Use of Ethnicity-Specific Sequence Tag Site Markers for Y Chromosome Microdeletion Studies

Abstract

Introduction: Microdeletions in the azoospermia factor region on the long arm of Y chromosome are associated with spermatogenic failure. There are many markers for the diagnosis of Y chromosome microdeletion analysis, but in routine practice only a limited set of markers can be tested. Objective: The objectives of this study were to determine the frequency of Y chromosome microdeletion in idiopathic cases of male infertility in India, to attempt genotype-phenotype correlation, and to evaluate whether markers to be tested for diagnosis of Y chromosome microdeletion should be ethnicity specific. Methods: Microdeletions in the Y chromosome were analyzed in 200 infertile males. The six sequence tag site (STS) markers prescribed by the European Academy of Andrology (EAA) were used initially. Patients in whom no deletions were detected by use of these markers were tested by markers selected from other studies from India. Results: The STS markers prescribed by EAA detected deletions in only 6 (3%) of 200 infertile males. However, markers selected from previous Indian studies showed deletions in an additional 15 (7.5%) of infertile males. Overall, Y chromosome microdeletions were observed in 21 (10.5%) of 200 patients. Of these, 13 were cases of azoospermia and 8 were cases of severe oligospermia. Conclusion: The markers prescribed by EAA alone are not suitable for the diagnosis of Y chromosome microdeletions in infertile males. The protocol for identification of Y chromosome microdeletions in cases of nonobstructive azoospermia/severe oligospermia would have to include a different set of STS markers.

Introduction

Infertility is defined as an inability to conceive after 1 year of unprotected intercourse. Infertility affects about 15% of couples, with the male being responsible in approximately half of the cases (Foresta et al., 2001). Molecular studies have suggested that deletions in the azoospermia factor (AZF) present in interval 5 and 6 of the human Y chromosome play an important role in the causation of male infertility (Krausz et al., 2001). The AZF area is divided into three nonoverlapping regions AZFa, AZFb, and AZFc. Microdeletions are reported in all the three regions. Each AZF locus acts at a different phase of spermatogenesis (Pryor et al., 1997). Deletions in AZFa are associated with complete absence of germ cells in the seminiferous tubules and leads to Sertoli cell only syndrome (SCOS). Deletions in AZFb are associated with developmental arrest of germ cells at the pachytene stage. Deletions in AZFc result in developmental arrest of germ cells at the spermatid stage, but can also be associated with hypospermatogenesis and maturation arrest. The frequency of microdeletions in infertile males shows a wide variation in different studies varying from 0.9% to 55.5% (Van et al., 1997; Foresta et al., 1998).

The total number of markers that have been shown to be deleted in various studies is more than 80 (Najmabadi et al., 1996; Vogt et al., 1996; Foresta et al., 1997; Pryor et al., 1997; Selva et al., 1997; Van et al., 1997; Foresta et al., 1998; Foresta et al., 1999; Simoni et al., 1999; Martinez et al., 2000; Tse et al., 2000; Krausz et al., 2001; Babu et al., 2002; Peterlin et al., 2002; Ambasudhan et al., 2003; Dada et al., 2003; Pedro et al., 2003; Swarna et al., 2003; Thangaraj et al., 2003; Rao et al., 2004; Kumar et al., 2006; Vutyavanich et al., 2006; Mitra et al., 2008). However, in practice, only a limited number of markers can be analyzed. The European Academy of Andrology (EAA) recommended six sequence tag site (STS) markers for detecting Y chromosome microdeletions, which enabled the detection of over 90% of the deletions in the AZF loci (Simoni et al., 1999). We aimed to study the frequency of Y chromosome microdeletion in idiopathic cases of male infertility and to evaluate the utility of EAA recommended STS markers to detect the Y chromosome microdeletions in Indian infertile males. To improve the yield and develop a cost-effective protocol, we also analyzed other markers that have been documented to be deleted among Indian patients (Babu et al., 2002; Ambasudhan et al., 2003; Dada et al., 2003; Swarna et al., 2003; Thangaraj et al., 2003; Rao et al., 2004; Kumar et al., 2006; Mitra et al., 2008). We compared the data with published results from other countries to examine the issue whether the markers to be selected for analysis should be ethnicity specific. We also attempted to establish a correlation between the genotype and phenotype in the infertile males.

Materials

Two hundred infertile males were enrolled in the study who visited the In Vitro Fertilization Unit at the Centre for Human Reproduction, Sir Ganga Ram Hospital, New Delhi, from January 2007 through December 2009. The age range of the patients was 28-36 years and the mean age of the patients was 32.7 years. Of the 200 cases, 150 were azoospermic males and 50 were severely oligospermic males. Individuals were defined as azoospermic when there was complete absence of sperms and as severely oligospermic when sperm count was less than 5 × 10⁶, as per the criteria of the World Health Organization (1999) manual. A written informed consent was obtained from each individual. They were subjected to clinical examination, and laboratory investigations including semen analysis, endocrinological studies, and when available, results of aspiration of sperms from the testis were also documented.

Methods

Peripheral blood samples were collected in EDTA vials from all the 200 subjects and DNA was isolated using salt precipitation method (Miller et al., 1988). The STS markers initially used for analysis were those prescribed by the European Academy of Andrology—AZFa sY84, sY86; AZFb sY127, sY134; and AZFc sY254, sY255 (Simoni et al., 1999). In patients who did not show any deletion, additional STS markers were tested (AZFa sY742, 746, 87; AZFb sY113, 117, 120, 125, 129, 130, 131, 132; AZFc sY146, 148, 149, 153, 156, 158, 243, 269). These have been reported to be deleted among Indian infertile males (Babu et al., 2002; Ambasudhan et al., 2003; Dada et al., 2003; Swarna et al., 2003; Thangaraj et al., 2003; Rao et al., 2004; Kumar et al., 2006; Mitra et al., 2008).

The PCR mixture included 50-250 ng of DNA sample, 1 × PCR buffer, 1.5 μM MgCl₂, 200 μM deoxynucleotidetriphosphates (dNTPs), 50 pmol of each primer pair, and 1 U Taq DNA polymerase. Thermal cycling conditions included initial denaturation for 5 min at 95°C followed by 35 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min 30 s, with a final extension for 5 min at 72°C. Primer sequences of STS markers used for characterization of deletion breakpoints were selected from previous studies (Babu et al., 2002; Ambasudhan et al., 2003; Dada et al., 2003; Swarna et al., 2003; Thangaraj et al., 2003; Rao et al., 2004; Kumar et al., 2006; Mitra et al., 2008). Serum follicle-stimulating hormone FSH, LH, and total testosterone levels were assayed by ELISA technique. The control group consisted of 100 males who had fathered children.

If the deletion of a marker was detected, the PCR amplification was repeated thrice to rule out failure of reaction. Absence of amplification in three reactions was considered to confirm deletion of the STS marker. With each run, samples of genomic DNA from a female and a normal fertile male (control group) were also tested. The PCR products were then analyzed in 2% agarose gel containing ethidium bromide and visualized under UV light.

Results

Using STS markers recommended by the EAA, only six (3%) of 200 infertile males were detected to carry microdeletions and they were all azoospermic. Using additional STS markers selected from Indian studies, deletions were detected in another 15 cases (7.5%) (Fig. 1). These 15 cases included seven azoospermic and eight oligospermic cases. Altogether Y chromosome microdeletions were observed in 21 cases (10.5%).

FIG. 1.

Results of PCR amplification of sets of sequence-tagged sites in Y-chromosome DNA from a normal man (A, B, C, D, E, F) and a man with infertility (A1, B1, C1, D1, E1, F1).

In all the 21 cases found to carry microdeletions, the span of deleted regions included the AZFc region. Deletions restricted only to the AZFc region were detected in 10 cases (47.6%); of these, 8 were severely oligospermic (sperm count between 1 and 4 million) and 2 were azoospermic (1-2 motile sperm high per field through TESA). Deletions of the AZFc region combined with AZFb region were detected in nine infertile males (42.8%), of which seven were azoospermic males (sperm count 1-2 motile sperm high per field on TESA) and two were having Sertoli cell only syndrome (SCOS). Deletions of AZFc combined with AZFa region were observed in two azoospermic males with SCOS (9.5%). The deletions observed in these two cases had a normal intervening region between the two markers (Fig. 2).

FIG. 2.

Y chromosome deletion map of azoospermic/severe oligospermic men generated by STS markers. Black solid line shows the region with no deletion. Thin black line shows deletion and extents of deletion. SCOS, Sertoli cell only syndrome; TESA, testicular sperm aspiration; STS, sequence tag site.

None of the control men showed deletions of the 27 STS markers spanning the entire AZFa, AZFb, and AZFc regions.

Among 150 azoospermic males, interstitial deletions of Yq11 were recorded in 13 (8.6%) patients; in 50 severely oligospermic males, deletions were observed in 8 (16%) patients (p-value <0.05). FSH level in patients with deletions was found to be mildly elevated (of 11 ± 2.7 mIU/mL) when compared with subjects with no deletions (6 ± 1.5 mIU/mL) (Table 1). All the patients had normal serum hormone levels (LH, prolactin, and total testosterone).

Table 1.

Clinical Features of Men with Microdeletion in Azoospermia Factor Region

S. no.	Patient	Type of infertility	Sperm count (million/mL)	Testicular sperm aspiration/extraction (TESA)	FSH (1.5-11.0 mIU/mL)	LH (1.1-8.0 mIU/mL	Prolactin (2.7-17.0 ng/mL)	Testosterone (8.69-54.69 pg/mL)	Free testosterone (2.5-8.5 ng/mL)	Deletion
1	P5	Oligozoospermic	3	Not done	11	3	4	12.5	4	sY156 and sY158 of AZFc
2	P6	Oligozoospermic	4	Not done	10	4	3	14	3.5	sY158 of AZFc
3	P7	Oligozoospermic	1	Not done	8	5	5	34	5	sY153, sy148, sY149, sY158 and sY156 of AZFc
4	P10	Oligozoospermic	2	Not done	8	4	3	28	4	sY153, sy148, sY149, sY156 and sY158 of AZFc
5	P11	Oligozoospermic	4	Not done	8	3	4	29	4	sY156 and sY158 of AZFc
6	P1	Oligozoospermic	3	Not done	12	4	5	30	4	sY153, sY148, sY149, sY156 and sY158 of AZFc
7	P2	Oligozoospermic	2	Not done	12	3	4	32	5	sY153, sY148, sY149, sY156 and sY158 of AZFc
8	P13	Oligozoospermic	2	Not done	11	3	4	40	4	sY153, sY 148, sY149, sY156, sY158 of AZFc
9	P3	Azoospermic	Nil	1-2 motile sperms/HPF	14	4	2	19	5	sY143 of AZFb region and sY153, sY153, sY148, sY149, sY156 and sY158 of AZFc
10	P4	Azoospermic	Nil	TESE only Sertoli cells	14	3	4	20	6	sY742 of AZFa and sY158 of AZFc
11	P8	Azoospermic	Nil	0-1 motile sperm/HPF	13	3	5	31	5	sY143 of AZFb and sY153, sY148, sY149, sY156, sY158of AZFc
12	P9	Azoospermic	Nil	1-2 motile sperms/HPF	14	4	4	41	39	sY143 of AZFb and sY153, sY148, sY149, sY156, sY158 of AZFc
13	P12	Azoospermic	Nil	no motile sperm/HPF	15	4	4	34	4.2	sY742 of AZFa and sY158 of AZFc
14	P14	Azoospermic	Nil	1-2 motile sperms/HPF	12	4	8	28	4.8	sY143 of AZFb and sY153, sY148, sY149, sY156 and sY158 of AZFc
15	P15	Azoospermic	Nil	0-1 motile sperm/HPF	15	3	8	33	5.5	sY143 of AZFb and sY153, sy148, sY149, sY156 and sY158 of AZFc
16	P16	Azoospermic	Nil	TESE only Sertoli cells	14	3	4	30	4	sY113, sY117, sY120, sY125, sY127, sY129, sY130, sY131, sY132, sY133, sY134, sY143 of AZFb and sY153, sY148, sY149, sY156,sY254, sY255, sY158, sY160 of AZFc
17	P17	Azoospermic	Nil	no motile sperm/HPF	15	5	11	32	6	sY113, sY117, sY120, sY125, sY127, sY129, sY130, sY131, sY132, sY133, sY134, sY143 of AZFb and sY153, sY148, sY149, sY156,sY254, sY255, sY158, sY160 of AZFc
18	P18	Azoospermic	Nil	1-2 motile sperms/HPF	11	3	4	28	5	sY153, sY 148, sY149, sY156, sY254, sYT255, sY158 of AZFc
19	P19	Azoospermic	Nil	0-1 motile sperm/HPF	16	4	10	29	5	sY153, sY 148, sY149, sY156, sY254, sYT255, sY158, sY160of AZFc
20	P20	Azoospermic	Nil	1-2 motile sperms/HPF	13	2.8	9	30	4	sY143 of AZFb and sY153, sY 148, sY149, sY156, sY254, sYT255, sY158, sY160 of AZFc
21	P21	Azoospermic	Nil	0-1 motile sperm/HPF	15	3	4	35	6	sY143 of AZFb and sY153, sY 148, sY149, sY156, sY254, sYT255, sY158, sY160 of AZFc

Mean ± SD: FSH = 11 ± 2.7; LH = 3.5 ± 0.6; prolactin = 5.1 ± 2.1; testosterone = 29 ± 6.9; free testosterone = 4.6 ± 0.7.

AZF, azoospermia factor.

Discussion

Deletions in the Y chromosome have emerged as an important cause of male infertility. In most studies, the frequency of Y chromosome deletions increases with the severity of spermatogenic defect (Foresta et al., 1997; Pryor et al., 1997; Seva et al., 1997; Van et al., 1997). Generally, about 15% of azoospermic males and 5%-10% of oligospermic males show deletions in Y chromosome (Foresta et al., 1999). However, in the present study, the frequency of Y chromosome microdeletions was high in oligospermic males when compared with azoospermic males (8.6% vs. 16%, respectively; p-value <0.05). It is likely that the oligospermic cases in our study belonged to the very severe group and were close to the azoospermic group.

The frequency of Y chromosome microdeletions has been reported from a number of developed countries such as the United States, Germany, France, Paris, Japan, Denmark, Spain, Taiwan, Slovenia, Finland, and Hong Kong as well as from some developing countries such as India, Brazil, Iran, Egypt, and Thailand (Najmabadi et al., 1996; Vogt et al., 1996; Pryor et al., 1997; Lin et al., 2000; Tse et al., 2000; Krausz et al., 2001; Bor et al., 2002; Aho et al., 2003; Ambasudhan et al., 2003; Pedro et al., 2003; Swarna et al., 2003; Thangaraj et al., 2003; Rao et al., 2004; Kihaile et al., 2005; Omrani et al., 2006; Vutyavanich et al., 2006; Viswambharan et al., 2007; Abol et al., 2009; Sheikh et al., 2009). These data are summarized in Table 2. The reported frequency of deletions has a wide variability (0.9%-55.5%) (Van et al., 1997; Foresta et al., 1998; Foresta et al., 1999; Rao et al., 2004). It is likely that the difference of frequency of Y chromosome microdeletions is related to sample size, differences in inclusion criteria for enrolling subjects into the study, and the number of markers tested. For example, in a study conducted by Foresta et al. (1998), 18 azoospermic males with Sertoli cell only syndrome, a highly specific group, was screened for Y chromosome microdeletion and deletions found in 55.5% of the cases. In their subsequent study of 130 infertile males, who were azoospermic/severely oligospermic (110) and moderately oligospermic (20), Y chromosome microdeletions were present in 28 cases (25.4%), but no deletions were found in moderately oligospermics males (Foresta et al., 1999). The former study had a homogenous group of patients, and maybe the frequency of Y chromosome microdeletion is higher in such cases. In contrast, in the latter study, despite a larger sample size, the patients included belong to different groups—azoospermia, severe oligospermia, and moderate oligospermia. Therefore, increasing the sample size with lack of homogeneity in enrolled patients may result in reduced frequency of microdeletions. This has also been reiterated by two other independent studies conducted by Rao and his associates in India and Van and his group in Germany (Van et al., 1997; Rao et al., 2004). In the study conducted by Rao and his associates, they enrolled 251 infertile males, which included 104 azoospermia, 41 severely oligospermia, 49 mild oligospermia, and 57 oligoasthenoteratozoospermics, and found a very low frequency of microdeletions. Van et al. (1997) in a group of 171 infertile males (13 azoospermia, 79 severely oligospermia, 52 mild oligospermia, 27 moderately oligospermia) detected microdeletions in only 0.9%. The low frequency of deletions in both these studies may be due to inclusion of large number of mild as well as moderate oligospermic males. The higher the number of patients with azoospermia, Sertoli cell only syndrome, or severe oligospermia, the better are the chances of detecting Y chromosome microdeletions; in contrast, if the patients have sperm count more than 8 millions/mL, the chance of having Y chromosome microdeletions decreases markedly.

Table 2.

Studies of Y Chromosome Microdeletions in Different Countries

S. no.	Countries	Total subject studied	Distribution	Patients with Y microdeletion No.	%	Total STS markers studied	EAA STS markers used in these studies	% of deletion if only EAA STS markers were used	Studies in developed countries
1	Germany	370	Azoo and severely oligo	26	7	76	sY84, 86, 127, 134	3.5	Vogt et al. (1996)
2	Germany	204	13 Azoo, 79 severely oligo, 52 mild oligo, 27 moderately oligo, 33 normal control	2	0.9	23	sY84, 86, 127, 134, 255, 254	0.49	Van et al. (1997)
3	USA	200	26 Azoo, 30 obstructive azoo, 42 oligo, 102 normozoospermics	14	7	85	sY84, 86, 127, 134, 255, 254	2.5	Pryor et al. (1997)
4	France	18	Azoospermics	10	55.5	29	sY84, 86, 127, 255	50	Foresta et al. (1998)
5	Paris	131	89 Azoo, 39 severely oligo, 7 oligo, 2 OAT	15	15.3	23	sY86, 127, 254, 255	11.4	Krauz et al. (1999)
6	France	130	110 Azoo and severely oligo, 20 moderately oligo	28	25.4	31	sY84, 86, 127, 255, 254	20.7	Foresta et al. (1999)
7	Spain	128	57 Azoo, 71 severely oligo	9	7.0	9	sY84, 86, 127	3.9	Martinez et al. (2000)
8	Hong Kong	58	35 Azoo, 9 severely oligo, 14 obstructive azoo	4-	6.9	6	sY86, 84, 127, 254, 255	6.9	Tse et al. (2000)
9	Taiwan	94	94 Azoo	11	11.7	27	sY86, 84, 127, 255, 254	7	Lin et al. (2000)
10	Slovenia	226	92 Azoo, 134 OAT	10	4.4	42	sY84, 86, 134, 254	3.9	Peterlin et al. (2002)
11	Denmark	400	48 Azoo, 322 oligo, 30 normozoospermic	3	0.75	25	sY84, 86, 127, 134, 255, 254	0.25	Bor et al. (2002)
12	Finland	201	68 Azoo, 133 severely oligo	18	9	20	sY84, 86, 127, 134, 255, 254	5	Aho et al. (2003)
13	Japan	162	47 Azoo, 115 severely oligo	7	4.3	24	sY84, 254	4.3	Kihaile et al. (2005)
	Studies in Developing Countries
14	Kolkata (India)	340	Azoo	29	8.5	32	sY84, 86, 127, 134, 255, 254	6.5	Thangaraj et al. (2003)
15	Hyderabad (India)	50	10 Azoo, 12 severely oligo, 28 oligo	4	8.0	2	sY254	2	Swarna et al. (2003)
16	Mumbai (India)	100	27 Azoo, 73 OAT	12	12.0	18	sY127, 254	6	Athalye et al. (2004)
17	Hyderabad (India)	251	104 Azoo, 41 severely oligo, 49 mild oligo, 57 OAT	10	3.9	17	sY86, 127, 134, 254, 255	1.5	Rao et al. (2004)
18	Brazil	60	29 Azoo, 31 severely oligo	4	6.6	14	sY84, 86, 254, 255	6.6	Pedro et al. (2003)
19	Thailand	130	50 Azoo, 80 severely oligo	6	4.6	11	Different STS used	NA	Vutyavanich et al. (2006)
20	Iran	99	39 Oligo, 60 azoo	24	24.2	18	sY254, sY255	2	Omrani et al. (2006)
21	Egypt	100	68 Azoo, 32 oligo	4	4	6 EAA recommended STS markers	sY84, 86, 127, 134, 255, 254	4	Shiekh et al. (2009)
22	Egypt	40	25 Azoo, 15 oligo	0	0	6 EAA recommended STS markers	sY84, 86, 127, 134, 255, 254	0	Abol et al. (2009)
23	Delhi (India)	200	150 Azoo, 50 severely oligo	21	10.5%	25	sY84, 86, 127, 134, 255, 254	3	Present study

Azoo, azoospermics; oligo, oligospermics; OAT, oligoasthenoteratozoospermics; NA, not applicable; EAA, European Academy of Andrology; STS, sequence tag site.

In the present study, the most commonly deleted region of Y chromosome is the AZFc region. Indeed, all the 21 infertile males showed deletions in this region. Previous studies also demonstrated a high frequency of deletions of the AZFc region (88.8%), when compared with AZFa and AZFb regions (Martinez et al., 2000; Peterlin et al., 2002). This may be due to the presence of many repetitive sequences in this region (Kawaguchi et al., 2001). The AZFc region alone was involved in 47.6% of individuals with deletions. A varying frequency of deletion of AZFc alone (44.4%, 27.5%, and 38.4%) has been reported in infertile men in different studies (Foresta et al., 1999; Martinez et al., 2000; Thangaraj et al., 2003).

We did not observe deletions of AZFb region alone in any of the infertile men analyzed. However, such deletions have been reported with a frequency of 11.1%, 14.1%, and 21.1% (Foresta et al., 1999; Martinez et al., 2000; Kumar et al., 2006). Deletions of AZFb combined with deletions of AZFc were observed in 42.6% of the cases in the present study. Such combined deletions were also observed in other studies with a frequency of 44.4%, 44.8%, 33.3%, and 25% (Martinez et al., 2000; Pedro et al., 2003; Thangaraj et al., 2003; Mitra et al., 2008).

The frequencies of deletions in the AZFa region are, in general, less when compared with AZFb and AZFc regions (Peterlin et al., 2002). This may be due to the small size of AZFa (800 kb) region when compared with AZFb (3.2 Mb) region and AZFc (3.5 Mb) region. In our study, two patients showed deletions in the AZFa region and both of them had Sertoli cell only syndrome. In these cases, deletion involved the AZFa region and the AZFc region but with a normal intervening sequence. Similar findings have also been reported by two other independent studies (Kamp et al., 2001; Thangaraj et al., 2003). It is interesting that none of the azoospermic men studied showed the deletion of sY84 and/or sY86. Surprisingly, these two markers have been recommended by the EAA for the detection of microdeletion in the AZFa region (Simoni et al., 1999).

We attempted to make genotype-phenotype correlations by comparing the size of deletion in the Y chromosome and the sperm count. It was observed that if the deletion was very large and covered entire AZFb and AZFc regions (sY113, 117, 120, 125, 127, 129, 130, 131, 132, 133, sY134, 143 of AZFb and sY153, 148, 149, 156, 254, 255, 158, 160 of AZFc), it was associated with the phenotype Sertoli cell only syndrome (SCOS). When AZFb and beyond were missing, there was predictably no detectable completion of spermatogenesis. Therefore, deletion of functional members of the RBM gene families or other genes found in AZFb region (EIF1AY, PRY, TTY2) and DAZ gene family in AZFc region might contribute to the enhanced severity of spermatogenetic defect observed in patients whose deletions are very large and cover the entire AZFb and AZFc regions (Chai et al., 1997). If deletions involved a small part of the AZFb region (sY143) and the AZFc region (sY153, 148, 149, 156, 158, 254, 255, and 160), it was associated with 1-2 motile sperm high per field on TESA (Table 1; Fig. 2). If the deletion involves only the AZFc region (sY156, 158, 148, 149, and 153), a sperm count between 1 and 4 million was observed (Table 1; Fig. 2). The absence of these markers indicates deletion of the AZFc region, which removes all copies of the members of the DAZ gene family. The DAZ gene family is the most important candidate gene for male infertility in the AZFc region and consists of four functional copies including DAZ1, DAZ2, DAZ3, and DAZ4 arranged in two clusters. Deletions in DAZ genes may have different effects: partial or complete deletions in the AZFc region may result in different phenotypes varying from oligozoospermia to azoospermia, so there may be a chance for retrieving sperm from testis and TESE/ICSI can be attempted in these patients (Mirzajani et al., 2010).

In all 21 infertile males with deletions of Y chromosome, FSH levels were found to be mildly elevated (11 ± 2.7 mIU/mL when compared with subjects with no deletions, 6 ± 1.5 mIU/mL) (Table 1). However, the value of FSH was not as high as that observed in patients with testicular failure.

The STS markers prescribed by the European Academy of Andrology claim to detect over 90% of the microdeletions in the AZF loci (Simoni et al., 1999). However, in the present study, only 3% of subjects with deletions were identified using the STS markers recommended by EAA. Using STS markers selected from Indian studies, microdeletions were observed in another 7.5% of the infertile cases. A study conducted in the Egyptian population also reports a lower frequency of detection of Y chromosome microdeletion, wherein 100 infertile men were screened and deletion was found only in 4 cases (4%) (Sheikh et al., 2009). Another independent study was carried out in the same population who reported absence of microdeletions in their study (40 cases) and compared their finding with other published reports (Abobakr et al., 2009). The authors explain why they failed to identify any Y chromosome microdeletion, which could be due to the fewer number of STS markers, heterogeneity of markers used among different studies, and also ethnic variations (Abobakr et al., 2009).

Only four of six STS makers (sY127, sY134, sY255, sY254) recommended by the EAA showed a deletion frequency of 2.8%-4%. Of 16 markers that were used in Indian studies and not included in the EAA, six STS markers (sY113, sY143, sY148, sY153, sY156, sY158) showed deletion with a frequency range of 2.6%-6.8% (Table 3). Thus, it is clear that the use of markers recommended by the EAA alone are not enough to screen the deletions in Y chromosome in Indian subjects and additional markers should be used. We suggest that each country should examine Y chromosome microdeletions in a large number of sample with homogenous phenotypes and using large number of STS makers, which will eventually identify markers specific for their population to increase the detection of Y chromosome microdeletions.

Table 3.

Sequence Tag Site Markers Used in Indian Studies from 2002 to 2010

S. no.	STS marker	Studied in number of patients	Deletion frequency No. (%)
1	sY84^a	365	3 (0.8)
2	sY86^a	435	6 (1.1)
3	sY127^a	1032	31 (3)
4	sY129	200	2 (1)
5	sY130	200	2 (1)
6	sY134^a	1086	31 (2.8)
7	sY255^a	1313	50 (3.8)
8	sY254^a	1283	52 (4)
9	sY243	200	4 (2)
10	sY269	200	4 (2)
11	sY742	200	2 (1)
12	sY746	500	6 (1.2)
13	sY113	752	20 (2.6)
14	sY117	377	3 (0.7)
15	sY120	200	2 (1)
16	sY131	377	2 (1)
17	sY132	200	3 (3.6)
18	sY143	930	34 (3.6)
19	sY148	377	24 (6.3)
20	sY153	370	21 (5.6)
21	sY156	377	36 (6.8)
22	sY158	971	48 (4.9)

STS markers are EAA recommended.

Footnotes

Acknowledgments

The authors thank Drs. M. Kochar, S. Mittal, K.K. Ghautam, and G. Majumdar, who referred the patients, and Dr. S. Kohli for technical advice. K. Singh, J. Singh, and D. Gupta are thanked for valuable technical assistance.

Disclosure Statement

No competing financial interests exist.

References

Abobakr

, Mostafa

, Mahmoud

et al. 2009. Detection of azoospermia factor (AZF) microdeletion on Y chromosome in infertile men with azoospermia and severely oligispermia. J Derm Androl, 29:100-104.

Aho

, horkonen

, Suikkari

et al. 2003. Y chromosome microdeletions among infertile Finnish men. AOGS, 80:652-656.

Ambasudhan

, Singh

, Agarwal

et al. 2003. Idiopathic cases of male infertility from a region in India show low incidence of Y-chromosome microdeletion. J Biosci, 28:605-612.

Babu

, Swarna

, Padmavathi

et al. 2002. PCR analysis of Yq microdeletions in infertile males, a study from South India. Asian J Androl, 4:265-268.

Bor

, Hindkjaer

, Kolvraa

et al. 2002. Y chromosome microdeletions and cytogenetic findings in unselected ICSI, candidates at a Danish fertility clinic. J Asst Reprod Genet, 19:224-231.

Chai

, Salido

, Yen

. 1997. Multiple functional families copies of RBM gene family, a spoermatogenesis candidate on the human Y chromosome. Genomics, 45:355-361.

Dada

, Gupta

, Kucheria

. 2003. Molecular screening for Yq microdeletion in men with idiopathic oligospermia and azoospermia. J Biosci, 28:163-168.

Foresta

, Bettella

, Moro

et al. 2001. Sertoli cell function in infertile patients with and without microdeletions of the azoospermia factors on the Y chromosome long arm. J Clin Endocrinol Metab, 86:2414-2419.

Foresta

, Ferlin

, Garolla

. 1997. Y chromosome deletion in idiopathic servere testiculopathies. J Clin Endocrinol Metab, 82:1075-1080.

10.

Foresta

, Ferlin

, Garolla

et al. 1998. High frequency of well-defined Y-chromosome deletions in idiopathic Sertoli cell-only syndrome. Hum Reprod, 13:302-307.

11.

Foresta

, Moro

, Garolla

et al. 1999. Y chromosome microdeletions in cryptorchidism and idiopathic infertility. J Endocrinol Metab, 84:3660-3665.

12.

Kihaile

, Yasui

, Shuto

. 2005. Prospective assessment of Y chromosome microdeletions and reproductive outcome among infertile couples of Japanese origin. J Exp Clin Asst Reprod, 2:1-7.

13.

Krausz

, Quintana-Murci

, Barbaux

et al. 2001. A High frequency of Y chromosome deletions in males with nonidiopathic infertility. J Clin Endo Metab, 84:3606-3612.

14.

Kumar

, Dada

, Gupta

et al. 2006. Serum FSH levels and testicular histology in infertile men with non obstructive azoospermia and Y chromosome microdeletions. Ind J Urol, 22:332-336.

15.

Kuroda-Kawaguchi

, Skaletsky

, Brown

et al. 2001. The AZFc region of Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat Genet, 29:279-286.

16.

Lin

, Chin

, Sun

et al. 2000. Y chromosome microdeletions and its effect on reproductive decisions in Taiwanese patients presenting with nonobstructive azoospermia. Urology, 56:1041-1046.

17.

Martinez

, Bernabe

, Gomez

et al. 2000. Screening for AZF deletion in a large series of severely impaired spermatogenesis patients. J Androl, 21:651-655.

18.

Miller

, Dykes

, Polesky

. 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res, 16:1215.

19.

Mirzajani

, Kalantar

, Montazeri

. 2010. High prevalence of AZFb microdeletion in Iranian patients with idiopathic nonobstructive azoospermia. Indian J Med R, 132:265-270.

20.

Mitra

, Dada

, Kumar

et al. 2008. Screening for Y-chromosome microdeletions in infertile Indian males: utility of simplified multiplex PCR. Indian J Med Res, 27:124-132.

21.

Najmabadi

, Huang

, Yen

et al. 1996. Substantial prevalence of microdeletion of the Y chromosome in infertile men with idiopathic azoospermia or oligozoospermia detected using a sequence tagged site mapping strategy. J Clin Endocrinol Metab, 81:1347-1352.

22.

Omrani

, Samadyadae

, Bagheri

et al. 2006. Y chromosome microdeletions in idiopathic infertile men from west Azarbaijan. Urology, 3:38-43.

23.

Pedro

, Fraietta

, Spaine

et al. 2003. Prevalence of Y chromosome deletion in Brazilian population of non-obstructive azoospermia and severely oligospermic men. J Med Bio Res, 3:287-293.

24.

Peterlin

, Kunej

, Sinkovec

et al. 2002. Screening for Y chromosome microdeletions in 226 Slovenian subfertile men. Hum Reprod, 17:17-24.

25.

Pryor

, Kent-First

, Muallem

. 1997. Microdeletions in the Y-chromosome of infertile men. N Engl J Med, 336:534-539.

26.

Rao

, Babu

, Kanakavalli

et al. 2004. Chromosomal abnormalities and Y chromosome microdeletions in infertile men with varicocele and idiopathic infertility of South Indian origin. J Androl, 25:47-53.

27.

Selva

, Kanafani

, Prigent

et al. 1997. Incidence of AZF (azoospermia factor) deletion and familial forms of infertility among patients requiring intracytoplasmic spermatozoa injection (ICSI) J Assist Reprod Genet, 14:593-595.

28.

Sheikh

, Nower

, Rasol

et al. 2009. Prevalence of Y chromosome microdeletions in males with azoospermia and severe oligospermia in Egypt. Kasr Aini Med J, 12:47-50.

29.

Simoni

, Bakeker

, Eurling

MCM

et al. 1999. Laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions. Int J Androl, 22:292-299.

30.

Swarna

, Babu

, Reddy

. 2003. AZFc deletions in idiopathic infertile males from south India. Int J Hum Gene, 3:1-4.

31.

Thangaraj

, Gupta

, Pavani

et al. 2003. Y chromosome deletions in azoospermic men in India. J Androl, 24:588-597.

32.

Tse

, Yeung

, Lau

et al. 2000. Deletions within the azoospermia factor subregions of the Y chromosome in Hong Kong Chinese men with severe male factor infertility: controlled clinical study. HKMJ, 6:143-146.

33.

Van der Van

, Montag

, Peschka

et al. 1997. Combined cytogenetic and Y chromosome microdeletion screening in males undergoing intracytoplasmic sperm injection. Mol Hum Reprod, 3:699-704.

34.

Viswambharan

, Suganthi

, Simon

et al. 2007. Male infertility: polymerase chain reaction- based deletion mapping of genes on the human Y chromosome. Singapore Med J, 48:1140-1147.

35.

Vogt

, Edelmann

, Kirsch

et al. 1996. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet, 5:933-943.

36.

Vutyavanich

, Promletarmoen

, Siriungsi

et al. 2006. Frequency of Y chromosome microdeletion and chromosome abnormalities in infertile Thai men with oligospermia and azoospermia. Asian J Androl, 9:68-75.

37.

World Health Organization. 1999. Laboratory Manual for the Examination of Human Semen, 4th. Cambridge University Press: Cambridge.