The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

STUDY QUESTION: Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? SUMMARY ANSWER: In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. WHAT IS KNOWN ALREADY: In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype. STUDY DESIGN, SIZE, AND DURATION: We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. PARTICIPANTS/MATERIALS SETTING METHODS: Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes. MAIN RESULTS AND THE ROLE OF CHANCE: Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature. WIDER IMPLICATIONS OF THE FINDINGS: We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests.

Detection of sperm in men with Y chromosome microdeletions of the AZFa, AZFb and AZFc regions.

BACKGROUND: Y chromosome microdeletions are associated with severe male factor infertility. In this study, the success rate of testicular sperm retrieval was determined for men with deletions of AZF regions a, b or c. METHODS: AZF deletions were detected by PCR of 30 sequence-tagged sites within Yq emphasizing the AZFa, b and c regions. Semen analysis and diagnostic testis biopsy or testicular sperm extraction (TESE) findings were correlated with the specific AZF region deleted. RESULTS: A total of 78 men with AZF deletions included three with AZFa deletion, 11 with AZFb, 42 with AZFc, 16 with AZFb+c and six with Yq (AZFa+b+c). All men with AZFa, AZFb, AZFb+c and Yq deletions were azoospermic and no sperm were found with TESE or biopsy. Of men with isolated AZFc deletion, sperm were found in 75% (9/12) by TESE and 45% (9/20) on biopsy (56% overall); 62% (26/42) were azoospermic and 38% (16/42) severely oligozoospermic. A total of 7 patients with deletion patterns that included the complete AZFa region and 23 that included the complete AZFb region who underwent TESE or biopsy did not have sperm detected by these surgical measures. CONCLUSIONS: Microdeletion of the entire AZFa or AZFb regions of the Y chromosome portends an exceptionally poor prognosis for sperm retrieval, whereas the majority of men with AZFc deletion have sperm within the semen or testes available for use in IVF/ICSI.

High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome.

We have developed a rapid screening protocol for deletion analysis of the complete AZFa sequence (i.e. 792 kb) on the Y chromosome of patients with idiopathic Sertoli-cell-only (SCO) syndrome. This Y deletion was mapped earlier in proximal Yq11 and first found in the Y chromosome of the SCO patient JOLAR, now designated as the AZFa reference patient. We now show that similar AZFa deletions occur with a frequency of 9% in the SCO patient group. In two multiplex polymerase chain reaction experiments, deletions of the complete AZFa sequence were identified by a typical deletion pattern of four new sequence-tagged sites (STS): AZFa-prox1, positive; AZFa-prox2, negative; AZFa-dist1, negative; AZFa-dist2, positive. The STS were established in the proximal and distal neighbourhoods of the two retroviral sequence blocks (HERV15yq1 and HERV15yq2) which encompass the break-point sites for AZFa deletions of the human Y chromosome. We have found deletions of the complete AZFa sequence always associated with a uniform SCO pattern on testicular biopsies. Patients with other testicular histologies as described in the literature and in this paper have only partial AZFa deletions. The current AZFa screening protocols can therefore be improved by analysing the extension of AZFa deletions. This may provide a valuable prognostic tool for infertility clinics performing testicular sperm extraction, as it would enable the exclusion of AZFa patients with a complete SCO syndrome.

Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men.

The evident ability of the intracytoplasmic sperm injection (ICSI) procedure to achieve high fertilization and pregnancy rates regardless of semen characteristics has induced its application with spermatozoa surgically retrieved from azoospermic men. Here, ICSI outcome was analysed in 308 cases according to the cause of azoospermia; four additional cycles were with cases of necrozoospermia. All couples were genetically counselled and appropriately screened. Spermatozoa were retrieved by microsurgical epididymal aspiration or from testicular biopsies. Epididymal obstructions were considered congenital (n = 138) or acquired (n = 103), based on the aetiology. Testicular sperm cases were assessed according to the presence (n = 14) or absence (n = 53) of reproductive tract obstruction. The fertilization rate using fresh or cryopreserved epididymal spermatozoa was 72.4% of 911 eggs for acquired obstructions, and 73.1% of 1524 eggs for congenital cases; with clinical pregnancy rates of 48.5% (50/103) and 61.6% (85/138) respectively. Spermatozoa from testicular biopsies fertilized 57.0% of 533 eggs in non-obstructive cases compared to 80.5% of 118 eggs (P = 0.0001) in obstructive azoospermia. The clinical pregnancy rate was 49.1% (26/53) for non-obstructive cases and 57.1% (8/14) for testicular spermatozoa obtained in obstructive azoospermia, including three established with frozen-thawed testicular spermatozoa. In cases of obstructive azoospermia, fertilization and pregnancy rates with epididymal spermatozoa were higher than those achieved using spermatozoa obtained from the testes of men with non-obstructive azoospermia.

Preoperative screening for genetic abnormalities in men with nonobstructive azoospermia before testicular sperm extraction.

PURPOSE: Men with nonobstructive azoospermia may be treated with testicular sperm extraction and assisted reproduction. However, in men with severely impaired spermatogenesis genetic anomalies may be a cause of low sperm production. We present the results of genetic screening of men with nonobstructive azoospermia who are testicular sperm extraction candidates. MATERIALS AND METHODS: Genetic evaluation was performed in a sequential series of 190 men with nonobstructive azoospermia who were candidates for testicular sperm extraction, including standard peripheral karyotypic analysis and detailed polymerase chain reaction based evaluation of the Y chromosome. All men found to harbor genetic defects were referred for genetic counseling. RESULTS: Of the 190 patients 33 (17%) had genetic abnormalities. Of the 101 men who underwent karyotyping 21 (21%) had cytogenetic abnormalities, including 13 with Klinefelter's syndrome. Of the 183 men who underwent Y chromosome partial deletion analysis defects were detected in 17 (9%). Five men had karyotypic anomalies as well as evidence of partial Y chromosome deletions on polymerase chain reaction based analysis. Genetic counseling was done in 31 men with defects before testicular sperm extraction. Knowledge of the specific genetic defect resulted in a change of clinical management in 7 of the 33 couples (21%) in which the man had a genetic abnormality. CONCLUSIONS: These results indicate that preoperative genetic screening is important in men with nonobstructive azoospermia who are candidates for testicular sperm extraction with intracytoplasmic sperm injection. Genetic testing provides men with reassuring etiological information regarding the infertile condition as well as a means to assess the risk of passing infertility traits to male offspring. It allows specific preimplantation genetic testing of embryos during assisted reproduction to minimize the risk of transmitting genetic defects to offspring.

AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test.

Genetic abnormalities, including partial deletions of the Y-chromosome, are commonly detectable in men with non-obstructive azoospermia (NOA). NOA can be treated using testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI). Recent studies have shown that the presence of deletions involving the AZFc region do not appear to affect the chance of retrieving spermatozoa or have a significant impact on fertilization or pregnancy rates with ICSI. We investigated the effect of Y-chromosome partial deletions on the chance of sperm retrieval with TESE. Eighty attempts at sperm retrieval were performed using TESE on men who were previously evaluated for Y-chromosome partial deletions. Y-chromosome analysis was performed using a polymerase chain reaction (PCR)-based technique with 35 sequence-tagged-sites. Of the 80 men, nine (11%) had partial Y-chromosome deletions detected. Two azoospermic men with AZFc deletions had successful sperm retrieval, ICSI and a subsequent clinical pregnancy. Seven men had deletions involving the AZFb region (three men had isolated AZFb deletions, one had AZFa, AZFb and AZFc deleted, and three had AZFb and AZFc deleted). None of the seven men had spermatozoa extracted by TESE, a result that is significantly different from the overall 64% (47/73) sperm retrieval rate achieved at our centre (P = 0.001). Two men with AZFb deletions had cells consistent with round spermatids identified and injected into oocytes without effecting any normal fertilizations. Although preliminary, these results suggest that the presence of an AZFb deletion is a significantly adverse prognostic finding for TESE. Men with AZFb deletions should be apprised of these results before attempting TESE-ICSI. Alternatives such as donor insemination or adoption should be considered or therapy delayed until improved results with round spermatid injections are likely.

Submicroscopic deletions in the Y chromosome of infertile men.

Recent investigations have suggested a high prevalence of Y chromosome submicroscopic deletions in men with severely impaired spermatogenesis. We evaluated the frequency of Y chromosome deletions in 160 infertile men using a series of 36 sequence-tagged-sites, emphasizing intervals 5 and 6 of the long arm of the Y chromosome. Peripheral leukocyte DNA was extracted and amplified with two parallel techniques to minimize potential overestimation of the frequency of deletions. The presence of deletions was evaluated relative to patient's sperm concentration, testis volume, and hormonal parameters. Men with sperm concentration <5 x 10(6)/ml had a 7% prevalence of submicroscopic Y chromosome deletions. Deletions were detected in 7% of azoospermic men, 10% of men with <1 x 10(6) spermatozoa/ml, and 8% of men with >1 x 10(6) but <5 x 10(6) spermatozoa/ml. Other clinical parameters did not identify men with Y chromosome deletions prior to polymerase chain reaction (PCR)-based testing for the presence of sequence-tagged-sites. Two distinct regions of Y chromosome deletions were detected, approximately 3.6 Mb and 1.4 Mb in length respectively. These deleted regions are present in AZFb and AZFc respectively. No deletions were detected in AZFa. The loss of these two distinct areas is supported by the finding of highly repetitive sequences along the Y chromosome, predisposing to deletion of specific intervals on the Y chromosome during meiosis. Men with severe male infertility are at high risk for Y chromosome deletions. Testing of men for these genetic abnormalities is indicated prior to treatment with assisted reproduction.

POU-domain gene expression during spermatogenesis.

Spermatogenesis is an orderly process whereby male germ cells pass through sequential phases of differentiation to develop into mature spermatozoa. At present, little is known about the factors that regulate this process. The POU domain represents a group of transcription-activator proteins with well-described roles in development. These proteins have been shown to regulate the tissue-specific expression of genes involved in cellular differentiation. The recent identification of Sperm-1, a testis-specific POU-domain protein expressed in primary spermatocytes just prior to the onset of meiosis, suggests that this protein may be involved in the regulation of male germ-cell differentiation. Continued evaluation of Sperm-1 and other related transcriptional activators may shed some light on the regulation of spermatogenesis.