Abstract

Microdeletions in the azoospermia factor (AZF) regions on the long arm of the human Y chromosome are known to be associated with spermatogenic failure. Although AZFc is recurrently deleted in azoospermic or oligozoospermic males, no definitive conclusion has been reached for the contribution of different partial AZFc deletions to spermatogenic failure. To further investigate the roles of partial deletions in spermatogenic failure and the relationship between the complete and partial AZFc deletions, we performed deletion typing and Y chromosome haplogrouping in 756 idiopathic infertile Han-Chinese and 391 healthy Han-Chinese. We found that both the b2/b3 partial deletion and the DAZ3/4+CDY1a deletion pattern were associated with spermatogenic failure. We also confirmed that two previously reported fixations, the b2/b3 deletion in haplogroup N1 and the gr/gr deletion in haplogroup Q1. Remarkably, the frequency of the complete AZFc deletion in haplogroup N1 was significantly higher than that in the haplogroup Q1. These results suggest that the b2/b3 partial deletion was associated with a higher risk of complete AZFc deletion compared with the gr/gr partial deletion. Compared with the gr/gr deletion, the b2/b3 deletion presents a shorter distance among recombination targets and longer recombination substrates, which may be responsible for the increased incidence of subsequent recombination events that can lead to the complete AZFc deletion in this Chinese study population. The susceptibility of the b2/b3 partial deletion to the complete AZFc deletion deserves further investigation in larger and diverse populations, especially those with a relatively high frequency of b2/b3 and gr/gr partial deletions.

INTRODUCTION

Infertility is estimated to affect 10–15% of couples, and roughly half of these cases are due to the man’s problem (1). Spermatogenic failure is the most common form of male infertility, and the important role of the Y chromosome in male infertility is increasingly recognized. The male-specific region of the Y chromosome (MSY) consists of long, Y-specific repeats called amplicons. Homologous recombination between amplicons has been shown to generate deletions, commonly resulting in spermatogenic failure. Three azoospermia factors (AZFa, AZFb and AZFc) have been mapped to Yq11, and the AZFc region completely comprising amplicons is particularly susceptible to deletions, the most commonly known genetic factor that leads to azoospermia or oligozoospermia (2,3). The complete AZFc deletion (referred to as the b2/b4 deletion), which loses a segment of about 3.5 Mb with eight testis-specific expressed gene families, has long been known to cause spermatogenic failure with few exceptions (4,5) (Fig. 1A).

Figure 1.

Structures and rearrangements at the AZFc region in human Y chromosome. The deleted regions are shown as broken lines. (A) The amplicon structure, three protein-coding gene families (DAZ, CDY1 and BPY2) and five non-coding gene families (TTTY17, TTTY4, TTTY3, GOLGA2LY and CSPG4LY) at the AZFc region based on the GenBank reference sequence (4,40). The recombination between two b amplicons (shown in black) can lead to complete deletion of AZFc (∼3.5 Mb deleted). (B) The gr/gr deletion can occur between two g or two r amplicons which has a deletion of about 1.6 Mb fragment of AZFc (6). (C) The b2/b3 deletion can be derived from the gr/rg inversion or b2/b3 inversion, which has a deletion of about 1.8 Mb fragment of AZFc (19,22). (D) Comparison of the distance of the recombination targets of the complete AZFc deletion in the gr/gr-deleted AZFc and b2/b3-deleted AZFc. (E) Comparison of the length of recombination substrates in the gr/gr-deleted AZFc and b2/b3-deleted AZFc.

Figure 1.

Structures and rearrangements at the AZFc region in human Y chromosome. The deleted regions are shown as broken lines. (A) The amplicon structure, three protein-coding gene families (DAZ, CDY1 and BPY2) and five non-coding gene families (TTTY17, TTTY4, TTTY3, GOLGA2LY and CSPG4LY) at the AZFc region based on the GenBank reference sequence (4,40). The recombination between two b amplicons (shown in black) can lead to complete deletion of AZFc (∼3.5 Mb deleted). (B) The gr/gr deletion can occur between two g or two r amplicons which has a deletion of about 1.6 Mb fragment of AZFc (6). (C) The b2/b3 deletion can be derived from the gr/rg inversion or b2/b3 inversion, which has a deletion of about 1.8 Mb fragment of AZFc (19,22). (D) Comparison of the distance of the recombination targets of the complete AZFc deletion in the gr/gr-deleted AZFc and b2/b3-deleted AZFc. (E) Comparison of the length of recombination substrates in the gr/gr-deleted AZFc and b2/b3-deleted AZFc.

After the physical maps of the AZFc region were constructed, several types of AZFc partial deletions had been identified including the gr/gr subdeletion, the b2/b3 subdeletion (also referred to as the g1/g3 deletion or the u3-gr/gr deletion) and the b1/b3 subdeletion. The gr/gr subdeletion and the b2/b3 subdeletion are the most commonly detected.

The gr/gr partial deletion, which is caused by non-allelic homologous recombination between g1/r1/r2 and g2/r3/r4 amplicons, leads to an excision of 1.6 Mb DNA segment from the AZFc region (Fig. 1B). This deletion is reported to be fixed in Y chromosome haplogroup D2b and Q1. The results of studies on the roles of this deletion with spermatogenic failure have not been consistent. In Dutch, Spanish, Italian and Australian populations, the gr/gr partial deletion has been reported as a significant risk factor for spermatogenic failure (6–10). However, in other populations such predisposition has not been found to date (11–18).

The b2/b3 partial deletion, which removes a 1.8 Mb DNA segment, is preceded by an inversion event. The gr/rg inversion is followed by a b2/b3 deletion, while the b2/b3 inversion is followed by a gr/gr deletion (19) (Fig. 1C). This deletion is found to be fixed in N, a haplogroup widely distributed in Northern Eurasia (20,21). The association of the b2/b3 partial deletion with male infertility has been recently reported in Chinese men, whereas no such a predisposition was detected in other populations (10–12,18,19,22). Other genetic events, such as the b1/b3 deletion, have a much lower frequency in the general population than the gr/gr and b2/b3 deletion, and their influence on spermatogenesis have not been identified.

Few studies have investigated the possible association between complete AZFc deletion and partial AZFc deletion. Our previous studies have found that the haplogroups abundant in partial deletions showed a higher frequency of complete deletions than other haplogroups. Thus, we hypothesized that there was a two-step process leading to a complete AZFc deletion and we showed that the partial AZFc deletions could increase the incidence of complete AZFc deletions (23).

Recently, several studies have suggested the predisposition of genetic background to spermatogenic failure in Danish, Japanese and Chinese populations (24–26). To verify the consistency of genetic background, the distribution differences of Y chromosome haplogroups between the case group and the control group need to be tested in this association study. Therefore, in the present study, we typed 14 binary markers on the Y chromosome and subdivided 14 Y chromosome haplogroups. Our aim was to better clarify the prevalence and characteristics of AZFc subdeletions, to evaluate their associations with spermatogenic failure, and to elucidate the real association between the partial and complete AZFc deletions in a Han-Chinese population.

RESULTS

Detection of potential distribution differences of Y chromosome haplogroups

To eliminate the influence of genetic backgrounds in our association study, we typed 14 Y chromosome markers that helped define 14 haplogroups in patients and normal subjects (controls) with different spermatogenic phenotypes. No significant difference in the Y chromosome haplogroup distribution was found among the groups with different spermatogenic status (Table 1). This result suggests that genetic background may not affect our results of the present association study.

Table 1.

Distribution of the patients and controls in Y chromosome haplogroup

Group Number Y chromosome haplogroup
 
DE F* K* N1 Q1 Q1 O2a O2* O2b O* O3* O3e* O3e1 
Fertility/normozospermia 391 39 16 13 66 13 100 54 68 
Infertility/abnormospermiaa 419 31 10 29 20 59 20 115 52 59 
Azoospermia 220 18 17 11 34 10 52 27 29 
Oligozoospermia 199 13 12 25 10 63 25 30 
Infertility/normospermia 337 22 20 15 49 21 93 42 46 
All infertilityb 756 53 14 15 49 35 108 18 41 11 209 94 105 
Group Number Y chromosome haplogroup
 
DE F* K* N1 Q1 Q1 O2a O2* O2b O* O3* O3e* O3e1 
Fertility/normozospermia 391 39 16 13 66 13 100 54 68 
Infertility/abnormospermiaa 419 31 10 29 20 59 20 115 52 59 
Azoospermia 220 18 17 11 34 10 52 27 29 
Oligozoospermia 199 13 12 25 10 63 25 30 
Infertility/normospermia 337 22 20 15 49 21 93 42 46 
All infertilityb 756 53 14 15 49 35 108 18 41 11 209 94 105 

aThe sum of azoospermia and oligozoospermia.

bThe sum of infertility/abnormospermia and infertility/normospermia.

Distributions of deletion in groups with different spermatogenic status

Overall, 45 out of 756 infertile men were found to have classical AZF deletions in the AZF region, using the STS analysis of genomic DNA recommended by EAA/EMQN. These classical deletions included seven AZFa deletions, four AZFb deletions, four AZFbc (AZFb+AZFc deletion) deletions and five AZFabc deletions (AZFa+AZFb+AZFc deletion) found only in azoospermia patients, and 25 AZFc deletions with 14 azoospermia and 11 oligozoospermia. However, there was no classical deletion in the groups of infertility/normospermia and fertility/normospermia (Table 2). These results are consistent with those previously reported, suggesting that complete AZFc deletions may lead to azoospermia or oligozoospermia with few exceptions (4,5).

Table 2.

The distribution of gr/gr and b2/b3 partial deletion in groups with different spermatogenic status

Group Number AZFa/b/bc/abc AZFc Without classical AZF deletion gr/gr subdeletion
 
b2b3 subdeletion
 
Number (%) OR (95%CI) P-value Number (%) OR (95%CI) P-value 
Fertility/normozospermia 391 391 40 (10.23) 1.00  20 (5.12) 1.00  
Infertility/abnormospermiaa 419 20 25 374 54 (14.44) 1.48 (0.96–2.29) 0.076 37 (9.89) 2.04 (1.16–3.58) 0.012 
Azoospermia 220 20 14 186 25 (13.44) 1.36 (0.80–2.32) 0.254 19 (10.22) 2.11 (1.10–4.06) 0.023 
Oligozoospermia 199 11 188 29 (15.43) 1.60 (0.96–2.67) 0.071 18 (9.57) 1.96 (1.01–3.81) 0.042 
Infertility/normospermia 337 337 35 (10.39) 1.02 (0.63–1.64) 0.945 29 (8.61) 1.75 (0.97–3.15) 0.061 
All infertilityb 756 20 25 711 89 (12.52) 1.26 (0.85–1.86) 0.258 66 (9.28) 1.90 (1.13–3.18) 0.014 
Group Number AZFa/b/bc/abc AZFc Without classical AZF deletion gr/gr subdeletion
 
b2b3 subdeletion
 
Number (%) OR (95%CI) P-value Number (%) OR (95%CI) P-value 
Fertility/normozospermia 391 391 40 (10.23) 1.00  20 (5.12) 1.00  
Infertility/abnormospermiaa 419 20 25 374 54 (14.44) 1.48 (0.96–2.29) 0.076 37 (9.89) 2.04 (1.16–3.58) 0.012 
Azoospermia 220 20 14 186 25 (13.44) 1.36 (0.80–2.32) 0.254 19 (10.22) 2.11 (1.10–4.06) 0.023 
Oligozoospermia 199 11 188 29 (15.43) 1.60 (0.96–2.67) 0.071 18 (9.57) 1.96 (1.01–3.81) 0.042 
Infertility/normospermia 337 337 35 (10.39) 1.02 (0.63–1.64) 0.945 29 (8.61) 1.75 (0.97–3.15) 0.061 
All infertilityb 756 20 25 711 89 (12.52) 1.26 (0.85–1.86) 0.258 66 (9.28) 1.90 (1.13–3.18) 0.014 

aThe sum of azoospermia and oligozoospermia.

bThe sum of infertility/abnormospermia and infertility/normospermia.

χ2 test (P < 0.05) when compared with fertility/normozospermia group.

After having eliminated 45 classical AZF deletions, we evaluated the distributions of partial AZFc deletions in 711 infertile patients and 391 healthy controls (Table 2). Overall, we found 89 gr/gr deletions (25 with azoospermia, 29 with oligozoospermia and 35 with normospermia) and 66 b2/b3 deletions (19 with azoospermia, 18 with oligozoospermia and 29 with normospermia) in the patients and 40 gr/gr and 20 b2/b3 deletions in the fertile men (Table 3). There was a statistically significant difference in the frequency distribution of b2/b3 subdeletions (OR 1.90; 95% CI 1.13–3.18) (P = 0.014) between the controls and infertile patients (Table 3). However, there was no significant difference in the frequency distribution of the gr/gr deletion between patients and controls. To unmask the exact role of b2/b3 in male infertility, we compared different groups of spermatogenic status with the controls. Compared with the fertility/normospermia, we found that b2/b3 subdeletions were associated with significantly increased risk of azoospermia (OR 2.11; 95% CI 1.10–4.06) (P = 0.023), oligozoospermia (OR 1.96; 95% CI 1.01–3.81) (P = 0.042) and infertility/abnormospermia (subtotal of azoospermia and oligozoospermia) (OR 2.04; 95% CI 1.16–3.58) (P = 0.012), while we did not find any difference between the groups of infertility/normospermia and fertility/normospermia. Interestingly, there was no significant difference in the frequency of the gr/gr subdeletions between subdivided groups with different spermatogenic status and the controls.

Table 3.

The distribution of the deletion types of DAZ and CDY1 genes in gr/gr and b2/b3 subdeletions

Group All Without classical deletion gr/gr b2/b3 DAZ1/2+CDY1a
 
DAZ1/2+CDY1b
 
DAZ3/4+CDY1a
 
DAZ3/4+CDY1b
 
gr/gr b2/b3 gr/gr b2/b3 gr/gr b2/b3 gr/gr b2/b3 
Fertility/normozospermia 391 391 40 20 14 10 10 14 
Infertility/abnormospermiaa 419 374 54 37 24 13 30 
Azoospermia 220 186 25 19 16 
Oligozoospermia 199 188 29 18 15 14 
Infertility/normospermia 337 337 35 29 15 21 
All infertilityb 756 711 89 66 16 31 28 51 14 
Group All Without classical deletion gr/gr b2/b3 DAZ1/2+CDY1a
 
DAZ1/2+CDY1b
 
DAZ3/4+CDY1a
 
DAZ3/4+CDY1b
 
gr/gr b2/b3 gr/gr b2/b3 gr/gr b2/b3 gr/gr b2/b3 
Fertility/normozospermia 391 391 40 20 14 10 10 14 
Infertility/abnormospermiaa 419 374 54 37 24 13 30 
Azoospermia 220 186 25 19 16 
Oligozoospermia 199 188 29 18 15 14 
Infertility/normospermia 337 337 35 29 15 21 
All infertilityb 756 711 89 66 16 31 28 51 14 

aThe sum of azoospermia and oligozoospermia.

bThe sum of infertility/abnormospermia and infertility/normospermia.

χ2 test (P < 0.05) when compared with fertility/normozospermia group.

To further characterize the partial deletion subtypes, we typed for DAZ and CDY1 gene status for the subjects with gr/gr or b2/b3 partial deletions, using the SFV or SNV analysis. We found that there were four types of deletion patterns in both gr/gr and b2/b3 subdeletions: DAZ1/2+CDY1a, DAZ1/2+CDY1b, DAZ3/4+CDY1a, DAZ3/4+CDY1b. The distributions of these four deletion pattern types are shown in Table 3.

In the b2/b3 subdeletion group, 60 out of 66 deletion cases did not have DAZ3/4 SFV, a finding consistent with that in previous reports (18,23). Combined with the deletion of CDY1a, the DAZ3/4+CDY1a deletion pattern was associated with risk of infertility (OR 2.08; 95% CI 1.14–3.81) (P = 0.015) when comparing the subtotal of male infertility group with the controls. Specifically, the frequencies of DAZ3/4+CDY1a deletion pattern between subdivided groups of male infertility and the controls were significantly different in azoospermia group (OR 2.53; 95% CI 1.21–5.31) (P = 0.011), oligozoospermia group (OR 2.17; 95% CI 1.01–4.64) (P = 0.042) and infertility/abnormospermia (subtotal of azoospermia and oligozoospermia) group (OR 2.35; 95% CI 1.22–4.50) (P = 0.008). However, no significant difference was found between infertility/normospermia group and the controls. Although there were more DAZ1/2 SFV absent in patients than in the controls (none was found), no significant difference was found in the frequencies of partial deletion subtypes.

In the gr/gr subdeletion group, the distribution of the four types of deletion pattern was more balanced than the b2/b3 deletion, and no significant difference of these types was found among these subpopulations.

Distributions of complete and partial AZFc deletions in Y chromosome haplogroups

To explore the relationship between deletions and Y chromosome haplogroup, we analyzed the distribution of complete/partial AZFc deletions in the 14 Y chromosome haplogroups (Table 4).

Table 4.

Distributions of the subjects and partial/complete AZFc deletions in Y chromosome haplogroups

Group Number Y chromosome haplogroup
 
DE F* K* N1 Q1 O1 O2a O2* O2b O* O3* O3e* O3e1 
All 
 Fertility/normozospermia 391 39 16 13 66 13 100 54 68 
 All infertility 756 53 14 15 49 35 108 18 41 11 209 94 105 
gr/gr deletion only 
 Fertility/normozospermia 40     13   
 Infertility/azo-/oligospermia 54    20   10 
 Infertility/normozospermia 35     15   
 Subtotal/infertility 89    35  12 10 
b2/b3 deletion only 
 Fertility/normozospermia 20   16         
 Infertility/azo-/oligospermia 37   23        
 Infertility/normozospermia 29   20        
 Subtotal/infertility 66   43       
Complete AZFc deletion only 
 Fertility/normozospermia               
 Infertility 25         
Group Number Y chromosome haplogroup
 
DE F* K* N1 Q1 O1 O2a O2* O2b O* O3* O3e* O3e1 
All 
 Fertility/normozospermia 391 39 16 13 66 13 100 54 68 
 All infertility 756 53 14 15 49 35 108 18 41 11 209 94 105 
gr/gr deletion only 
 Fertility/normozospermia 40     13   
 Infertility/azo-/oligospermia 54    20   10 
 Infertility/normozospermia 35     15   
 Subtotal/infertility 89    35  12 10 
b2/b3 deletion only 
 Fertility/normozospermia 20   16         
 Infertility/azo-/oligospermia 37   23        
 Infertility/normozospermia 29   20        
 Subtotal/infertility 66   43       
Complete AZFc deletion only 
 Fertility/normozospermia               
 Infertility 25         

Overall, 25 complete AZFc deletions were found in six haplogroups of infertility/abnormospermia, whereas none was found in healthy men and infertility/normospermia (Table 4).

Two deletion-fixed haplogroups were found: b2/b3-deleted haplogroup N1 and gr/gr-deleted haplogroup Q1, which are consistent with the reported fixation of b2/b3 and gr/gr deletions in these haplogroups (19,22,23). In haplogroup N1, all 16 control men and 43 of 49 patients carried b2/b3 deletions, and the other six patients carried a complete AZFc deletion. In haplogroup Q1, all 13 control men and all 35 patients carried the gr/gr deletion, and no complete AZFc deletion was found in this haplogroup. Therefore, we speculated that the ancestors of haplogroup N1 and Q1 may be the b2/b3 and gr/gr deletion carriers, respectively, and that complete deletions of deletion-fixed haplogroups may be derived from the gr/gr-deleted Y chromosomes of N1 or the b2/b3-deleted Y chromosomes of Q1. Then, we compared the distributions of the patients of complete AZFc deletions between the b2/b3-deleted haplogroup N1 (fixation of b2/b3 partial deletion) and the gr/gr-deleted haplogroup Q1 (fixation of gr/gr partial deletion), and we found a significant difference (P = 0.034) in the frequency distributions of complete AZFc deletions (Table 5). Considering the fixation of partial AZFc deletions in haplogroups N1 and Q1, our data suggested that the b2/b3-deleted haplogroups showed a higher frequency of complete deletion than gr/gr-deleted haplogroups. Namely, the b2/b3 partial deletion was more likely to suffer from complete AZFc deletion in the deletion-fixed haplogroups.

Table 5.

Distribution of the patients of complete AZFc deletion between b2/b3-deleted haplogroup N1 and gr/gr-deleted haplogroup Q1

Haplogroup Group b2/b4 deletion Without b2/b4 deletion P-value 
gr/gr-deleted haplogroup Q1 Infertility/azo-/oligospermia 20  
Infertility/normozospermia 15  
Subtotal 35  
b2/b3-deleted haplogroup N1 Infertility/azo-/oligospermia 23 0.034 
Infertility/normozospermia 20  
Subtotal 43 0.034 
Haplogroup Group b2/b4 deletion Without b2/b4 deletion P-value 
gr/gr-deleted haplogroup Q1 Infertility/azo-/oligospermia 20  
Infertility/normozospermia 15  
Subtotal 35  
b2/b3-deleted haplogroup N1 Infertility/azo-/oligospermia 23 0.034 
Infertility/normozospermia 20  
Subtotal 43 0.034 

Indicates significant difference by Fisher’s exact test, one-sided (P < 0.05) when compared with infertility/azo-/oligospermia group of gr/gr-deleted haplogroup Q1.

Fisher’s exact test, one-sided (P < 0.05) when compared with infertility/subtotal group of gr/gr-deleted haplogroup Q1.

Through a further screening of AZFc deletions in all patients, we found that in haplogroups K* and N1, only b2/b3 deletion existed, while in haplogroups DE, Q1, O1, O2a, O3e* and O3e1, only gr/gr deletion existed and in haplogroups C, O2*, O* and O3*, both gr/gr and b2/b3 deletion existed (Table 4). As haplogroups F* and O2b contained few samples and no partial deletion was found, the two haplogroups were not included in the association analysis. Then, we compared the frequency distributions of complete deletions in three groups: haplogroups with both gr/gr and b2/b3 deletions, haplogroups with only the gr/gr deletion and haplogroups with only the b2/b3 deletion. A significant difference (OR 7.33; 95% CI 2.45–21.95) (P < 0.001) in the frequency distributions of complete deletion was found in only b2/b3 deletion existed group when compared with the group of both gr/gr and b2/b3 deletions existed, while no significant difference was found in the group of only gr/gr deletion existed group (Table 6).

Table 6.

Distribution of the patients with complete AZFc deletion in Y chromosome haplogroups with both or only gr/gr, b2/b3 partial deletion

Subgroup Haplogroup Complete AZFc deletion Without complete AZFc deletion OR (95%CI) P-value 
Both gr/gr and b2/b3 partial deletion existed haplogroupa C, O2*, O*, O3* 308 1.00  
Only gr/gr partial deletion existed haplogroupb DE, Q1, O1, O2a, O3e*, O3e1 11 363 1.56 (0.57, 4.25) 0.386 
Only b2/b3 partial deletion existed haplogroupc K*, N1 56 7.33 (2.45, 21.95) <0.001 
Subgroup Haplogroup Complete AZFc deletion Without complete AZFc deletion OR (95%CI) P-value 
Both gr/gr and b2/b3 partial deletion existed haplogroupa C, O2*, O*, O3* 308 1.00  
Only gr/gr partial deletion existed haplogroupb DE, Q1, O1, O2a, O3e*, O3e1 11 363 1.56 (0.57, 4.25) 0.386 
Only b2/b3 partial deletion existed haplogroupc K*, N1 56 7.33 (2.45, 21.95) <0.001 

aHaplogroups with both gr/gr and b2/b3 partial deletion including haplogroup C, O2*, O* and O3*.

bHaplogroups with only gr/gr partial deletion including haplogroup DE, Q1, O1, O2a, O3e* and O3e1.

cHaplogroups with only b2/b3 partial deletion including haplogroup K* and N1.

χ2 test when compared with the group of both the gr/gr and b2/b3 partial deletion existed haplogroup, P < 0.001. The significance level was set to 0.025, using a Bonferroni correction.

To identify the correlation between b2/b3 partial deletion and complete deletion, further analysis was performed in the only b2/b3 deletion existed subgroups (including haplogroups N1 and K*) (Table 7). A significant difference (P < 0.001) in the frequency distributions of complete AZFc deletions was found between N1 (only b2/b3 deletion existed) and non-N1. In contrast to haplogroup N1, no significant difference was found between K*(only b2/b3 deletion existed) and non-K* groups.

Table 7.

Distribution of the patients with complete AZFc deletion in haplogroups with only b2/b3 deletion

Subgroup Haplogroup Complete AZFc deletion Without complete AZFc deletion P-value 
Only b2/b3 partial deletion existed haplogroupa K* 13 0.085b 
Non- K* 23 718  
N1 43 0.0003 
Non-N1 19 688  
Subgroup Haplogroup Complete AZFc deletion Without complete AZFc deletion P-value 
Only b2/b3 partial deletion existed haplogroupa K* 13 0.085b 
Non- K* 23 718  
N1 43 0.0003 
Non-N1 19 688  

aIncluding the haplogroups of N1 and K*.

bIndicates no significant difference by Fisher’s exact test, one sided.

χ2 test when compared with haplogroup N1 and Non-N1, P < 0.001. Post-hoc test, based on Table 6, considering multiple-test, we use a Bonferroni correction and set the significance level to 0.0125.

DISCUSSION

A clear association between classical AZF deletions and spermatogenic failure has been established based on well-designed cause–effect studies (27–29). In this study, we found a total of 45 cases with classical deletions among 419 infertile men with abnormospermia. Almost all of these patients with such deletions were azoospermic, except for 11 AZFc deleted cases who were all oligozoospermic, and no such deletions were found in normospermic men. These findings confirm the specific relationship between Y chromosome deletions and spermatogenic failure in a Chinese population (30).

In contrast to classical AZF deletions, the possible susceptibility of the different partial AZFc deletions to spermatogenic failure varied among different populations. In our previous study, the b2/b3 deletion showed a strong association with idiopathic male infertility but not with spermatogenic failure, and the gr/gr deletion did not show an increase in the risk of male infertility or spermatogenic failure in the men from East Asians (18). In the present larger study, the distribution difference of studied population was assessed with the Rousset’s exact test, and no significant difference in the Y chromosome haplogroup distribution was found between different groups of spermatogenic status and controls (Table 1).

Consistent with our previous study, the frequency of the gr/gr deletion in the men from East Asians was higher than other populations reported to date (6,9,11,12). The gr/gr partial deletion was not found to be associated with male infertility, and no substantial difference was observed in the degree of spermatogenic failure. Considering the different populations we studied, it is likely that the genetic background, other genetic or environmental factors may play important roles in spermatogenesis.

The b2/b3 subdeletion with a relatively low frequency in a study population was initially described by Repping et al. (19). A significant difference (P = 0.014) between cases of completely infertile men and healthy controls was detected, which indicates that the b2/b3 deletion may have some effect on male infertility. To clarify the exact role of the b2/b3 deletion in male infertility, we subdivided the patients into three groups according to the degree of spermatogenic status: non-obstructive azoospermia, oligozoospermia and infertility/normospermia. The b2/b3 deletion frequencies in the groups of azoospermia and oligozoospermia were 10.22 and 9.57%, respectively, which correlate with the severity of the spermatogenesis impairment. In the group of infertility with normospermia, the frequency of b2/b3 deletion was 8.61%, which was not different from the frequency of b2/b3 deletion in the controls. Thus, we can conclude that the b2/b3 partial deletion was significantly associated with spermatogenic failure in male infertility in the Chinese population.

In addition, the analysis of DAZ and CDY1 gene copy number in these deletion samples allowed us to identify four types of deletion pattern, with possible different impacts on spermatogenesis. In the gr/gr deletion group, no significant association between deletion pattern and infertility was found, while in the b2/b3 deletion group, the frequencies of DAZ3/4+CDY1a deletion pattern were higher in the groups of azoospermia, oligozoospermia and infertility/normospermia than in the control group, with significant differences only found in the groups of azoospermia (P = 0.011) and oligozoospermia (P = 0.042) but not in the group of infertility/normospermia (P = 0.095). Thus, the DAZ3/4+CDY1a deletion pattern is likely to be associated with spermatogenic failure and closely correlated with the degree of spermatogenesis impairment in this study population. However, the effect of DAZ3/4 or CDY1a to spermatogenesis may be limited because the frequency of DAZ3/4 or CDY1a deletion in the gr/gr subdeletion group was not statistically significant.

In this study, we also studied the deletion distribution in Y chromosome haplogroups, which may help uncover the effect of the genetic background of each Y haplogroup in determining the incidence of deletions. A total of 25 complete AZFc deletions were found in 419 patients with azoospermia or oligozoospermia, which is consistent with the frequency previously estimated in azoospermia or in severe oligozoospermia (31). Furthermore, two deletion-fixed haplogroups were confirmed: the haplogroup Q1 (fixation of gr/gr partial deletion) and the haplogroup N1 (fixation of b2/b3 partial deletion). Significantly more complete AZFc deletions were found in the b2/b3-deleted haplogroup N1 (6 out of 49) than in the gr/gr-deleted haplogroup Q1 (none). This finding suggests that the complete deletions of AZFc are more likely to occur in the presence of the b2/b3 partial deletion. Therefore, we further analyzed this possibility by three groups: only gr/gr deletion existed haplogroups (DE, Q1, O1, O2a, O3e and O3e1), only b2/b3 deletion existed haplogroups (K* and N1) and both gr/gr and b2/b3 deletion existed haplogroups (C, O2*, O* and O3*). Compared with the group of both the gr/gr and b2/b3 deletion-existed haplogroups, significant difference (P < 0.001) was found in the group of only b2/b3 deletion-existed haplogroups, but not in the group of only gr/gr deletion-existed haplogroups. Subsequently, we analyzed the frequency of complete AZFc deletion in haplogroup K* and N1 (only b2/b3 deletion-existed haplogroups), respectively. Significant difference (P < 0.001) was also found between N1 and non-N1, while no significant difference was found between K* and non-K*. These data suggest that the significant difference (compared with only b2/b3 deletion existed group and both b2/b3 /gr/gr deletion existed group) was attributed to the b2/b3-deleted haplogroup N1. Taken together, all these results indicate that the b2/b3 partial deletion contributes to a higher risk of complete AZFc deletion than the gr/gr subdeletion at least in the b2/b3-deleted haplogroup N1.

Although the mechanism of the susceptibility of the b2/b3 partial deletion to the complete AZFc deletion requires further scrutiny, the comparison of the structures of the b2/b3 deletion with the gr/gr partial deletion suggested that differences in the lengths of recombination substrates and the distance between them may be responsible for this predisposition. The complete AZFc deletion is known to be caused by non-allelic homologous recombination between b amplicons at opposite ends of the AZFc region (4) (Fig. 1A). As described in the introduction, the b2/b3 deletion arised from the gr/rg or b2/b3 inversion. After the b2/b3 partial deletion occurred, the gray amplicon adjacent to the blue-b3 amplicon relocated next to the blue-b2 amplicon, and homologous recombination can occur between b2-gray and b4-gray combination units, subsequently resulting in the complete AZFc deletion (4) (Fig. 1C). Contrary to the b2/b3 deletion, in the process of the gr/gr deletion, the gray amplicon was excised and not participated in the subsequent process of the formation of the complete AZFc deletion (Fig. 1B). The larger recombination substrates of b2/b3 (b+gray amplicons, about 0.33 Mb) than that of gr/gr (b amplicon, about 0.22 Mb) may account for the susceptibility of the b2/b3 partial deletion to the complete AZFc deletion (Fig. 1E). Another possible explanation is the distance between recombination targets. The distance between recombination targets is 1.9 Mb in the gr/gr deletion (two b amplicons) and 1.7 Mb in the b2/b3 deletion (two b+gray recombination units) (Fig. 1D). According to recent genomic survey of deletion polymorphisms in humans reported that the deletion length followed an L-shaped distribution of the deletion frequency, suggesting that small deletions may have a higher prevalence than larger ones (32). Therefore, the shorter distance between recombination targets in the b2/b3 deletion may consequently increase the incidence of the complete AZFc deletion, although the extent of its effects is still uncertain.

In summary, by examining 745 patients with male infertility and 391 healthy controls, we found an association of the b2/b3 partial deletion with spermatogenic failure but not with male infertility. In addition, the deletion pattern of DAZ3/4+CDY1a was also found to be correlated with spermatogenic failure, although the effect of DAZ3/4 or CDY1a on spermatogenesis may be limited. Furthermore, by screening the distribution of partial and complete AZFc deletions in Y chromosome haplogroups, two deletion-fixed haplogroups were confirmed: b2/b3-deleted haplogroup N1 and gr/gr-deleted haplogroup Q1, whose ancestors may be the deletion carriers. A significantly higher frequency of the complete AZFc deletion was observed in the b2/b3-deleted haplogroup N1 than in the gr/gr-deleted haplogroup Q1. It was suggested that the b2/b3 partial deletion contributed to a higher risk of the complete AZFc deletion than gr/gr subdeletion, at least in the b2/b3-deleted haplogroup N1. The enlarged recombination region and shortened recombination targets were the possible reasons for this predisposition. However, further studies with larger and better selected populations are needed to validate this association and to obtain further insights into the mechanism by which the b2/b3 partial deletion to complete AZFc deletion.

MATERIALS AND METHODS

Study subjects

In this study, a total of 835 infertile men and 391 fertile men were sampled from the infertility clinic at the Affiliated Hospitals of Nanjing Medical University at Jiangsu (NJMU Infertile Study). All the subjects were genetically unrelated ethnic Han-Chinese from East China. The patients with infertility were candidates seeking treatment in the Center of Clinical Reproductive Medicine from April 2004 to April 2007 and recruited into this study by a retrospective design. The fertile men (controls) were healthy young men who had fathered at least one healthy child without assisted reproductive measures during the same period as those of the infertile men recruited in the same hospital. This study was approved by the Institutional Ethics Committees of Nanjing Medical University and Institutional Review Board. The informed consent was obtained from all the participants.

A total of 835 unrelated patients were recruited in this study. All patients underwent at least two semen analyses according to the World Health Organization guidelines (33), a series of physical examinations, and serum determination, which helped us to exclude 79 individuals: 8 obstructive azoospermia, 18 with abnormal karyotype (8 of them with Klinefelter’s syndrome), 9 with cryptorchidism and 44 secondary sterility cases. In the final analysis 756 idiopathic infertility patients aged from 24 to 36 years were included and were divided into three subgroups: 220 with non-obstructive azoospermia (no sperms in the ejaculate even after centrifugation), 199 with oligozoospermia (sperm counts from 0.1 to 20 × 106/ml) and 337 with normal spermatogenesis (sperm counts ≥20 × 106/ml).

A total of 391 healthy men, who were 26–40 years old, were sampled as the controls at the same hospitals where the patients were seen. All these subjects had normal sperm concentration, motility and morphology, and had fathered one or more healthy children. The semen analysis for sperm concentration, motility and morphology was performed following the World Health Organization criteria (33).

Mapping of Yq classical deletions

Genomic DNA was extracted from peripheral leukocytes by means of proteinase K digestion followed by phenol–chloroform extraction and ethanol precipitation. All idiopathic infertility patients perform AZF deletion in Yq11. According to the European Academy of Andrology/European Molecular Genetics Quality Network (EAA/EMQN) guidelines, two multiplex PCR systems were carried out: System A (ZFY, SRY, sY86, Sy127, sY254) and System B (ZFY, SRY, Sy84, sY134, sY255) (34). This set of markers allows identification of deletions in AZFa, AZFb and the complete AZFc (b2/b4) deletion. To confirm the role of AZF classical deletion in spermatogenesis, mapping of Yq classical deletion was also performed in all of the control men.

Typing of AZFc subdeletions

All subjects including both patients and controls without AZFa, AZFb and AZFc deletions underwent this assessment for AZFc subdeletions. Overall, nine AZFc-specific sequence-tagged sites (STSs) (sY1191, sY1291, sY1206, sY1201, sY142, sY1258, sY1197, sY1054, sY1161) were used to identify the types of AZFc deletions (6,19). The absence of amplification of the STSs markers sY1191, sY1291, sY1206 and sY1054 combined with the presence of other STSs indicated the complete AZFc deletion (b2/b4). The absence of sY1191 (specific for b2/b3) and sY1291 (specific for gr/gr) represented b2/b3 and gr/gr deletions, respectively.

Quantitative analysis for the copies of deleted in azoospermia (DAZ) gene and Chromodomain of Y (CDY1) gene was performed to further characterize partial AZFc deletion according to previously described methods (11). For DAZ analysis, we typed the sequence family variant (SFV) at sY587, which can distinguish DAZ1/2 from DAZ3/4. For CDY1 analysis, we used an SFV situated 7750 bp 5′ of the CDY1 translation start codon (CDY7750), which distinguishes CDY1a from CDY1b.

Y chromosome haplogroup and molecular analysis

In this study, 14 binary markers were used to define Y chromosome haplogroups which were highly informative in East Asians (35,36). The markers are as the follows: M130, YAP, M89, M9, LLY22g, M120, M119, M268, M95, M176, M175, M122, M134 and M117. LLY22g was typed using the protocol kindly provided by Y. Xue and C. Tyler-Smith (the Wellcome Trust Sanger Institute). As shown in Figure 2, a total of 14 haplogroups were defined following the nomenclature recommended by the Y Chromosome Consortium and its update (37,38).

Figure 2.

The phylogenetic tree of Y-chromosomal haplogroups in the study population. The phylogeny is based on that of the Y Chromosome Consortium (2002). The binary markers typed in this study are indicated by the defined branches.

Figure 2.

The phylogenetic tree of Y-chromosomal haplogroups in the study population. The phylogeny is based on that of the Y Chromosome Consortium (2002). The binary markers typed in this study are indicated by the defined branches.

Statistical analysis

The distributions of Y chromosome haplogroup between infertile patients and healthy control men that were used to test the population differentiation were assessed by using the Arlequin software (39). Differences in deletion frequencies between cases and controls or among Y chromosome haplogroups were calculated with χ2 test using the Intercooled Stata 7.0 or Fisher’s exact test. Probability (P) values <0.05 were regarded as statistically significant.

FUNDING

This study was supported by grants from National Basic Research Program of China (973 Program) (no. 2009CB941703); Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (no. IRT0631); National Natural Science Foundation of China (no. 30571582) and Graduate Student Grants of Jiangsu Province (no. CX08B-182Z).

ACKNOWLEDGEMENTS

We thank Drs Y. Xue and C. Tyler-Smith for kindly providing the unpublished information of the LLY22g marker. We thank Dr Q. Wei, Department of Epidemiology, the University of Texas M. D. Anderson Cancer Center for English-language editing. We thank Drs J. Liang, Y. Han, Z. Bian, P. Zhu and X. Ding for their efforts in sample collection.

Conflict of Interest statement. None declared.

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