Chapter 3 To investigate the Y chromosome AZFc partial deletion types and its association in spermatogenic impairment and male infertility

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1 Chapter 3 To investigate the Y chromosome AZFc partial deletion types and its association in spermatogenic impairment and male infertility 3.1 Introduction Classically, infertility is defined as the inability to conceive naturally after a year of unprotected intercourse. In the global counterpart, 10-15% of couple suffers from infertility or sub-fertility each year of which the male factor is responsible for approximately 50% of the cases with low sperm production or poor quality as a key factor in substantial part of the cases (MacLeod and Gold 1951; Zuckerman et al., 1977; MacLeod and Wang 1979; Hull et al., 1985). Moreover, impairment in spermatogenesis is the most common cause of male factor infertility where, individual exhibits different infertile conditions such as, azoospermia, oligozoospermia, asthenozoospermia, teratozoospermia or any combination of these conditions (Razvi et al., 1999). Excluding the sperm disorders, there are several contributing factors for example, varicocele, obstruction of spermatic ducts, agglutination of sperms, impotency, hormonal imbalance, and genetic defects are known to cause infertility in males (Pryor et al., 1997). However, the genetic factors are found to be associated with approximately 10% of male infertility where, structural and numerical chromosomal abnormalities are in minor and Y chromosome AZF gene deletions are considered to be crucially involved (de Kretser, 1997). Chapter 3 79

2 3.1.1Y chromosome and AZF structure The human Y chromosome plays a vital role in fertility factor comprises of several male-specific gene families, which are exclusively expressed in the testis (Lahn and Page, 1997). Tiepolo and Zuffardi in 1976 identified deletions in the long arm of the Y chromosome associated with spermatogenic failure. Specifically, the Yq11 segment consisting of three AZF encoding regions namely, AZFa, AZFb and AZFc that accounts for structural abnormalities in the male-specific region of MSY. These regions contain several genes or gene families that are expressed in the testis and involved in spermatogenesis (Pryor et al., 1997). Deletions in the AZFa region are associated with Sertoli Cell Only (SCO) type-i syndrome, which is characterized by the total absence of germ cells in seminiferous tubules and may be linked with non-obstructive azoospermia. Complete deletion of AZFb region is usually associated with meiotic arrest, whereas total AZFc deletion is associated with variable phenotypes with significant reduction in sperm count, motility, morphology and progressive decline in semen quality (Mitra et al., 2008). The clinical prognosis of men with complete AZFc deletions include variable seminal and testicular phenotypes, with sperm production levels ranging from azoospermia to severe oligozoospermia (Saut et al., 2000; Ferlin et al., 2007). Though, the presence of sperm is observed in the ejaculate, the count varies from normal that result in the failure of natural conception (Chang et al., 1999; Calogero et al., 2002; Xia et al., 2006). Interestingly, successful fertilization may occur even in the presence of various partial AZFc deletions. For example, the AZFc deletions are transmitted to the male offspring, and surprisingly, the sons who have phenotypes not necessarily similar to their fathers (Zhang et al., 2007). Taken together, till date the role of these deletions in spermatogenesis is controversial. Chapter 3 80

3 3.1.2 Genetic cluster of the AZFc region: the palindromes and the amplicons In general, the AZFc region encompasses of eight gene families including BPY2, CDY, DAZ, CSPG4LY, GOLGAZLY, TTY3.1, TTY4.1, and TTY7.1, of which, former five genes codes for proteins that are essential for spermatogenesis. While, the AZFc region is more prone to de novo mutations than AZFa and AZFb regions, the emerging findings of AZFc functionality and polymorphism in spermatogenesis has gained renewed scientific interest in these years (Sun et al., 1999; Skaletsky et al., 2003). Despite, the identification of different types of AZFc subdeletions/ partial deletions such as, gr/gr, b1/b3, b2/b3 and b1/b3; b2/b3 deletions, their influence on spermatogenic impairment is still remains unclear (Figure 3.1). Among these, 1.6Mb of gr/gr partial deletion has been frequently reported with prevalence of 2.1% to 12.5% among infertile individuals and 0 to 10.2% in the normozoospermic men (Stouffs et al., 2011) AZFc partial deletions and male infertility: global scenario Essentially, AZFc partial deletions are the group of different deletions caused by homologous recombination flanking g1/g2, r1/r3 and r2/r4 amplicons (Figure 3.1) in P1 and P2 palindromes and accounts for reduction in half of the AZFc region that results in loss of DAZ, CDY1 and BPY2 gene copies in combination, which is anticipated as a risk factor for decreased sperm count observed among Dutch, Spanish, Italian and Australian population but not in others (Yen et al., 2001;Repping et al., 2003; Frenandes et al., 2004; Noordam et al., 2006). On contrary, inspite of losing most of the AZFc gene copies (1.8 Mb deletion), the b2/b3 partial deletion (Figure 3.1) appears to be polymorphic without any obvious effect on fertility. Although, the deletion frequency of b2/b3 is less common compared to gr/gr partial Chapter 3 81

4 deletion and its association with infertility are documented in Chinese and Moroccan population, the effect of this deletion on spermatogenesis among different population remains to be elusive (Eloualid et al., 2012; Giachini et al., 2008; Hucklenbroich et al., 2005; Shahid et al., 2011; Shaqalaih et al., 2009; Wu et al., 2007). Interestingly, the AZFc partial deletions that include gr/gr, b1/b3 and b2/b3 are most often seen in men with azoospermia and oligozoospermia. Previous studies reports the frequency of AZFc partial deletions especially gr/gr deletion is higher in infertile men as compared to fertile controls, whereas some studies are failed to confirm this association (Giachini et al., 2005; Navarro-Costa et al., 2010; Stouffs et al., 2010; Ghorbel et al., 2012; Krausz et al., 2013; Lo Giacco et al., 2014). The metaanalysis studies with a large population involving >20,000 showed that the gr/gr deletion is a risk factor for male infertility (Tutttelmann et al., 2007; Visser et al., 2009; Navarro-Costa et al., 2010; Stouffs et al., 2010; Rozen et al., 2012). The other type of AZFc partial deletions namely b1/b3 and b2/b3 occur at a lower frequency and their association with male infertility is unclear. While, the association of different types of AZFc partial deletions with male infertility is in general observation, ethnicity is predicted to be a most important determinant for such association. From several global studies, it has been observed that the Europeans and the Han Chinese showed a strong association of gr/gr deletion with male infertility (Giachini et al., 2005; Yang et al., 2008; Lo Giacco et al., 2014) however, the American and African men with gr/gr deletion do not seem to be susceptible to azoospermia or oligozoospermia (Stahl et al., 2011; Carvalho et al., 2006; Imken et al., 2007). In the Asian counter part, the gr/gr deletion is not associated with male infertility as seen in Malaysian and Japanese men meanwhile, a significant association are observed in the Chapter 3 82

5 Korean and Chinese population (de Carvalho et al., 2006; Yang et al., 2008;Choi et al., 2012; Almeamar et al.,2013) Classical AZFc deletions and male infertility: Indian context Despite several studies have reported the risk factors for the AZF deletions, the precise nature as well as the size of these deletions with possible impact on spermatogenesis and fertility impairment across different ethnic groups are yet to be stated clearly (Frenandes et al., 2002). For instance, in India the Y chromosome microdeletion investigation in various classes of infertile men has been reported by different groups with wide variation ranging 0-28% (Table 3.1 and 3.2). Babu et al., (2000) first reported the occurrence of Yq classical deletion in the Indian population, with 5% of prevalence rate. However, Sakthivel and Swaminathan (2008) failed to detect any deletions in their study and Nangevenkar et al., (2005) observed deletions in only 1% in idiopathic infertile men, whereas Dada et al., (2003, 2004) reported 6.01 and 9.63% from each independent studies. Further, Thangaraj et al., (2003) reported 8.52% of classical AZF deletions among North East Indians. In the context of Indian population, the association of AZFc partial deletions and male infertility is still unclear. One of the studies from North part of India illustrated that the gr/gr and b1/b3 are associated with male infertility but not b2/b3 deletion (Shahid et al., 2011). However, in another study from South India, only b2/b3 deletion is found to be associated with male infertility but not other deletions (Vijesh et al., 2015). Thus, extensive research is essential to determine the association of AZFc partial deletions and male infertility in Indian population. Chapter 3 83

6 If only classical AZFc deletions are considered, Indian studies records 2% % from North India and 1.13% % from South India (Sen et al., 2013). Such wide variation of Yq AZF deletions is not observed in any other population due to following reasons: (i) India accounts for various ethnic backgrounds with geographic and racial divergence in the occurrence of Y deletions among infertile men (Tse et al., 2002). (ii) Extensive variation in the sample size across different studies. (iii) Moreover, apart from the sample size and ethnic background, the STS markers employed for Y deletion assessment also influence the frequency. Authors have deviated in uniformity usage in same number and types of STS markers for Y deletion assays that are reported from Indian sub-population (Khan et al., 2011; Mahanta et al., 2011; Vishwambhran et al., 2007). (iv) Further, in some studies non EAA/EQMN markers have been utilized that may be possible reason for the observed variations and the true frequency of Y chromosome AZFc deletions in infertile men from Indian population remains unclear (Sachdev et al., 2011; Simoni et al., 1999) (Table 3.1 and 3.2). As stated above, the Y chromosome is highly variable and these dissimilarities may generate a genetic background for vulnerable to Y chromosome related spermatogenic failure. Secondly, states like Delhi, Uttar Pradesh, Mumbai, Assam, Kolkatta, Andhra Pradesh, Tamil Nadu and Kerala have an ample data, whereas there is no single evident data regarding Yq deletion specially AZFc partial deletion from Karnataka state, South India. Therefore, the present study was designed to investigate the Y chromosome AZFc partial deletions among 239 infertile subjects, 244 normozoospermic controls (fertile) and in 200 Siddi tribal men of South and West Karnataka population, (i) to determine the different types of infertile/sub-conditions Chapter 3 84

7 with their frequencies, (ii) comparative evaluation of AZFc partial deletion and its effect on semen parameters as well as the functional status of sperm among study subjects (iii) to assess the association of spermatogenic failure and to characterize the genetic association between the AZFc partial deletions among precise number. To the best of our knowledge, this is the pioneer report from Karnataka state that investigated the association of AZFc partial deletions with spermatogenic impairment. 3.2 Materials and Methods 3.2.1IHEC clearance statement: As mentioned in the previous chapter 2 the IHEC has approved the current project. Further, after briefing the objectives of the present study written informed consent forms were obtained from all the study subjects Study population and geographic location details: In addition to urban fertile individuals/ normozoospermic controls (Group 1) and urban infertiles (Group 2) that were studied for various semen parameters in the preceding chapter of this thesis, a third group has been analysed for various AZFc partial deletions. Group 3 includes a distinctive tribal community called Siddi s that has an African ancestry. Around 1100 AD, the existence of this tribal group in Western India has been first documented and later, in 13 th century Nawabs and Sultans of India imported large group of Siddi individuals from Africa as their slaves (Lodhi, 1997). Currently, this tribal group has been majorly settled in and around Western Ghats forest of Karnataka as well as meagerly in some parts of Gujarat and Andhra Pradesh, with 0.25 to 0.30 million of total population size. Given that Siddi s practice agriculture and daily waging as their primary occupation, they migrate less often compared to other tribal groups and out Chapter 3 85

8 breeding has not practiced in this community, which allowed us to reach them easily considering their lineage has been more conserved and unique from rest of the admix Indian population. In the current study group 3 consisted of 200 volunteer Siddi tribal men with an age ranging from years and the study was carried out in different places of Siddi tribal settlement situated close to Western Ghats forest in and around Uttar Kannada district, South India. The predesigned proforma was prepared to gather information about the age, health status, food habits, marital status as well as reproductive history in case of married individuals, and other relevant details necessary for this study. Men who were above 45 years of age and the individuals who had history of any sort of health issues were excluded. In total, 104 healthy Siddi tribal men who were married and fathered one or more children with proven fertility without any medical assistance and 96 unmarried Siddi men with unknown fertility status were examined for AZFc partial deletions. In spite of several continues attempts and negotiation with Siddi individuals it was not possible to retrieve the semen sample for assessment of semen morphological changes, if any. For that matter, due to strong mythological belief and other religion barriers the tribal subjects were denied to provide their semen sample. Thus, group 3 study cohorts were excluded in the previous chapter Collection of intravenous blood sample: All the subjects were briefed about the purpose of the present study as well as about the sample collection. In addition to several rounds of interaction especially with the tribal community, we have implemented power point presentation module for effective communication and for Chapter 3 86

9 open discussion with the study subjects. Later, with the help of a clinician 3mL venous blood was collected from the each subject in EDTA vaccutainer tubes by intravenous puncture from all the three groups Extraction of genomic DNA: Genomic DNA was extracted from the peripheral blood sample using QiAmp Blood DNA mini kit (Qiagen, Germany) by employing below mentioned protocol. i. 200μL of collected blood sample to was added into a 1.5mL microcentrifuge tube containing 20μL QIAGEN Protease. ii. 200μL Buffer AL was pipetted into the sample and mixed by pulse vortexing for 15 seconds. iii. iv. The mixture was incubated in a hot water bath for 10 minutes at 56 C. Briefly, the sample tubes were centrifuged to remove a drop from the inside of the lid. v. 200μL of ethanol (96 100%) was added to the sample and mixed again by pulse vortexing for 15 seconds and centrifuged for a brief period. vi. The mixture was carefully transferred to the QIAamp Mini spin column without wetting the rim. After closing the cap, the samples were centrifuged at 8000 rpm for 1 minute. Now, by placing the QIAamp Mini spin column in a clean 2mL collection tube and the microcentrifuge tube containing the filtrate was discarded. vii. Without wetting the rim, 500μL of Buffer AW1 was added to all the tubes and centrifuged at 8000 rpm for 1 minute. Again, by positining the QIAamp Mini spin column in a clean 2mL collection tube and the collection tube containing the filtrate was removed. Chapter 3 87

10 viii. 500μL of Buffer AW2 was pipette into the tubes, without wetting the rim and centrifuged the tubes at 14,000 rpm for 3 minutes. ix. Next, by placing the QIAamp Mini spin column in a clean 1.5mL microcentrifuge tube and the filtrate containing tube was discarded. After adding 200μL of Buffer AE, the tubes were incubated at room temperature (15 C to 25 C) for 1 minute. Following, 1 minute of centrifugation at 8000 rpm and the extract was stored at -20 C deep freezer until further use Quantification of DNA by spectrophotometry: The amount of DNA extracted from the above mentioned protocol was quantified by using Nanodrop spectrophotometer (ND 2000c, Thermo Scientific, USA) and the presence of DNA was further confirmed by running an agarose gel electrophoresis. Approximately 1-2µL of the extracted DNA was used and the absorbance of the sample was read at 260nm in a spectrophotometer. For purity of the DNA, the extract was measured using ratio of absorption at 260nm:280nm and the value were expected to be Based on the obtained concentration all the DNA samples were diluted to ~ ng/µL for further downstream process Sequence Tagged Site (STS) markers and oligo synthesis: For the present analysis, five different sets of STS markers have been employed for screening of Y chromosome AZFc region. The information pertaining to the STS were collected from the National Centre for Biotechnology Information (NCBI) using UniSTS data base and also from Y chromosome consortium portal. The selected markers encompass hotspot deletion loci that include single copy and multicopy genes, which involved in spermatogenesis process. All the five set of STS oligo markers were synthesised and Chapter 3 88

11 acquired from Integrated DNA Technology (IDT, USA). Thus, obtained markers were validated using gradient polymerase chain reaction (PCR) for each set of primer at specific reaction temperature. For the details of the STS markers, deletion loci, PCR product size and Genbank accession number refer Table Y chromosome AZFc deletion analysis a. DAZ gene deletion analysis: According to the European Academy of Andrology (EAA) and European Molecular Genetics Quality Network (EMQN) (Simoni et al., 2004) guidelines, we have carried out standard PCR reactions for DAZ gene deletions. All the subjects were tested for the AZFc DAZ gene deletion using low resolution STS markers, sy254 and sy255. b. Screening of AZFc partial deletions: Using specific STS markers we have screened for the presence and absence of AZFc partial deletions in all the study subjects. The STS marker sy1291 corresponds to gr/gr ampliconic region, sy1191 correspond to b2/b3 region, sy1197 to b1/b3 region similarly, sy1291 and sy1197 indicates the b1/b3 and b2/b3 target regions (Repping et al., 2003). c. Reaction mixture and reaction conditions for analysing AZFc partial deletions: 25µL of standard PCR reaction was used to perform a gradient PCR amplification by using gradient thermal cycler (Super Cycler, Kyratec, Australia). Compositions of PCR reaction mixture are as follows: i. 12µL of PCR ready Go Taq colourless master mix (Promega, USA) ii. For sy254 marker, 0.50µL of forward and reverse primer (IDT, USA) For sy255 marker, 0.40µL of forward and reverse primer (IDT, USA) Chapter 3 89

12 For sy1291, sy1191 and sy1197 marker, 0.30µL of forward and reverse primer (IDT, USA) iii. iv. 0.30µL of bovine serum albumin solution 9.7µL of triple distilled water v. 2.0µL of template DNA The reaction conditions for the standard PCR reaction are as follows: Step i. Initial denaturation Step ii. Denaturation - 94 C for 3 minutes - 94 C for 30 seconds Step iii. Annealing for sy1291 & C for 30 seconds Annealing for sy254/sy255, sy C for 30 seconds X 30 Cycles Step iv. Extension Step v. Final extension Step vi. Hold - 72 C for 30 seconds - 72 C for 5 minutes - 10 C for infinite time For PCR amplification, the reaction from denaturation to extension step was repeated 30 times (30 cycles) before holding the reaction mixture at 10 C. d. Agarose gel electrophoresis: To prepare 2% agarose gel, 2gms of agarose (SRL, Mumbai, India) was mixed in 2mL of 40X TAE buffer (Promega, USA) and final volume was made upto 100mL using double distilled water. Following thorough mixing, the solution containing conical flask was heated in a microwave for 2-5 minutes by swirling the solution. After cooling 6µL of ethidium bromide (EtBr) (SRL, Mumbai, India) was added and mixed well. Immediately, the solution was poured inside a casting tray and allowed to solidify for 30 minutes at room temperature. Before, loading the samples in to the casted gel 2µL of gel loading dye Chapter 3 90

13 was added to all the PCR reaction mixtures. In order to estimate the PCR product size, along with the samples 100bp DNA ladder (Merck Millipore GeNeI, India) was loaded in a separate lane. Subsequently, the PCR products were separated at 85V constant voltage until the loading dye migrates three by fourth of the gel. Finally, the DNA bands were visualized and image was documented using software based gel documentation system (UVItec, Cambridge, UK). e. AZFc partial deletion analysis: For the above mentioned AZFc partial deletion screening, the analysis was performed using male positive control (M), female negative control (F) and water as a blank (B). The samples in which the deletions were observed in the first round of PCR reaction were further reconfirmed by repeating the PCR analysis at least twice. As stated before, the partial deletion of DAZ gene was screened by the absence of both sy254 & sy255 markers. Further, gr/gr partial deletion was examined by the absence of marker sy1291 and by presence of other STSs. Similarly, b2/b3 deletion are detected by the absence of sy1191, whereas the lack of sy1197 indicates only b1/b3 deletion and absence of sy1291 and sy1197 indicates b1/b3; b2/b3 deletion in the study subjects Statistical analysis: The obtained data was analyzed using the statistical software SPSS version 21. The mean ± SD was calculated for continues variables between normozoospermic controls and infertile group. The Chi-square test (Fisher Exact test) was applied between the case and control groups to calculate the significant difference in frequency. Independent t-test analysis employed to calculate Chapter 3 91

14 the significance difference between AZFc partial deletion pattern and variation in semen profile and sperm function test among case and control group. Further, odds ratio and 95% confidence of interval was calculated using Binary logistic regression analysis to calculate the risk factor. For all the statistical tests, p < 0.05 was considered as significant. 3.3 Results A total of 244 normozoospermic controls, 239 clinically proven infertile individuals and 200 Siddi tribal men with a respective mean age of 34.6±6.61 years, 34.6±5.36 years and 30.4±9.73 years are subjected to Y chromosome AZFc partial deletion analysis. As expected, the higher frequency of partial AZFc deletions are observed in infertile subjects compared to controls. 27 (11.06%) normozoospermic controls and 44 (17.81%) infertile individuals demonstrated random deletions patterns for partial AZFc deletions STS markers namely, gr/gr, b1/b3, b2/b3, b1/b3; b2/b3 and gr/gr; b2/b3 that are employed in the current study (Figure ). In this study we recorded 16 (36.3%) gr/gr, 7 (15.9%) b1/b3, 6 (13.6%) b2/b3, 5 (11.4%) b1/b3; b2/b3 and 10 (22.7%) gr/gr; b2/b3 deletions, where in the occurrence of gr/gr deletion is more frequent compared to gr/gr; b2/b3 deletion combination in infertile subjects (Figure 3.3; 3.5 and Table 3.4). On contrary, among normozoospermic controls 8 (29.6%) gr/gr, 3 (11.1%) b1/b3, 12 (44.4%) b2/b3, 1 (3.9%) b1/b3; b2/b3 and 2 (7.4%) gr/gr; b2/b3 deletions are recorded with higher occurrence of b2/b3 deletion (Figure 3.2; 3.5 and Table 3.4). Surprisingly, DAZ cluster deletion for sy254 and sy255 markers are absent among all the study subjects. Chapter 3 92

15 Interestingly, out of 200 Siddi tribal males only one individual (0.5%) exhibited the b2/b3 deletion (Figure 3.4 and 3.5), whereas rest of the subjects showed no deletions for any other markers (Table 3.4). Due to fall of short of semen sample the correlation between the observed b2/b3 deletion to variation in semen profile and sperm dysfunction if any, is not conclusive. In spite of continues attempts the tribal volunteer s denied to provide their semen samples owing to strong mythological belief and other religious reasons. Hence, as secondary approach the individual s personal information are robustly cross examined and reconfirmed the previously gathered details. Interestingly, the performa reveals that the subject is married and fathered two children, suggesting the b2/b3 deletion in this particular individual seems to have no much impact on fertility impairment. Taken together, in the absence of semen profile our data is solely interpreted based on molecular approaches. Excluding the tribal subjects the comparison of the AZFc deletion data with individual semen characteristics is carried for control and infertile cohorts. Compared to normozoospermic controls carrying AZFc partial deletions infertile individuals showed statistically significant reduction in mean values for sperm vitality, count and motility (Figure 3.6A and Table 3.5). Additionally, lower scores are observed in infertile men with AZFc deletions for sperm function assay, moreover independent t- test showed significant differences for HOS test (p = 0.001), but not for NCD and AIT (Figure 3.6B and Table 3.5). Furthermore, the affect of different types of Y chromosome AZFc partial deletions such as, gr/gr, b1/b3, b2/b3, b1/b3; b2/b3 and gr/gr; b2/b3 deletions, on semen variables and sperm dysfunction are carefully examined among control and Chapter 3 93

16 infertile individuals (Figure and Table 3.6). Interestingly, although the frequency of gr/gr deletion is higher in infertile subjects compared to control men, the association of deletion with semen variables showed insignificant results, whereas NCD and AIT tests displayed statistically significant differences (p = 0.011) (Figure 3.7 and Table 3.6), suggesting gr/gr deletion possibly has lesser impact on spermatogenic impairment and male infertility in the current study population. Similarly, the higher percentage of b2/b3 deletion among normozoospermic controls showed no effect on semen profile or sperm dysfunction, but the average values for sperm vitality (p = 0.002), count (p = 0.003) and motility (p = 0.003) are significantly reduced in infertiles (Figure 3.9 and Table 3.6). Nonetheless, the independent t-test analysis revealed that the b2/b3 deletion appears to have no influence on sperm dysfunction in our study subjects (Figure 3.9 and Table 3.6). On the other, gr/gr; b2/b3 deletion exhibited highly significant difference only for sperm count (p = ) (Figure 3.11 and Table 3.6). In addition, although the mean scores for semen profile and sperm function tests are relatively reduced in infertiles compared to WHO reference values and control group, the semen characteristics remain statistically insignificant (p > 0.05) in the other AZFc partial deletion conditions (b1/b3 and b1/b3; b2/b3 deletion) (Figure 3.8; 3.10 and Table 3.6). Thus, the gr/gr, b2/b3 and gr/gr; b2/b3 deletions showed high risk factor for spermatogenic impairment and sperm dysfunction in the current study population. However, this conclusion is further examined by employing binary logistic regression analysis to determine the risk factor via odds ratio calculation (Table 3.7). The statistical analysis revealed that the b2/b3 deletion is significantly associated with increased risk of spermatogenic impairment in infertile individuals (OR 5.06; 95% CI ) (p = 0.006) and compared to other AZFc partial deletions (gr/gr, b1/b3, b1/b3; b2/b3 and gr/gr; b2/b3) that are Chapter 3 94

17 investigated, b2/b3 deletion is a possible risk factor for male infertility in our study cohorts. Next, the AZFc partial deletions and semen variables between normozoospermic controls and different infertile sub-conditions are carefully analysed using one way ANOVA. The statistical test revealed that the sperm vitality (p = 0.002), count (p = ) and motility (p = ) values are significantly different (Figure 3.12A and Table 3.8). Meanwhile, the sperm function test showed significant values for HOS (p = 0.002) and AIT (p = 0.05), but not for NCD (Figure 3.12B and Table 3.8). To our surprise, yet again gr/gr and gr/gr; b2/b3 deletion combination was observed to be in higher percentage among different infertile sub conditions showing collective scores of 40.7% (gr/gr) and 25.9% (gr/gr; b2/b3) respectively (Figure 3.5). The binary logistic regression test showed that b2/b3 deletion followed by gr/gr deletion is risk factors for spermatogenic impairment and male infertility among different infertile sub-conditions (Table 3.9). For instance, compared to normozoospermic controls, asthenospermia (OR 4.21; 95% CI ; p = 0.03), oligoasthenospermia (OR 8.84; 95% CI ; p = 0.009), azoospermia (OR 4.21; 95% CI ; p = 0.03) and oligospermia (OR 8.84; 95% CI ; p = 0.009) with gr/gr deletion showed significant differences. Similarly, b2/b3 deletion in asthenospermia (OR 16.8; 95% CI ; p = 0.001), oligoasthenospermia (OR 34.4; 95% CI ; p = 0.001), azoospermia (OR 34.4; 95% CI ; p = 0.001) and oligospermia (OR 10.93; 95% CI ; p = 0.001) displayed significance values compared to normozoospermia controls (Table 3.9). Thus, our study suggests that b2/b3 deletion and gr/gr; b2/b3 deletion are possible high risk factors for impaired spermatogenesis and male Chapter 3 95

18 infertility. However, absence of other STS s deletions and presence of b2/b3 deletion in a Siddi tribal male has no influence on individual s fertility status. While, in the absence of semen profile and sperm function data in Siddi males, perhaps it may be difficult to draw any strong positive conclusion by possibly suggesting negative correlation of AZFc partial deletion and impairment in fertility among Siddi tribes. 3.4 Discussion In the present investigation, systematic analysis of AZFc partial deletion has been carried out in the South Indian sub-population that includes Siddi tribal population. Our study demonstrates that the higher frequency of AZFc partial deletions b2/b3 and gr/gr with b2/b3 deletion combination in the study cohorts. Additionally, the present study illustrates those men with AZFc partial deletion in infertile are highly predisposed to azoospermia or oligospermia with oligospermic sub conditions. Moreover, our study observed significant decrease in semen variants and sperm function assay with individuals carrying gr/gr, b2/b3 and gr/gr with b2/b3 deletion combination in infertiles and no such variations were observed in controls. Interestingly, in the current study we have not observed any striking correlation between AZFc partial deletions and their effect on reproductive status among Siddi tribal men as it was recorded only in single study subject. Thus, at this point, by reporting the negative co-relationship of AZFc partial deletion and fertility impairment, the current study has made an attempt and significant contribution in understanding the frequency of AZFc deletion and its non-deleterious effect on fertility among Siddi men. Chapter 3 96

19 In this perspective, it is noteworthy to mention that most accounted studies in Indian tribal population are predominantly restricted to reproductive health care and fertility related issues in women, however male-specific reproductive literature are very limited may be due to various unapproachable reasons. For example, effects of inbreeding on fertility and sterility among urban and rural population of South India has been studied by assessing the type of consanguineous marriage, marital age, duration of marriage, number of pregnancies, live births and child mortality (Rao and Inbaraj, 1979). Kumar et al., (2006) conducted a factorial experiment to determine the outcome of sex and survivorship to fertility in Kamara tribes from Chhattisgarh. To quote an another example, age of menarche, marriage and menopause along with nutritional status of mother during prenatal and post pregnancy periods are well documented in Dhur and Gond tribal women of Chhattisgarh (Chandraker et al., 2009) Taken together, to best of our knowledge for the first time in South India, the current study systematically screened 200 Siddi tribal men and analysed their genomic samples for the presence or absence of Y chromosome AZFc partial deletions. Finally, we demonstrate that these deletions also occur naturally in Siddi tribal men with comparatively in very lower frequency (0.50%) with no functional or deleterious impact on their reproductive fitness. The observation that the low frequency occurrence of AZFc subdeletion, which has no impact on fertility in Siddi tribal men is in partial agreement with AZFc deletion studies carried out in an African population. Total of 49 infertile individuals comprise of 21 non-obstructive azoospermics and 28 oligospermics conditions are screened for Y chromosome microdeletions in AZFa, AZFb and AZFc regions (Kihaile et al., 2005). Surprisingly, the absence of deletion in entire AZF region Chapter 3 97

20 among all the study subjects suggests that the observed infertility condition may possibly arise from deletion in other unknown subregions of Y chromosome or may be due to secondary sexual infections. Compared to non-african countries, 50% of African couples display secondary infertility due to sexual transmitted diseases (STD) (Cates et al., 1985). In addition, cultural practices such as polygamy, hinders the analysis of precise frequency of infertility in African population (Ness et al., 1997). In general, the tribal communities are known for sexual practices that vary from main stream population, however in the Siddi tribes polygamy is not in practice and there are no reports of STD. Nevertheless, it is too early to draw any robust conclusions regarding the AZFc deletion analysis in Siddi tribe considering the sample size, employed STS s markers and population with geographic variation. Further, this investigation necessitates a comprehensive analysis of AZFc deletion screening with gene copy dosage detection in large and well selected different tribal groups in India to unmask the genetic mechanism behind the association. Meanwhile, the observed low frequency AZFc partial deletions (0.50%) matches to previous studies that accounts for 0% 5.93% of b2/b3 deletion in Siddi tribal men with no association between this deletion on fertility (Lu et al., 2009, 2011; Eloualid et al., 2012). As stated earlier, the current study is mainly focused on the Y chromosome AZFc partial deletion types, which includes gr/gr, b1/b3 and b2/b3 deletions that reduces the overall dosage copy between Mb of the AZFc genes (DAZ, CDY and BPY copies) and several global studies also suggests that these deletions, may results in male infertility that vary according to ethnicity and geographic location. Therefore, we have evaluated the correlation between the AZFc partial deletion and its association on spermatogenic impairment and male infertility in South and West Chapter 3 98

21 Karnataka population of South India. In an unexpected observation all our study subjects (n=683, Siddi tribal, controls and infertiles) demonstrated the absence of deletion for DAZ gene (sy254 and sy255) cluster that encodes RNA-binding protein, which is exclusively expressed in the germ cells (Kleiman et al., 2007). The deletion of each member of DAZ gene family (DAZ1, DAZ2, DAZ3, and DAZ4) is shown to have differential effect on male fertility and recent studies have reported various DAZ deletions in fertile men. For example, expression of DAZ1 seems to be essential for spermatogenesis, but these deletions are also known to occur in fertile individuals. Similarly, deletion of DAZ2, DAZ3, and DAZ4 gene copies are found both in fertile as well as in infertile men and are described as familial variants (Fernandes et al., 2004). Thus, the requirement of DAZ gene cluster for normal reproductive function and impact of deletion on fertility impairment across different ethnic groups remained as a major unsolved problem in human reproductive genetics. In agreement with previous studies, the gr/gr deletion is found to be most prevalent in infertile subjects (16/44, 36.3%) (Giachini et al., 2005; Navarro-Costa et al., 2010; Stouffs et al., 2011). Furthermore, screening of 12,000 patients and controls for the presence of gr/gr partial deletion demonstrate that this deletion occur more recurrently in infertile men than fertile men, but prevalence may vary widely among fertile individuals. These gr/gr deletions are also detected in normozoospermic controls, who have fathered at least one child without any medical aid. Therefore, having gr/gr partial deletion does not necessarily result in having fertility issues. However, the couples anticipating to have a child should be intimated about the risk factor, as no prediction can be made for their sons who can have the same deletion as their fathers (Stouffs et al., 2011). Our study analyses among controls showed 29.6% Chapter 3 99

22 of gr/gr deletion (8/27), which are comparatively lesser than infertile subjects. Similar to DAZ deletion, the frequency of gr/gr deletion and its impact on fertility varies from one ethnic group to another. For example, 8% to 10% of deletions are observed in East Asian men with spermatogenic failure suggesting an increased risk of infertility (Zhang et al.,2006; Wu et al., 2007), whereas, 1.4% of normozoospermic Chinese men with this deletion are normal for fertilization (Navarro-Costa et al., 2010). The incidence of gr/gr partial deletion observed in our study group is lower than the reports from West coast of India (Sen et al., 2015) and is on higher side compared with the populations of North India, South India, Han-Chinese, USA and European populations (Repping et al., 2003; Machev et al., 2004; Hucklenbroich et al., 2005; Wu et al., 2007; Shahid et al., 2011; Vijesh et al., 2015). However, the observed wide variations in the frequency of gr/gr partial deletion in our study may be due to genetic background of the study participants. Moreover, a few global studies have illustrated that gr/gr is more common in infertile men with azoospermia or oligospermia suggesting that gr/gr deletion may be a significant genetic risk factor for spermatogenesis and male infertility (Giachini et al., 2005; Lynch et al., 2005; Yang et al., 2008; Shahid et al., 2011). Meanwhile, other reports have failed to show any genotypic and phenotypic association of gr/gr deletion to the spermatogenic impairment and male infertility in which the gr/gr deletion are fixed on haplogroups D2b and Q1, which are prominent in Japan and China (Repping et al., 2003; Lu et al., 2009 and Yang et al., 2010). Surprisingly, data from our study cohort indicate nonsignificant differences for the gr/gr deletion frequency between infertiles and controls that are studied for failure of spermatogenic process. However, sperm function assay showed positive association for NCD and AIT (Figure 3.7 and Table 3.6). Our observation further supports the previous studies, which demonstrated negative Chapter 3 100

23 correlation between the gr/gr deletion and failure of spermatogenesis (Zhang et al., 2006; Wu et al., 2007; Stahl et al., 2011). On the contrary, odds ratio analysis between different infertile sub-conditions such as, azoospermia, oligospermia, asthenospermia and oligoasthenospermia exhibited gr/gr deletion as a risk factor for spermatogenic impairment. Furthermore, the b2/b3 deletion occurred as a result of gr/gr or b2/b3 inversion. In the light of previous studies, compared to gr/gr deletion b2/b3 deletion is considered as a potential genetic risk factor for spermatogenic impairment and male infertility in different ethnic populations. On the other, b2/b3 deletion increases the risk of complete AZFc (b2/b4) deletion and this predisposition may be due to the size and distance of the recombination substrate (Lu et al., 2009). Besides, the b1/b3 and b2/b3 deletions are rare and literature is very limited in Indian population (Shahid et al., 2011). However, the higher frequency of gr/gr but not b1/b3 and b2/b3 deletions are reported in Indian and Chinese population (Lu et al., 2009; Shahid et al., 2011; Sen et al., 2015). Interestingly, in current our study 36.3% of gr/gr deletion and 44.4% of b2/b3 deletion are respectively recorded in infertile and control individuals. Given that, the role of b1/b3 and b2/b3 deletion in male infertility is still unclear, the b1/b3 deletion appears to be associated with male infertility as Shahid et al., studies found a significant higher number of infertile subjects with b1/b3 deletion (Shahid et al., 2011; Sen et al., 2015). In contrast, we recorded lower incidence of b1/b3 (infertile - 7/44, 15.9%; controls -3/27, 11.1%) and b1/b3; b2/b3 (infertile - 5/44, 11.3%; controls - 1/27, 3.70%)) deletions in total AZFc partial deletion conditions in our study subjects (Figure 3.5 and Table 3.7). Chapter 3 101

24 Previously, Repping et al., (2004) and Lu et al., (2009) demonstrated that the b2/b3 partial deletion removes 1.8Mb of DNA segment and Northern European as well as Chinese population with a haplogroup N1 and Q1 are fixed for this specific deletions. Whereas, the other studies also screened for the presence of b2/b3 deletion in different Y haplogroups other than the N1 haplogroup and reported that the b2/b3 partial deletion seems to have no significant impact on spermatogenic failure and male infertility (Imken et al., 2007; Zhang et al., 2007; Shahid et al., 2011). In contrast to this observation, a significant correlation between the b2/b3 partial deletion and spermatogenic failure are observed in the Moroccan and Han-Chinese populations (Wu et al., 2007; Lu et al., 2009; Eloualid et al., 2012). Till this date, there is no agreement in findings regarding the etiology of b2/b3 deletion. In the present analysis, we found 6 infertiles with b2/b3 deletion with a frequency of 13.2% corresponding to azoospermia (one case), oligoasthenospermia (one case), oligospermia (two cases) and asthenospermia (two cases). On contrary, 12 (44.4%) of the control samples also showed b2/b3 deletion surprisingly with no affect on spermatogenic impairment and male infertility. Among infertile men with b2/b3 deleted genotype, our study records significantly reduced sperm vitality, count and motility compared to control group (Figure 3.9 and Table 3.6) and a strong association between b2/b3 deletion and spermatogenic failure has been observed in our study cohort. The observed frequency of b2/b3 subdeletion in the present study (infertile, 13.6% and 44.4%, controls) is relatively higher in comparison to North Indian (1.44%), West Indian (11.1%) and South Indian population (7.21%) as well as Han-Chinese (9.2%), Moroccan (1.34%) and Italian (0.5%) population (Imken et al., 2007; Wu et al., 2007; Giachini et al., 2008; Shahid et al.,2011; Vijesh et al., 2015; Sen et al., 2015). In addition, the frequency of b2/b3 partial deletion is found to be Chapter 3 102

25 higher in different infertile sub- conditions. The current findings supports the hypothesis that the b2/b3 deletion increases the risk of complete AZFc deletion, thereby leads to severe spermatogenic impairment resulting in male infertility (Lu et al., 2009). Finally, the admixture South Indian sub-population is divergent from the rest of the Indian population and may have precise pattern of Y haplogroups, which is still not known. Moreover, the presence of haplogroup N1 is susceptible to partial AZFc deletions and it has been traced in the South Indian population with an estimated frequency range of 5% to 42% (Suhasini et al., 2011). In our study, the frequency of b2/b3 deletion is relatively higher compared to previous studies (Repping et al.,2004; Eloualid et al., 2012; Rozen et al., 2012; Vijesh et al., 2015; Sen et al., 2015). Such wide variation in the frequency and phenotype is not exactly known or may be due to several reasons such as, experimental design, strict selection criteria, impact of environmental and genetic factors, and geographic background of the study population and study area. Chapter 3 103

26 Figure 3.1: Map of the AZFc region. The AZFc subdeletions pattern in Y chromosome: illustrating the palindromes and amplicons in the AZFc region. Locations of different STS markers that are employed to screen the subdeletions are indicated below the ampliconic bar, where the colour boxes depict the protein encoding genes in the AZFc region (gene names are presented inside the colour box). The AZFc subdeletion patterns due to homologous recombination between the amplicons indicates the b1/b3 deletion, b12b3 subdeletion and gr/gr deletion with g1/g2, r1/r3 and r2/r4 that removes different set of genes are indicated with open bar. Chapter 3 104

27 Figure 3.2: Agarose gel showing amplified PCR products for different STS markers that are used to screen the Y chromosome AZFc subdeletions in normozoospermic control men. White arrow in indicates the deletion of sy1291, sy1191 and sy1197 markers in respective sample lane. Lane 1 represents 100bp marker. M, F and B corresponds to male control genomic DNA, female genomic DNA and water blank respectively Chapter 3 105

28 Figure 3.3: Agarose gel showing amplified PCR products for different STS markers that are used to screen the Y chromosome AZFc subdeletions in infertile males. White arrow in indicates the deletion of sy1291, sy1191 and sy1197 markers in respective sample lane. Lane 1 represents 100bp marker. M, F and B corresponds to male control genomic DNA, female genomic DNA and water blank respectively. Chapter 3 106

29 Figure 3.4: Agarose gel showing amplified PCR products for different STS markers that are used to screen the Y chromosome AZFc subdeletions in Siddi tribal men. Red arrow in (D) indicates the deletion of sy1191 marker in sample number T92. Lane 1 represents 100bp marker. M, F and B corresponds to male control genomic DNA, female genomic DNA and water blank respectively. Chapter 3 107

30 Figure 3.5: The prevalence of different types of Y chromosome AZFc partial deletions in Siddi tribes, normozoospermic controls and infertile males. Figure 3.6: Comparison of mean scores for (A) semen variables and (B) sperm function assay in control and infertile group with Y chromosome AZFc partial deletions. Error bars indicate mean ± SE. Chapter 3 108

31 Figure 3.7: Histogram shows the mean scores for (A) semen variables and (B) sperm function assay in control and infertile group exhibiting AZFc gr/gr partial deletion. Error bars indicate mean ± SE. Figure 3.8: Illustration of mean values for (A) semen variables and (B) sperm function assay in control and infertile group with AZFc b1/b3 partial deletion. Error bars indicate mean ± SE. Chapter 3 109

32 Figure 3.9: Histogram shows the mean scores for (A) semen variables and (B) sperm function assay in control and infertile group showing AZFc b2/b3 partial deletion. Error bars indicate mean ± SE. Figure 3.10: Illustration of mean values for (A) semen variables and (B) sperm function assay in control and infertile group with AZFc b1/b3; b2/b3 partial deletion. Error bars indicate mean ± SE. Chapter 3 110

33 Figure 3.11: Histogram shows the mean scores for (A) semen variables and (B) sperm function assay in control and infertile group showing AZFc gr/gr; b2/b3 partial deletion. Error bars indicate mean ± SE. Chapter 3 111

34 Figure 3.12: Representation of mean values for (A) semen variables and (B) sperm function assay in control and different infertile sub-conditions with AZFc partial deletion. Error bars indicate mean ± SE. Chapter 3 112

35 Table 3.1: Frequency of Y chromosome AZF and AZFc deletions and STS markers employed for the study reported by different authors from North India Author Year Study area Total number of cases Total AZF deletions Total AZFc deleted number Frequency Thangaraj K et al., 2003 Kolkatta, WB % Dada R et al., 2003 New Delhi % sy254, sy255 Ambasudhan et al., 2003 Varanasi, UP % sy254, sy255 Dada R et al., 2004 New Delhi % sy254, sy255 Mittal R. D et al., 2004 Lucknow, UP % sy254, sy255 Dada R et al., 2007 New Delhi % sy254, sy255 Dada R et al., 2007 New Delhi % sy254, sy255 Sachdeva K et al., 2011 New Delhi % Mahantha et al., 2011 Assam % sy254, BPY2 Shahid et al., 2011 New Delhi 418 N/A % Shamsi et al., 2012 New Delhi % sy254, sy255 STS Markers Employed sy152, sy148, sy156, sy255, sy581, sy254, sy247, and sy158 sy254, sy 255, sy146, 148, 149, 153, 156, 158, 243, 269 sy142, sy1258, sy1161, sy1197, sy1206, sy1201,sy1291, sy1191 Sen S et al., 2013 New Delhi % sy254, sy255,sy145,sy160 Chapter 3 113

36 Table 3.2: Y chromosome AZF and AZFc deletions frequency and STS markers employed for the study reported by different authors from South India Total Total Total Author Year Study number AZF AZFc STS Markers Employed area of cases deletions deletions Frequency Arundathi et al., 2004 Mumbai % sy 157(DYS240) Laksmi Rao et al., 2004 Hyderabad 251 Nil % sy153, sy205, sy232, sy254, sy255, sy277, sy283, sy624 Swarna et al., 2004 Hyderabad % sy 254, sy158 Nagavenkar et al., 2005 Mumbai 88 nil % sy 254 sy 255 Viswambhran 2007 Coimbatore % sy1291, sy1161, sy1191, sy1206,sy1201,sy1197,sy1258,s Y277, sy254, sy283,sy255 Poongothai et al., 2008 Coimbatore % sy 254, BPY2 Abhilash et al., 2010 Vellore % sy 152, sy146, sy156, sy254, sy255,sy158 Sunganthi et al., 2013 Coimbatore % sy158, sy160, sy240, sy254, sy255, sy277 and CDY Vijayalakshmi et al., 2013 Chennai % sy158, sy283, sy254, CDY Prafulla et al., 2014 Wardha sy 254 sy 255 Chapter 3 114