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ORIGINAL ARTICLE
Year : 2006  |  Volume : 22  |  Issue : 4  |  Page : 332-336
 

Serum FSH levels and testicular histology in infertile men with non obstructive azoospermia and Y chromosome microdeletions


1 Department of Urology, All India Institute of Medical Sciences, New Delhi, India
2 Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India

Correspondence Address:
Rajeev Kumar
Assistant Professor of Urology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-1591.29119

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   Abstract 

Objectives: Men with nonobstructive azoospermia may father a child through intracytoplasmic injection of testicular sperms. This can result in transmission of genetic defects such as Y chromosome microdeletions which primarily caused the infertility and occur in up to 55% such men. It may not be feasible to screen all patients for Y chromosome microdeletions due to the cost and technical difficulty. A correlation with existing markers such as follicle stimulating hormone (FSH) and testicular histology may help identify a subgroup for screening. We therefore studied the association between regions of azoospermia factor (AZF) deletion, testicular histology and serum FSH level in men with nonobstructive azoospermia. Materials and Methods: One hundred and nine men presenting with primary infertility and diagnosed as nonobstructive azoospermia based on standard guidelines were included in the study. Fasting blood sugar, serum FSH, testosterone and prolactin estimation was done and testicular fine needle aspiration biopsy was performed where clinically indicated. Patients with normal karyotype on standard Q banding (n=82) were evaluated for microdeletions in the Y chromosome. Eight AZF loci which mapped to interval 5 and 6 of the Y chromosome were evaluated. Results: Microdeletions were found in seven of 82 men with normal karyotype (8.5%). Three patients had deletions in both AZFa and AZFb regions with Sertoli cell only (SCO) histology. Two had AZFc deletion with hypospermatogenesis and maturation arrest in one each. Two patients had cryptorchidism. FSH levels were higher (mean 38.77 mIU/ml) in patients with deletions in the AZFa and AZFb regions than in those with AZFc deletions (mean 5.86 mIU/ml). In patients without a deletion, FSH was higher in the group with SCO (mean 18.28 mIU/ml) compared to those with hypospermatogenesis or maturation arrest (mean 6.83 mIU/ml). Conclusions: Serum FSH is raised in patients with severely depleted germ cell function, irrespective of the presence or absence of a microdeletion. The levels may correlate with the region of deletion in that the patients with AZFa and AZFb deletion had a sertoli cell only picture on histology and raised FSH.


Keywords: Azoospermia, male infertility, Y chromosome


How to cite this article:
Kumar R, Dada R, Gupta NP, Kucheria K. Serum FSH levels and testicular histology in infertile men with non obstructive azoospermia and Y chromosome microdeletions. Indian J Urol 2006;22:332-6

How to cite this URL:
Kumar R, Dada R, Gupta NP, Kucheria K. Serum FSH levels and testicular histology in infertile men with non obstructive azoospermia and Y chromosome microdeletions. Indian J Urol [serial online] 2006 [cited 2019 Jun 18];22:332-6. Available from: http://www.indianjurol.com/text.asp?2006/22/4/332/29119


With a 15% prevalence among all reproductive age group couples, infertility constitutes a significant public health problem. The male partner is believed to be contributory in up to 50% cases.[1] Among the male causes of infertility, nonobstructive azoospermia is not amenable to therapy.[2] However, it has also been documented that in about 50% of patients with nonobstructive azoospermia, foci of spermatogenesis may be found in the testis and these may yield sperms through testicular extraction, sufficient for intracytoplasmic injection (ICSI).[3] One of the major issues with ICSI for male partner infertility and nonobstructive azoospermia is the vertical transmission of genetic abnormalities into the male offspring. Among these genetic abnormalities, Y chromosome microdeletions have been found in 1-55% cases.[4],[5],[6],[7],[8]

Patients with Y chromosome microdeletions show a variety of histological changes in the testis.[9],[10] These changes are clinically represented by changes in serum reproductive hormone profile including inhibin-B and follicle stimulating hormone (FSH) levels.[11],[12] While it may not be feasible to screen all patients of nonobstructive azoospermia for Y chromosome microdeletions, all such men routinely undergo an FSH assay and, often, a testicular biopsy during their infertility evaluation. If a significant association could be found to exist between Y chromosome microdeletions, FSH and testicular histology, it may be possible to identify a subset of patients for Yq microdeletion assay. With this hypothesis, we evaluated a series of infertile men with nonobstructive azoospermia for Yq microdeletions.


   Materials and Methods Top


Cases

Subjects for this study presented to our urology clinic with the inability to contribute to a pregnancy despite regular, unprotected intercourse for at least a year with a female partner who had been evaluated by a gynecologist and found to have no anatomic or functional reproductive abnormality. None of the subjects were related to each other and were not on any medication that could affect their reproductive function. No patient had a history of scrotal or inguinal surgery that could injure the vas deferens. All patients underwent a detailed physical examination including a genital exam to rule out congenital absence of vas deferens. Since none of the patients included in this study had absence of vas, a cystic fibrosis transmembrane regulator gene mutation analysis was not performed.

Semen analysis was performed according to the WHO criteria[13] on at least two occasions, four weeks apart. Patients with absolute azoospermia (absent sperm in the pellet after centrifugation) on both occasions and normal semen volume (>2 ml) and fructose underwent further investigations to evaluate the etiology of azoospermia. Fasting blood sugar was estimated to rule out diabetes mellitus and serum FSH, Leutenizing hormone and prolactin assays were performed in all patients in our centralized hormone laboratory that has an established quality control system. The assays were done using a micro particle enzyme immunoassay on the AxSYM Plus 5® system (Abbott Laboratories, Illinois, USA). The lower limit of quantification of FSH was 0.8 mIU/ml and an inter-assay and intra-assay coefficient of variance of <7.4% and <6% respectively. Inhibin B levels were not measured.

Fine needle aspiration biopsy of the testis was performed in all patients with a normal testicular size and serum FSH up to 2.5 times the upper limit of normal (10 mIU/ml). Biopsy was also done in patients with higher serum FSH if they consented for a biopsy after being informed that it was not required for diagnosis. Aspiration biopsy was performed under local anesthesia using a 23G (0.6 mm) butterfly needle and 10ml syringe using a single scrotal skin puncture on the palpable testes. The aspirate was air-dried and stained with the May-Grunwald-Giemsa stain and reported by a cytopathologist. The smear was considered adequate if at least 2000 cells or 100 clusters of 20 cells each were obtained. Normal spermatogenesis was defined as the presence of at least 10-20 mature sperms per high power field (400x).

Nonobstructive azoospermia was diagnosed in patients with both a raised serum FSH (> 2.5 times normal) and small or cryptorchid testis bilaterally (n=61) or normal FSH/ normal testis size but absence of complete spermatogenesis on testicular biopsy (n=48).

One hundred and nine consecutive patients who received a diagnosis of nonobstructive azoospermia based on the above investigations and consented for inclusion in the study were further evaluated for karyotype and Y chromosome microdeletions. The study was approved by the institutional ethics committee.

Molecular methods

Peripheral blood cultures were set up for chromosomal analysis and five well-spread G banded metaphases were karyotyped using standard protocols. Patients with normal karyotype (46, XY) underwent polymerase chain reaction (PCR) analysis for AZF loci microdeletion. Each of these patients was examined for eight AZF loci which mapped to interval 5 and 6 of the Y chromosome. Sex determining region (SRY) on the short arm of Y chromosome (sY14) was used as an internal control. The sequence tagged site (STS) primers used were sY84, sY86 (AZFa); sY113, sY116, sY127, sY134 (AZFb); sY254, sY255 (AZFc). This primer set suggested by Simoni et al[14] is prescribed by the European Academy of Andrology and enables the detection of over 90% deletions in the AZF loci. It allows for minimal standardization and comparison of the data on AZF deletions from different laboratories in different countries. Samples were subjected to PCR amplification using 35 cycles at 95 osub C for one minute, 56 osub C for 30 seconds and 72oC for30 seconds. Genomic DNA was isolated from the peripheral blood using the organic method. The PCR mix with DNA template was denatured for five minutes at 95OC in the thermal cycler and a final extension time of seven minutes at 72oC was given. An STS was considered absent only after at least three amplification failures in the presence of successful amplification of control (SRY -sY14). Fertile male and female samples were used as positive and negative controls and water was used as blank to check for false positive and negative results. The PCR products were analyzed on a 1.8% agarose gel containing ethidium bromide (0.5 ug/ml).

Statistics

The standard deviation of FSH levels in the group with and without microdeletions (21.00 and 12.34 respectively) was too large to permit application of parametric tests such as the t-test for comparison. On applying a logarithmic transformation, the mean and standard deviations were 2.80 ± 1.03 and 2.48 ± 0.77 respectively. Since the standard deviations were again very large, a nonparametric test (Wilcoxon rank sum test) was used for statistical analysis and the results are being reported based on this test. However, disregarding the standard deviation and assuming a normal population, we compared the groups using the t-test as well and found the results similar to those with the nonparametric test, considering a P value <0.05 as significant for both tests.


   Results Top


All patients included for the study had presented with primary infertility. Of the 109 patients, 27 had karyotypic abnormalities and are not included in this report. Of the remaining 82 patients with normal karyotype and nonobstructive azoospermia, Y chromosome microdeletions were found in seven cases (8.5%). The mean age of these seven subjects was 27 years (20-33 years). Two of these patients had bilateral cryptorchidism with abdominal testes which precluded biopsy examination. In the other five, biopsy revealed sertoli cell only picture (SCO) in three, maturation arrest in one and hypospermatogenesis in one [Table - 1]. The mean FSH level in this group of seven patients was 24.67 mIU/ml (range 4.6-60 mIU/ml).

Among the 75 men with nonobstructive azoospermia, normal karyotype and absence of microdeletions, biopsy reports were available for 35. Twenty-seven of these patients had SCO, six had maturation arrest and two had hypospermatogenesis. Mean FSH level of these 35 patients was 15.66mIU/ml (range 2.16-63.34mIU/ml) [Table - 2]. The FSH level of the seven patients with deletions and 35 without microdeletions was statistically similar.

In the group with microdeletions, FSH levels were higher (mean 38.77mIU/ml) in the four patients with deletions in the AZFa and AZFb regions than the three patients with AZFc deletions (mean 5.86 mIU/ml). The region of deletion was also reflected in the testicular histology with AZFa and AZFb deleted patients showing a SCO picture while the AZFc deleted patients had either hypospermatogenesis or maturation arrest.

In the group without microdeletions, 27 patients with SCO on the histology had higher FSH levels (mean 18.28 mIU/ml) compared to the eight cases with maturation arrest or hypospermatogenesis (mean 6.83 mIU/ml). This difference was statistically significant ( P =0.002).


   Discussion Top


The ICSI procedures form one of the principle modes of reproduction for couples where the men are diagnosed to have nonobstructive azoospermia. The risk of genetic transmission of an inheritable cause of infertility necessitates screening for these disorders before the use of sperm from such men. Y chromosome microdeletion is one of the transmissible causes of nonobstructive azoospermia and these patients have a variety of changes in the germ cell epithelium.[9],[10],[15],[16],[17] Follicle stimulating hormone production from the pituitary is under the feedback control of inhibin-B produced by the  Sertoli cells More Details. Patients with germ cell deficiency have decreased inhibin-B and raised FSH, reflecting a possible interaction between the germ cell and Sertoli cell function.[8],[12] Since serum FSH reflects the status of the germinal epithelium, a correlation can be assumed to exist between all three parameters i.e., FSH, region of Y chromosome microdeletion and testicular histology.

In a seminal study on Y chromosome microdeletions in infertile men, Pryor et al[17] evaluated the male partners in 200 infertile couples and found a 7% prevalence of such deletions and similar deletions in 2% controls. However, out of their 200 men, 51% had normal sperm counts and may not have been infertile. Among the 26 men with azoospermia, six (23%) had a deletion. Among the 14 men with microdeletions, only six had undergone a testicular biopsy making histological correlation difficult to assess. However, they noted that large deletions were associated with a more severe seminal abnormality and there was a trend towards correlation between the region of deletion and spermatogenesis. Men with small microdeletions may have sufficient sperms to father a child, particularly at a young age. The authors confirm this since the fathers of two of their infertile patients with microdeletions themselves had a microdeletion and also two of their patients with a microdeletion had previously fathered a child. This can explain the 2% incidence of microdeletion in their controls. Moreover, the authors' controls were drawn from a semen bank of men who had previously fathered a child. There is no data on when this child was fathered and whether these men were currently fertile.

The mean FSH in our seven patients with a microdeletion was greater than normal but similar to that in the 35 patients without a microdeletion. This precludes the use of a random FSH level in predicting the presence of a microdeletion. These findings are similar to those reported by Frydelund-Larsen[15] and Foresta et al[18] who also failed to find any difference in the FSH levels in the two groups as a whole.

This lack of association of FSH with Yq microdeletion is understandable in view of the fact that these microdeletions represent a heterogeneous group of disorders with varying findings on testicular histopathology. Patients with a deletion in the AZFa or AZFb region show a more severely affected germinal epithelium resembling the SCO syndrome while the AZFc deleted patients have a milder form of germ cell depletion ranging from hypospermatogenesis and oligospermia to SCO and azoospermia.[19],[20] Vogel and associates[20] postulated that the variation in patients with AZFc might be due to the variable penetrance and modifying effects of associated genes and environment.

Considering these facts, we separated our patients based on their testicular histology. We noted a trend towards a correlation between the biopsy findings, region of microdeletion and the FSH levels. Patients with AZFa and AZFb deletions had a more severely affected germ cell epithelium and raised FSH compared to those with AZFc deletion who had a normal FSH. None of our patients with AZFc deletion had raised FSH and all had some normal germ cell elements on biopsy. In patients without a microdeletion, high FSH levels were noted in patients with SCO compared to normal levels in those with milder impairment. These findings confirm that FSH levels reflect testicular histology rather than the presence of a microdeletion. However, there is an indirect correlation between region of microdeletion and FSH due to the associated histological changes.

Our findings are similar to earlier reports[15],[16] where an excellent association was noted between the hormonal profile and the testicular histology in both groups of men. These authors confirm that the hormone levels (serum inhibin-B and FSH) correlate only with the testicular histology and not merely with the presence or absence of microdeletion. However, they only evaluated men with deletions in the AZFc region and were therefore unable to comment on the possible association between histology, hormone levels and region of deletion as was seen in our cases.

Our hospital is a tertiary-care government-funded institution with free consultation and treatment and low or no charges for investigations. The consecutive recruitment of patients from the urology clinic of this hospital may not reflect the true prevalence in the general population because of the varying pattern of use of services by different patients. All patients were of Indian origin and therefore reflect prevalence only in this racial group.

The number of patients with deletions is too small to allow extensive statistical comparisons and definitive statements of association. However, this study justifies a larger study since the initial hypothesis of a possible association is emerging. We were able to obtain testicular biopsy in only 35 of the men without a microdeletion. Since an FSH level more than 2.5 times normal reflects severe germ cell depletion, it is not mandatory to obtain a testicular histology in these men to diagnose a testicular pathology. This may be a source of error since less than half the men without a microdeletion had their testicular histology assessed.


   Conclusions Top


Serum FSH levels reflect the functional state of the germinal epithelium. They are usually raised in patients with severely depleted germ cell function, irrespective of its cause. This is also true in patients with Y chromosome microdeletions as the cause of infertility. The FSH levels correlate with the region of deletion in that the patients with AZFa and AZFb deletion tend to have a SCO picture on histology and raised FSH. No such correlation can be detected for patients with AZFc deletions since these patients have a wide range of histological changes.


   Acknowledgement Top


The authors would like to thank Professor KR Sundaram, Head, Department of Biostatistics, All India Institute of Medical Sciences, New Delhi for his inputs on statistical methodology for this study.

 
   References Top

1.De Kretser DM, Baker HW. Infertility in men: Recent advances and continuing controversies. J Clin Endocrinol Metab 1999;84:3443-50.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Oates RD. The genetics of male reproduction. Infert Reprod Med Clin North Am 1999;3:411-27.  Back to cited text no. 2    
3.Faddy MJ, Silber SJ, Gosden RG. Intracytoplasmic sperm injection and infertility. Nat Genet 2001;29:131.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.van der Ven K, Montag M, Peschka B, Leygraaf J, Schwanitz G, Haidl G, et al . Combined cytogenetic and Y chromosome microdeletion screening in males undergoing intracytoplasmic sperm injection. Mol Hum Reprod 1997;3:699-704.  Back to cited text no. 4    
5.Foresta C, Ferlin A, Garolla A, Moro E, Pistorello M, Barbaux S, et al . High frequency of well-defined Y-chromosome deletions in idiopathic Sertoli cell-only syndrome. Hum Reprod 1998;13:302-7.  Back to cited text no. 5    
6.Chiang HS, Yeh SD, Wu CC, Huang BC, Tsai HJ, Fang CL. Clinical and pathological correlation of the microdeletion of Y chromosome for the 30 patients with azoospermia and severe oligoasthenospermia. Asian J Androl 2004;6:369-75.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Dada R, Gupta NP, Kucheria K. AZF microdeletions associated with idiopathic and non-idiopathic cases with cryptorchidism and varicocele. Asian J Androl 2002;4:259-63.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Dada R, Gupta NP, Kucheria K. Cryptorchidism and AZF microdeletion. Asian J Androl 2002;4:148.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Krausz C, McElreavey KY. Chromosome and male infertility. Front Biosci 1999;4:E1-8.  Back to cited text no. 9    
10.Krausz C, Quintana-Murci L, McElreavey K. Prognostic value of Y deletion analysis: What is the clinical prognostic value of Y chromosome microdeletion analysis? Hum Reprod 2000;15:1431-4.  Back to cited text no. 10    
11.Anawalt BD, Bebb RA, Matsumoto AM, Groome NP, Illingworth PJ, McNeilly AS, et al . Serum inhibin-B levels reflect Sertoli cell function in normal men and in men with testicular dysfunction. J Clin Endocrinol Metab 1996;81:3341-5.  Back to cited text no. 11    
12.von Eckardstein S, Simoni M, Bergmann M, Weinbauer GF, Gassner P, Schepers AG, et al . Serum inhibin B in combination with serum follicle stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. J Clin Endocrinol Metab 1999;84:2496-501.  Back to cited text no. 12    
13.World Health Organization laboratory manual for the examination of Human semen and sperm-cervical mucus interaction. 4th ed. Cambridge University Press: Cambridge, England; 1999.  Back to cited text no. 13    
14.Simoni M, Bakker E, Eurlings MC, Matthijs G, Moro E, Muller CR, et al . Laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions. Int J Androl 1999;22:292-9.  Back to cited text no. 14    
15.Frydelund-Larsen L, Krausz C, Leffers H, Andersson AM, Carlsen E, Bangsboell S, et al . Inhibin B: A marker for the functional state of the seminiferous epithelium in patients with azoospermia factor c microdeletions. J Clin Endocrinol Metab 2002;87:5618-24.  Back to cited text no. 15    
16.Luetjens CM, Gromoll J, Engelhardt M, Von Eckardstein S, Bergmann M, Nieschlag E, et al . Manifestation of Y chromosomal deletions in the human testis: A morphometrical and immunohistochemical evaluation. Hum Reprod 2002;17:2258-66.  Back to cited text no. 16    
17.Pryor JL, Kent-First M, Muallem A, Van Bergen AH, Nolten WE, Meisner L, et al . Microdeletions in the Y chromosome of infertile men. N Engl J Med 1997;336:534-9.  Back to cited text no. 17    
18.Foresta C, Bettella A, Moro E, Roverato A, Merico M, Ferlin A. Sertoli cell function in infertile patients with and without microdeletions of the azoospermia factors on the Y chromosome long arm. J Clin Endocrinol Metab 2001;86:2414-9.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]
19.Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al . Human Y chromosome azoospermia factors (AZF) mapped to different sub regions in Yq11. Hum Mol Genet 1996;5:933-43.  Back to cited text no. 19    
20.Vogel T, Speed RM, Teague P, Cooke HJ. Mice with Y chromosome deletion and reduced Rbm genes on a heterozygous Dazl1 null background mimic a human azoospermia factor phenotype. Hum Reprod 1999;14:3023-9.  Back to cited text no. 20  [PUBMED]  [FULLTEXT]



 
 
    Tables

  [Table - 1], [Table - 2], [Table - 3]

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