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Screening for Y-chromosome microdeletions in infertile Indian males: Utility of simplified multiplex PCR.

Background & objectives: Analysis of the microdeletions in the azoospermia factor (AZF) region of Y chromosome by PCR is an important screening tool in the work-up of infertile males opting for assisted reproductive techniques. In the present study, the Y chromosome microdeletions were analyzed by PCR using primers corresponding to 16 sequence tagged sites (STS) and three genes of the AZF region in infertile Indian men. Feasibility of developing a simplified multiplex PCR for screening of the Y chromosome microdeletions has been explored.

Methods: A total of 271 male subjects were analyzed, of which, 170 were infertile patients (51 oligospermic and 119 azoospermic) and 101 were fertile controls. Subjects showing normal karyotype only were included in the study. The semen analysis was done and plasma follicle stimulating hormone (FSH) concentrations were determined by radioimmunoassay. Testicular histopathology was analyzed by fine needle aspiration cytology (FNAC).

Results: Y chromosome microdeletions were observed in nine out of 170 (5.29%) infertile males all of whom were azoospermic. Of the nine subjects, two had deletions in AZFa, one in AZFb, three in AZFc and three in AZFb+c regions. No deletions were observed in the infertile severe oligospermic men (< 5 million sperm/ml semen) and fertile controls. No difference in the FSH concentrations of infertile patients with and without deletions (18.36 and 18.10 mlU/ml respectively) was observed. A clear relationship between Y chromosome microdeletions and testicular phenotypes could not be established. Two multiplex PCRs were designed using 7 STSs markers, which could detect Y chromosome microdeletions in infertile male subjects as efficiently as PCR based on larger number of PCR reactions.

Interpretation & conclusions: The multiplex PCRs described in the present study may be a suitable, cost-effective and less time consuming method for screening the Y chromosome deletions in infertile males in routine clinical diagnosis and counselling prior to assisted reproduction.

Key words AZF--FNAC--FSH--multiplex polymerase chain reaction--testicular phenotype--Y chromosome microdeletions

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Infertility is a major health problem affecting 1015 per cent of married couples and a male factor can be identified in half of these cases (1). Spermatogenesis may be affected by systemic disease, cryptorchidsm, endocrinological disorders, obstruction in seminal pathways, and infection or immunological factors. However, the cause of male infertility is unknown in up to 50 per cent of cases (1). In these cases, genetic aetiology may be associated with abnormal spermatogenesis. A genetic factor located at Yq11 has been established to be important for male germ cell development and the gene cluster is referred to as azoospermia factor (AZF)(2). In the AZF region, three loci termed as AZFa, AZFb and AZFc have been identified. In the Y chromosome, 300 sequence tagged sites (STS) have been generated and mapped for the above three AZF regions (1). STS are known sequences of genomic DNA that can be amplified by PCR. The role of Y chromosome microdeletions in male infertility has been well established (3-6). The frequency of reported Y chromosome microdeletions varies from 1 per cent in a cohort of men undergoing intracytoplasmic sperm injection (ICSI) for varying reasons (3) to 55 per cent in men with Sertoli cell only (SCO) syndrome (4). The variations among populations, the method of designing a particular study and the validation of real deletions may explain this wide range of variability. The recommendations of European Academy of Andrology (EAA) guidelines (7), suggest that over 90 per cent microdeletions can be detected by the use of 2 STS markers for each AZF loci. A study of Y chromosome deletions in 340 azoospermic Indian men employing 30 STS markers revealed an overall deletion of 8.5 per cent (6). Importantly, this study failed to find any deletion in the sY86 and sY84 STSs as recommended by EAA (7) for analysis. On the other hand, the deleted STSs were sY746, sY741, sY742, sY615 and the gene DFFRY; which are inter-spread between or around sY86 and sY84. The authors thus suggested that the chances of frequenting a deletion are more, if more sets of primers are used. These findings raise questions about the validity of the primer sets recommended in the EAA guidelines (7) in all racial populations.

The phenotypes associated with deletions in the different AZF regions are variable (8,9). Complete deletions of AZFb and AZFb+c are characterized by a histological picture of Sertoli cell only (SCO) or spermatogenic arrest resulting in azoospermia (9) and deletions in AZFa region have been associated with SCO I (complete absence of germ cells in the seminiferous tubules and presence of Sertoli cells only) syndrome (10,11). However, deletion of only AZFc region is found to be associated with a wide range of phenotypes ranging from hypospermatogenesis to SCO II (isolated foci of spermatogenesis along with Sertoli cells) syndrome (9). A study by Machev et a1 (12) represents the evidence that even partial AZFc deletion or gene conversion may frequently be the basis for spermatogenic failure. A plausible explanation for the variable phenotype is progressive regression of germinal epithelium over time which has been reported in patients with AZFc deletions (13). Among the three AZF regions, deletion of AZFc is more frequent, followed by AZFb and AZFa (14). This lack of accurate association of genotype with testicular histology has resulted in the absence of any clear clinical parameters that can be used to predict the presence of microdeletions in a given infertile male (15).

The ability to extract spermatozoa from the testes of men with non obstructive azoospermia followed by intracytoplasmic sperm injection (ICSI) offers this infertile population an efficacious therapeutic approach (16). These men, some of whom may harbor Y chromosome microdeletions, can contribute to a pregnancy and may transmit their genetic defect to a male offspring (17). Since the presence of microdeletions cannot be predicted on the basis of currently used routine investigations, it becomes imperative that all such men undergo screening for the presence of deletions not just for diagnosing the aetiology of their infertility but also for counselling prior to the use of assisted reproduction techniques (ART). The major deterrents to this universal screening are the cost and time involved in performing extensive polymerase chain reaction (PCR) procedure. In order to develop simplified techniques for screening, we analyzed the incidence of Y chromosome microdeletions among the Indian population using 16 different STS and three genes by PCR. We subsequently aimed to identify common deleted STS among all patients and regions in order to design and test simplified multiplex PCRs which would be more cost effective as primary screening tools.

Material & Methods

Idiopathic infertile male subjects with primary infertility attending the Infertility Clinic in the Urology Department of the All India Institute of Medical Sciences, New Delhi, were enrolled for the present study. All experiments using human samples were carried out with informed consent following clearance from the Institutional Bio-safety & Ethical Committee. Based on cytogenetic analysis, a total of 271 cases having a normal karyotype (46,XY) were included in this study, of which 170 were infertile patients and 101 were fertile males (with a child of less than 2 yr; sperm count > 20 million/ml). Karyotype analysis was performed for the fertile controls as well as the infertile cases. Idiopathic infertile cases with non obstructive azoospermia or oligospermia (counts <20 million) (18) were enrolled in the present study over a period of three years, i.e., from April 2003 to June 2006. Each patient was carefully examined to rule out known causes of infertility and a detailed family, occupational and reproductive history was collected in a pre-designed proforma. Specific inclusion criteria were: infertility, abnormal semen parameters (azoospermia or oligospermia) on at least two occasions, absence of normal spermatogenesis on fine needle aspiration cytology (FNAC) of testis or bilaterally small testes with follicle stimulating hormone (FSH) more than 3 times normal for azoospermic men and absence of varicocele in oligospermic men. Specific exclusion criteria included cytogenetic abnormalities, pyospermia, chemotherapy or radiotherapy, recent febrile illness, recent androgen administration, absence of vas deferens, hypogonadism or low volume ejaculate. The semen analysis was done according to the WHO guidelines (18). The plasma values of FSH were determined by radioimmunoassay using a commercial kit (FSH IRMA Kit, Immunotech-A Beckman Coulter Company, Prague, Czech Republic).

Fine needle aspiration cytology (FNAC): Whenever possible, testicular FNAC was performed in infertile cases using 23G (0.6 mm) butterfly needle and a 10 ml syringe by making multiple passes through skin puncture. The aspirate was confirmed to be adequate when at least 2000 cells or 100 clusters of 20 cells each were obtained. Air-dried slides were stained with the May-Grunwald-Giemsa stain (BDH, Merck Limited, Mumbai, India) and reported as normal spermatogenesis, hypospermatogenesis, maturation arrest or only Sertoli cells seen (19).

Karyotyping: Peripheral blood cultures were set up for chromosomal analysis in all the cases. The G-bands were obtained by standard trypsin technique (20) and twenty well spread G-banded metaphases were karyotyped using the automated karyotyping software (Cytovision 2.81, Applied Imaging Corp., Sanjose, CA, USA).

Analysis of Y-chromosome microdeletions by polymerase chain reaction (PCR): The genomic DNA from the infertile as well as normal subjects, were isolated by Genomic DNA Isolation Kit (Promega, USA) using the manufacturer's protocol. Thereafter, the concentration of the isolated genomic DNA was determined by spectrophotometric (Pharmacia Biotech, Uppsala, Sweden) analysis at 260 nm and the quality of the DNA was checked on agarose gel (0.9%).

All the DNA samples were processed for Yq microdeletions analysis using PCR. Each of these samples was analyzed using 19 sets of primers (custom made either by Microsynth GmbH, Hilden, Germany or Sigma Genosys Ltd., Bangalore, India).

The STS and genes used were as follows:

AZFa: sY746, sY84, sY86, DFFRY

AZFb: XKRY, sYll8, sYll3, sY127, sY134, sY143, RBM1Y

AZFc: sY153, sY148, sY157, sY158, sY254, sY255, sY 160 (heterochromatin region)

Sex determining region of Y (SRY i.e., STS sY14) was included as internal control. Detailed sequences of the primers are given in our previous study (21). The DNA from a fertile male subject was taken as positive control, the DNA from a female subject as negative control and water was used as blank in each reaction.

Initially, the Y-chromosome microdeletions analyses were performed employing four multiplex PCRs using 2 sets of primers each and eleven simple PCRs using 1 set of primers for each sample in a thermocycler (MJ Research, GMI Inc., Minnesota, USA). The multiplex PCRs used were (i) SRY and XKRY; (ii) sY86 and sY127; (iii) sY158 and sY254 at 57[degrees]C, and (iv) sY134 and sY153 at 52[degrees]C annealing temperature. The rest 11 primers were subjected to PCR amplification using initial denaturation for 5 min at 94[degrees]C and 35 cycles of 94[degrees]C for 1 min, annealing temperature varying from 50-64[degrees]C for 1 min and extension at 72[degrees]C for 1 min. The final extension time was 7 min at 72[degrees]C. Except for the annealing temperature the rest of the parameters in the multiplex PCRs were similar to the above conditions. The PCR reaction was performed in a reaction volume of 50 [micro]l consisting of 50 ng genomic DNA, 20 pM of forward and reverse primers each, 10 mM dNTPs and 25 mM Mg[Cl.sub.2] (Biotools, Madrid, Spain). The Taq polymerase (Biotools, Madrid, Spain) 1 U per tube was used in buffer (50 mM KC1, 10 mM Tris-HC1 pH 9.0, 0.1% Triton X-100) provided by the manufacturer.

All the PCR products were analyzed on a 2 per cent agarose gel containing ethidium bromide (0.5 [micro]g/ml).

If a STS or gene failed to amplify it was repeated in single PCR at least three times in the presence of internal control (SRY) as well as positive control. Moreover, the deletions with respect to the various STS markers were reconfirmed by performing temperature gradient PCR in absence or presence of 5 per cent dimethyl sulphoxide (DMSO; Sigma Chemical Co., St. Louis, MO, USA).

Multiplex PCR: Based on our findings of Y chromosome microdeletion through PCR with 16 STSs and 3 genes, simple multiplex PCRs spanning the AZFa, AZFb and AZFc regions were designed which can be performed by employing two tubes. The tube 1 comprised SRY, sY86, sY127 and sY254 STS markers, corresponding to SRY, AZFa, AZFb and AZFc regions respectively. Similarly, tube 2 comprised SRY, sY84, sY134 and sY153 STS markers, corresponding to SRY, AZFa, AZFb and AZFc regions respectively.

The genomic DNA samples from fertile and infertile males were given new code numbers and evaluated blindly by another person using the above multiplex PCR. The conditions of multiplex PCRs were same as previously described in general PCR, except that the dNTPs were used at 20 mM, instead of 10 mM and genomic DNA 500 ng instead of 50 ng. The annealing temperature of both the tubes was 57[degrees]C.

Results

The study included a total of 271 subjects, among these 170 were patients presented with primary infertility and 101 were healthy fertile controls. Of these 170 patients, 119 cases were azoospermic and 51 cases had severe oligozoospermia (< 5 million sperm/ml). All azoospermic patients were confirmed to have a non obstructive aetiology based on standard clinical evaluation. This included the finding of bilaterally small (< 5 ml) or cryptorchid testes with FSH >3x normal or FNAC showing testicular failure. The mean age of the fertile men was 24.35 yr whereas the mean age of the infertile patients was 29.12 yr (range 21-32 yr).

All subjects had a normal 46,XY karyotype. However, two azoospermic patients showed a very short long arm of the Y chromosome in all the metaphases analyzed for karyotyping (Fig. 1). These were not due to Y chromosome polymorphism as these two cases harboured a major Y chromosome deletion.

Nine of the 170 (5.29%) infertile men had a Y chromosome microdeletion (Table I). The mean age of patients with Y chromosome deletions was 27.78 yr (Table I). All Y chromosome deletions were present in azoospermic men and none were observed in the 51 oligospermic cases and the 101 fertile men. Of the nine azoospermic subjects harbouring Y chromosome microdeletions, two had deletions in AZFa, one in AZFb (Fig. 2), three in AZFc and three in AZFb+c regions. Of the three patients having AZFb+c deletions, two had deletions extended up to the heterochromatin region (sY160). One of these two had deletions starting from the sY113 STS, whereas the other had even longer deletion starting from the XKRYgene of the AZFb region (Fig. 2 & 3).

[FIGURE 1 OMITTED]

The FSH concentrations of the infertile subjects were in the range of 6.80-169.97 mIU/ml, while in the fertile men the FSH concentrations were in the range of 2-12 mIU/ml (data not shown). Among the infertile men, there was no difference in the FSH levels of men with or without Y chromosome microdeletions (18.36 and 18.10 mIU/ml respectively).

FNAC findings were available from all infertile men with the Y chromosome microdeletion. Testicular phenotype in these subjects ranged from maturation arrest to SCO I and SCO II syndrome (Table I). Similar variability in testicular histology was found among men without a deletion and no correlation could be established between the region of AZF deletions and testicular phenotypes (Table II).

According to our findings, of the four primers used for detecting deletion in the AZFa locus, STS sY86 was common in all cases. Among the seven primers used for detecting the deletions in the AZFb region, sY113, sYll8, sY127, sY134 and sY143 were common in all AZFb deletions. Similarly, of the seven primers used for detecting deletions in the AZFc region, sY153, sY158, sY254 and sY254 were common in all the AZFc as well as AZFb+c deleted subjects (Table I). After careful scrutiny of all the deletions, it was found that the deletions pertaining to the different AZF regions could be screened through simple multiplex PCRs in which 7 sets of primers were used. Using these multiplex PCRs, when Y chromosome microdeletion analysis was done blindly by another person, the same deletions of the particular AZF region were found (Fig. 4).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Discussion

Molecular genetic techniques have unveiled a number of aetiopathogenetic factors, including microdeletions of the long arm of the Y chromosome (Yq) associated with male infertility. Y chromosome deletions limited to intervals 6D-6F (the AZFc region) are generally associated with finding of some spermatozoa during testicular sperm extraction (TESE), while larger Y chromosome defects (extending more proximally and/or more distally) were associated with no spermatozoa (22). It has been reported that AZFc microdeletion constitute a progressive and deteriorating effect on spermatogenesis leading to a steady and methodical decline in spermatogenesis and deterioration in sperm quality (7, 13). But, a clinical study of azoospermic and oligospermic men, did not agree to this concept as they found that the AZFc deleted men had their baseline sperm production potential stable over time (23). All men with AZFa, AZFb, and AZFb+c Yq deletions were found to be azoospermic, while 62 per cent of men with isolated AZFc deletion were also azoospermic in a study conducted for the detection of sperm in men with Y chromosome microdeletions (24). This agrees to our study where the deletions of AZFa, AZFb, AZFb+c and all the AZFc deletions were found only in azoospermic males.

Although the Yq heterochromatic region predominantly consists of DYZ1 and DYZ2 classes of repeat sequences (25), a few studies have shown its association with infertility and reproductive failure. The deletions extending up to the heterochromatin region make the genome unstable, making it more prone to secondary deletions and this condition is promutagenic. In some earlier studies, subjects with complete AZFc deletions, sometimes extending to the heterochromatin region (sY159; sY160) have been reported (5). Though, our study results were in agreement (two patients with AZFb+c deletions had their deletions extending up to the heterochromatin region) with these findings, the severity of the deletion of heterochromatin region on spermatogenesis is not clear and needs further studies in larger population before concluding on its role during spermatogenesis. The highest per cent deletion in our study was found to be in the AZFc region, followed by AZFb and finally AZFa, which is consistent with some previous findings (1,3).

[FIGURE 4 OMITTED]

The FSH levels in AZF deleted males seem to vary in different studies. In our study FSH concentration in infertile men with or without deletions were not different. Similarly, significant differences in the mean FSH levels were not found in some of the studies (5,23) in infertile men with or without deletions in AZFc region. Thus, the FSH concentration is a good parameter for evaluation of testicular function but does not help differentiate cases with or without Yq microdeletions.

It is likely that the testicular phenotype may vary with the duration of the pathological lesion i.e., a progressive reduction of germ cells may occur with time (26), which is in agreement with our findings where a case with only AZFc deletions showed a phenotype of SCO II, which might have progressed over a period leading to its severity (age 32 yr). An alternative explanation for the variable phenotype may be that it is probably related to the influences of genetic background and environmental factors in different individuals (26). In our study, deletion of only AZFb region resulted in maturation arrest and men having AZFa or AZFb+c deletions had a much more severe phenotype like SCO I and SCO II. According to some studies (7,24), although it is difficult to establish precise genotype/phenotype correlations in patients with Y chromosomal microdeletions, deletion of AZFa/b and deletions of more than one region (AZFb+c or AZFa+b+c) have more severe deleterious effects on spermatogenesis than deletions of only AZFc. We had similar findings with AZFb+c and AZFa deletions having more severe effects on spermatogenesis than with only AZFc deletions. In a study (27), the patients having AZFc deletions showed severe phenotype of SCO I and maturation arrest, whereas, those having only AZFb deletions had less severe phenotype of maturation arrest and hypospermatogenesis in contrast to earlier studies (7) and the patients having AZFb+c deletions also showed severe phenotype like SCO I. In patients without Y chromosome microdeletions, the phenotype also ranged from maturation arrest to SCO I. Though some recent studies show that complete deletions of AZFb and AZFc may affect meiosis (28,29), deciphering an accurate relationship between all the AZF deletions and testicular phenotype is still awaited.

The frequent presence of Y chromosome deletions in infertile men and its potential transmission to future offspring makes screening prior to ICSI an important part of the work-up of the infertile male. However, the principal hurdles in its more widespread use are a lack of standardization of primers to be used, cost and technical availability. Our study aimed at finding solutions to these problems by first identifyirtg the deletions in infertile Indian men, identifying common STS markers and finally designing and testing multiplex PCRs to simplify the procedure. This became important since the previous study from this racial population (6) refuted the findings of existing guidelines for the use of only six primers suggested by EAA (7). While the study in southern India (6) found seven males with deletions in the AZFa region; surprisingly none had a deletion that would have been detected by the sY84 or sY86 markers recommended by EAA (7). On the contrary, our findings of two males with AZFa deletions, both with sY86 deletions, support the findings of Simoni et al (7). None of these two men had a deletion in the sY746 primer region in total contrast to the study in south India (6). To rule out that the non-amplification of sY86 in these two subjects was not due to mutations, repeat PCR employing additional set of adjacent primers also failed to PCR amplify sY86 STS. The two subjects belonged to different ethnic groups and had fertile siblings (brothers) borne after the patients in question. Hence, sY86 deletion in these two men was de novo and less likely to be from a common Y chromosome lineage. Although, it is difficult to ascribe the reasons for the discrepancy between these two studies from India, it may probably be due to ethnic variations in two populations.

Our multiplex PCRs were very similar to those recommended by EAA (7). Our reactions differed in only the sY153 primer which we used because the primer, sY255 used by EAA was from the same DAZ region as was sY254 and they are almost adjacent to each other and deletions in interval 6 of Y chromosome (AZFc), associated with azoospermia, are outside the DAZ gene (30). We invariably found men with sY254 deletions also having sY255 deletions. Hence, we chose sY153 which is towards the heterochromatin region of the Ychromosome (30) that will also help us to know the extent of AZFc deletion. However, association of the significance of deletion in the most proximal part of AZFc where gr/gr deletion is located with infertility, is still under investigation (31,32).

In conclusion, our findings showed that for diagnostic work-up of infertile males, the marker sets used in the simple multiple PCR as ,described in the present study, may be used for initial screening to determine Y chromosome microdeletion. Subsequently, the men with the deletion(s) may optionally be subjected to further evaluation with more STS markers to know the extent of deletion. This will help decrease the cost and technical difficulty of the procedure and allow a more widespread use of Y chromosome microdeletion screening in infertiiity clinics.

Reprint request: Dr. S.K. Gupta, Staff Scientiest-VII & Chief, Gamete Antigen Laboratory, National Institute of Immunology Aruna Asaf Ali Marg, New Delhi 110 067, India e-mail: skgupta@nii.res.in

Acknowledgment

This study was conducted as part of the Task Force on "Male Genomics" supported b), the Indian Council of Medical Research, New Delhi.

Received April 18, 2007

References

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Anurag Mitra, Rima Dada *, Rajeev Kumar **, N.P. Gupta **, Kiran Kucheria* & S.K. Gupta

Gamete Antigen Lab, National Institute of Immunology & *Departments of Anatomy & **Urology, All India Institute of Medical Sciences, New Delhi, India
Table I. Clinical details of the nine azoospermic men with different
azoospermia factor (AZF) deletions

Lab. No. Age (yr) FNAC/ Biopsy Karyotype FSH (mIU/ml)

267/03 21 Maturation arrest 46,XY 12.98

53/04 32 SCO II ** 46,XY 11.45

94/04 26 Maturation arrest 46,XY 10.26

2l3/04 34 SCO I * 46,XY 58.86

9/05 23 Maturation arrest 46,XY 7.52

13/05 28 SCO II** 46,XYde1(Y) 21.00

427/05 28 SCO I * 46,XY 10.59
434/05 28 SCO II ** 46,XY 12.00
440/05 30 SCO I * ([dagger]) 46,XYde1(Y) 20.61

Lab. No. STS deleted AZF deleted

267/03 sY153, sY148, sY157, AZFc
 sY158, sY254, sY255
53/04 sY153, sY148, sY157, AZFc
 sY158, sY254, sY255
94/04 sY153, sY148, sY157, AZFc
 sY158, sY254, sY255
2l3/04 sY113, sY118, sY127, AZFb, AZFc
 sY134, sY143, sYl53,
 sY148, sY254, sY255
9/05 sY113, SY118, sY127, AZFb
 sY134, sY143
13/05 sY113, sY118, sY127, AZFb, AZFc
 sY134, sY143, sY153,
 sY148, sY157, sY158,
 sY254, sY255, sY160 (hetero)
427/05 sY86 AZFa
434/05 sY86 AZFa
440/05 XKRY, RBMYl, sY113, AZFb, AZFc
 sY118, sY127, sY134, sY143,
 sY153, sY148, sY157, sY158,
 sY254, sY255, sY160 (hetero)

* SCO I- Sertoli cell only type I syndrome, complete absence of germ
cells in the seminiferous tubules and presence of Sertoli cells only;
** SCO II- Sertoli cell only type II syndrome, isolated foci of
spermatogenesis; ([dagger]) Additional phenotype on ultrasound-
remnants of mullerian duct FSH, follicle stimulating hormone; STS,
sequence tagged sites

Table II. Testicular phenotype and Y chromosome microdeletions

 Patients without deletions

Type of phenotype No Deletions

Maturation arrest 52
SCO II 8
SCO I 21
Others (Hypospermatogenesis,
germ cell aplasia, etc.) 4
FNAC not available 76

 AZF Region

 Patients with deletions

Type of phenotype AZFa AZFb AZFc AZFb+c

Maturation arrest -- 1 2 --
SCO II 1 -- 1 1
SCO I 1 -- -- 2
Others (Hypospermatogenesis,
germ cell aplasia, etc.) -- -- -- --
FNAC not available -- -- -- --

SCO I- Sertoli cell only type I syndrome, complete absence of germ
cells in the seminiferous tubules and presence of Sertoli cells only;
SCO II- Sertoli cell only type II syndrome, isolated foci of
spermatogenesis FNAC, Fine needle aspiration cytology
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Title Annotation:polymerase chain reaction
Author:Mitra, Anurag; Dada, Rima; Kumar, Rajeev; Gupta, N.P.; Kucheria, Kiran; Gupta, S.K.
Publication:Indian Journal of Medical Research
Article Type:Clinical report
Geographic Code:9INDI
Date:Feb 1, 2008
Words:5383
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