that loss of GOLGA2P3Y is an independent risk factor for oligozoospermia. We propose that GOLGA2P3Y deletion screening may be offered as a part of genetic evaluation to oligozoospermic men in a clinical setup. More populationbased studies, however, will be needed to determine the usefulness of this screening in a clinical setting. In conclusion, the results of the present study indicate that GOLGA2P3Y gene is an essential factor for the maintenance of spermatogenesis, and its deletion is a cause of oligozoospermia. It will be of interest to determine functionally how GOLGA2P2Y vs GOLGA2P3Y differ and determine the molecular mechanism by which loss of GOLGA2P3Y may lead to male infertility.
Acknowledgement SS and PA are thankful to Indian Council of Medical Research (ICMR) for Senior Research Fellowship. SS is also thankful to Lady Tata Memorial Trust (LTMT) for Junior Research Fellowship. RA is thankful to Indian Academy of Sciences (IAS) for Summer Research Fellowship. We are also thankful to the help extended by staff of Hinduja IVF Centre and INKUS IVF centre. The work included in this publication (RA/271/062015) has been supported financially by grants from the Indian Council of Medical Research (ICMR), New Delhi, India to JG, DM and AP.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.rbmo.2015.11.001.
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Declaration: The study was funded by the Indian Council of Medical Research (ICMR), New Delhi, India. The authors have no competing interests to declare.
Received 23 June 2015; refereed 16 October 2015; accepted 3 November 2015.
Please cite this article in press as: Sanjukta Sen, et al., Deletion of GOLGA2P3Y but not GOLGA2P2Y is a risk factor for oligozoospermia, Reproductive BioMedicine Online (2015), doi: 10.1016/j.rbmo.2015.11.001