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.

Genetic variants in the human glucocorticoid-induced leucine zipper (GILZ) gene in fertile and infertile men.

Sertoli cell only (SCO) syndrome is the predominant histology for men with non-obstructive azoospermia (NOA) and is usually of unexplained aetiology. Studies in mouse models indicated that the X-linked gene glucocorticoid-induced leucine zipper (GILZ) is essential for survival and differentiation of spermatogonia, and meiosis. GILZ deficiency results in a rapid and progressive loss of germ cells with SCO tubules and sterility in adults. The role of GILZ in human fertility has not been examined. Here we show that GILZ is localized to spermatogonia and spermatocytes in the human testis in a pattern analogous to that seen in mice. To assess the potential for an association between GILZ variants and human infertility, we sequenced the entire protein-coding regions of the GILZ gene in 65 SCO and 87 fertile Australian men. We identified six genetic variants, three of which had not been reported previously. Three variants, 107018665 G>A, 107018485 C>G and 106959283 C>T, were found at a low frequency only in SCO men. Although none of the identified variants changed the protein code, sequence analysis indicated that two variants, 107018665 G>A and 107018485 C>G, would completely abolish the exonic splicing enhancer (ESE)-binding motifs for the splicing factors SF2/ASF and SC35 respectively. This result prompted an assessment of whether these two variants were associated with male infertility in a separate population of men. We used a PCR-based SNP detection approach to screen an additional 52 NOA and 153 fertile Australian men, and 86 SCO and 54 fertile American men. None of these men carried either of these two variants. The cumulative allelic frequency of these variants is less than 1% in SCO men and no association with fertility status was observed. Our study suggests that GILZ variants are not common causes of SCO and NOA in Australian or American men.

Evaluation of male infertility.

Infertility is an emotionally charged problem affecting an estimated 15% of all couples. The man should be evaluated concurrently with the woman, since a male factor is the primary or contributing cause in 40% to 60% of cases. In addition to detecting treatable abnormalities, evaluation of the infertile man is critical to uncover life-threatening problems associated with the symptom of infertility, as well as genetic conditions associated with male infertility that could be transmitted to offspring with assisted reproduction. New diagnostic tests have been developed and surgical techniques refined resulting in improved treatment results and patient care. Dramatic advancements in, and widespread use of, assisted reproductive techniques such as intracytoplasmic sperm injection have created alternatives for couples who previously had little hope of reproductive success. The infertility practitioner should have a thorough understanding of the advantages and limitations of various laboratory tests as well as the indications, costs and success rates of all treatment options. The first step in evaluation is a thorough history and physical examination with initiation of basic laboratory studies.

A decision analysis of treatments for obstructive azoospermia.

BACKGROUND: Treatments for post-vasectomy obstructive azoospermia include vasectomy reversal, microsurgical epididymal sperm aspiration (MESA) or percutaneous testicular sperm extraction (TESE) with IVF/ICSI. We examined the cost-effectiveness of these treatments. METHODS: A decision analytic model was created to simulate treatment. Outcome probabilities were derived from peer-reviewed literature and the Society for Assisted Reproductive Technologies database. Procedural costs were derived from a sampling of high-volume IVF centers and the Medicare Resource Based Relative Value Scale. Indirect costs of complications, lost productivity and multiple gestation pregnancies were considered. Sensitivity analyses were performed. RESULTS: Vasectomy reversal was more cost-effective than either MESA or TESE under all probability conditions. In 1999, vasectomy reversal demonstrated superior cost-effectiveness to TESE and MESA ($19,633 versus $45,637 and $48,055, respectively, equivalent to $25,321 versus $58,858 and $61,977 in 2005 dollars). In 2005, vasectomy reversal ($20,903) remained the most cost-effective treatment over TESE ($54,797) and MESA ($56,861). The cost-effectiveness of all treatments improved over projections by inflation. The relative cost-effectiveness of the therapies was unchanged over time. CONCLUSIONS: Vasectomy reversal appears more cost-effective than percutaneous TESE and MESA for treatment of obstructive azoospermia when the impact of indirect costs is considered. The absolute cost-effectiveness of all therapies improved over time. These results may be tailored with institution-specific data to allow more individualized results.

Activation of p300 histone acetyltransferase activity and acetylation of the androgen receptor by bombesin in prostate cancer cells.

Androgen receptor signaling in prostate cancer cells is augmented by the androgen receptor (AR) coactivator p300, which transactivates and acetylates the AR in the presence of dihydrotestosterone (DHT). As prostate cancer (PC) cells progress to androgen independence, AR signaling remains intact, indicating that other factors stimulate AR activities in the absence of androgen. We previously reported that neuropeptide growth factors could transactivate the AR in the presence of very low concentrations of DHT. Here, we examine the involvement of p300 in neuropeptide activation of AR signaling. Transfection of increasing concentrations of p300 in the presence of bombesin into PC-3 cells resulted in a linear increase in AR transactivation, suggesting that p300 acts as a coactivator in neuropeptide-mediated AR transactivation. P300 is endowed with histone acetyltransferase (HAT) activity. Therefore, we examine the effect of bombesin on p300 HAT activity. At 4 h after the addition of bombesin, p300 HAT activity increased 2.0-fold (P<0.01). Incubation with neutral endopeptidase, which degrades bombesin, or bombesin receptor antagonists blocked bombesin-induced p300 HAT activity. To explore the potential signaling pathways involved in bombesin-induced p300 HAT activity, we examined Src and PKCdelta pathways that mediate bombesin signaling. Inhibitors of Src kinase activity or Src kinase siRNA blocked bombesin-induced p300 HAT activity, whereas PKCdelta inhibitors or PKCdelta siRNA significantly increased bombesin-induced p300 HAT activity suggesting that Src kinase and PKCdelta kinase are involved in the regulation of p300 HAT activity. As AR is acetylated in the presence of 100 nM DHT, we next examined whether bombesin-induced p300 HAT activity would result in enhanced AR acetylation. Bombesin-induced AR acetylation at the same motif KLKK observed in DHT-induced acetylation. Elimination of p300 using p300 siRNA reduced AR acetylation, demonstrating that AR acetylation was mediated by p300. AR acetylation results in AR transactivation and the expression of the AR-regulated gene prostate-specific antigen (PSA). Therefore, we examined bombesin-induced AR transactivation and PSA expression in the presence and absence of p300 siRNA and found inhibition of p300 expression reduced bombesin-induced AR transactivation and PSA expression. Together these results demonstrate that bombesin, via Src and PKCdelta signaling pathways, activates p300 HAT activity which leads to enhanced acetylation of AR resulting in increased expression of AR-regulated genes.

Causes of azoospermia and their management.

Azoospermia may occur because of reproductive tract obstruction (obstructive azoospermia) or inadequate production of spermatozoa, such that spermatozoa do not appear in the ejaculate (non-obstructive azoospermia). Azoospermia is diagnosed based on the absence of spermatozoa after centrifugation of complete semen specimens using microscopic analysis. History and physical examination and hormonal analysis (FSH, testosterone) are undertaken to define the cause of azoospermia. Together, these factors provide a >90% prediction of the type of azoospermia (obstructive v. non-obstructive). Full definition of the type of azoospermia is provided based on diagnostic testicular biopsy. Obstructive azoospermia may be congenital (congenital absence of the vas deferens, idiopathic epididymal obstruction) or acquired (from infections, vasectomy, or other iatrogenic injuries to the male reproductive tract). Couples in whom the man has congenital reproductive tract obstruction should have cystic fibrosis (CF) gene mutation analysis for the female partner because of the high risk of the male being a CF carrier. Patients with acquired obstruction of the male reproductive tract may be treated using microsurgical reconstruction or transurethral resection of the ejaculatory ducts, depending on the level of obstruction. Alternatively, sperm retrieval with assisted reproduction may be used to effect pregnancies, with success rates of 25-65% reported by different centres. Non-obstructive azoospermia may be treated by defining the cause of low sperm production and initiating treatment. Genetic evaluation with Y-chromosome microdeletion analysis and karyotype testing provides prognostic information in these men. For men who have had any factors potentially affecting sperm production treated and remain azoospermic, sperm retrieval from the testis may be effective in 30-70% of cases. Once sperm are found, pregnancy rates of 20-50% may be obtained at different centres with in vitro fertilisation and intracytoplasmic sperm injection.

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.

Testicular sperm extraction combined with intracytoplasmic sperm injection in the treatment of men with persistent azoospermia postchemotherapy.

BACKGROUND: Men who remain azoospermic long after undergoing chemotherapy have generally been considered sterile. The authors report their experience with testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) applied to azoospermic men who previously received chemotherapy for a variety of indications. METHODS: Among 231 cycles in 198 patients who underwent TESE-ICSI for nonobstructive azoospermia from 1995 to 2000, 20 TESE procedures in 17 patients who previously received chemotherapy were identified. All TESE procedures were performed with microsurgical control under local anesthesia with sedation or general anesthesia. The pretreatment hormonal profile, histology of testicular biopsies, and outcomes of TESE-ICSI in this subgroup of patients were analyzed. RESULTS: The mean patient age was 37.4 years (range, 28-54 years), and the mean follicle-stimulating hormone level was 21.8 mIU/mL (range, 7.1-43.1 mIU/mL). The mean age for female partners was 33.5 years (range, 22-43 years). Six patients had received chemotherapy for Hodgkin lymphoma (34%), four patients had received chemotherapy for testicular neoplasm (24%), two patients had received chemotherapy for non-Hodgkin lymphoma (12%), two patients had received chemotherapy for leukemia (12%), one patient had received chemotherapy for Wilms tumor (6%), one patient had received chemotherapy for mediastinal germ cell tumor (6%), and one patient had received chemotherapy for nephrotic syndrome (6%). Three patients (18%) received additional radiation therapy. The mean interval from chemotherapy to TESE was 16.3 years (range, 6-34 years). All patients had at least two semen analyses to confirm azoospermia. A total of 20 attempts of TESE-ICSI were performed (mean, 1.2 attempts per patient). Testicular histology revealed Sertoli cell-only pattern in 76% of patients. The remaining 24% of patients had hypospermatogenesis as their most advanced spermatogenic pattern. Among the men with Sertoli cell-only pattern, 23% had sperm retrieved by TESE. Sperm retrieval was accomplished in 9 of 20 attempts (45%), with biochemical pregnancy after sperm retrieval in 4 of 9 couples (45%) and clinical pregnancy in 3 of 9 couples (33%). Live deliveries were achieved in 2 of 9 couples (22%). Two healthy boys and one girl were delivered. No correlation was noted between the outcome of TESE-ICSI and the underlying conditions that were treated with chemotherapy nor with the chemotherapeutic agents used. CONCLUSIONS: Using TESE-ICSI, sperm retrieval leading to pregnancy and the delivery of healthy children is possible for men with long-standing azoospermia after chemotherapy. The prognosis for sperm retrieval was not influenced clearly by the chemotherapy regimen or the disease treated. Diagnostic biopsy also was of limited value in predicting the outcome of sperm retrieval. Despite prolonged nonobstructive azoospermia after undergoing chemotherapy, men no longer should be considered sterile in the era of advanced assisted reproductive techniques.

Effective long-term androgen suppression in men with prostate cancer using a hydrogel implant with the GnRH agonist histrelin.

OBJECTIVES: To evaluate the effectiveness of a hydrogel implant containing the gonadotropin-releasing hormone (GnRH) agonist histrelin in suppressing testosterone production in men with prostate cancer and to determine the effective dose (one, two, or four implants). METHODS: Forty-two men with prostate cancer and indications for androgen ablation were treated with one, two, or four implants. In two of the clinics, comprising 27 subjects, the treatment period was 12 months, with replacement with the same number of implants at 12-month intervals. In a third clinic, which treated 15 subjects, the implants were left in place for up to 30 months. The total experience was 605 treatment months. RESULTS: The histrelin levels were detected in serum proportional to the number of implants placed. The response, however, was similar among all three dose levels, with testosterone and luteinizing hormone essentially completely suppressed. Serum testosterone levels decreased from 21.9 +/- 17.6 nmol/L to 0.93 +/- 1.57 nmol/L within 1 month and were maintained at 0.55 +/- 0.24 nmol/L at 6 months and 0.60 +/- 0.28 nmol/L after 12 months of treatment. Of the 38 assessable patients, 35 (92%) had castrate levels of testosterone within 4 weeks of the initial implant placement. All patients followed for up for 12 months after placement of the initial set of implants maintained suppression of testosterone production while the implant was in place. CONCLUSIONS: The histrelin hydrogel implant provided adequate and reliable delivery of the potent GnRH agonist histrelin during at least 1 year using a single implant in men with prostate cancer. No apparent advantages were found in using more than one implant, and the question of the possible effectiveness of even lower doses remains open. This treatment modality appears to be both safe and effective.

Massive hematuria due to right renal artery mycotic pseudoaneurysm in a patient with subacute bacterial endocarditis.

A 40-year-old woman with recently diagnosed bacterial endocarditis was admitted to the hospital with gross hematuria and anemia. Computed tomography revealed a large right upper pole renal artery pseudoaneurysm, a wedge-shaped hypoperfused region of the left kidney, and a splenic abscess. Radiographic embolization of the right renal artery was performed to stabilize the bleeding. The splenic abscess was drained. Subsequent right nephrectomy and splenectomy were performed for persistent leukocytosis. This unusual presentation of a septic embolus and its management are discussed.

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.

Nitric oxide synthase activity in human seminal plasma.

OBJECTIVES: To determine whether human seminal plasma possesses nitric oxide synthase (NOS) activity and whether, if present, this activity correlates with standard semen analysis parameters. Recent studies have demonstrated the presence of NOS in the epithelium of the male reproductive tract and have shown that nitric oxide can influence human sperm function in vitro. METHODS: Semen samples were collected from 30 men presenting for an infertility evaluation at our institution. After a standard semen analysis, the semen samples were centrifuged at 10,000g, and the seminal plasma was collected and stored at -20 degrees C for later evaluation of the arginine concentration and calcium-dependent and calcium-independent NOS activity. NOS activity was measured by the conversion of [(3)H]L-arginine to [(3)H]L-citrulline. RESULTS: NOS activity was detected in 28 of 30 seminal plasma samples tested. The mean +/- SD calcium-dependent NOS activity was 1.6 +/- 0.8 nmol/min/mL and the mean calcium-independent NOS activity was 1.0 +/- 0.7 nmol/min/mL of the seminal plasma. The mean +/- SD arginine concentration in the seminal plasma was 7.0 +/- 1.9 mM. No significant correlations were found between the NOS activity in the seminal plasma and sperm concentration, sperm motility, or leukocyte concentration. The 2 men without detectable NOS activity in their semen had documented congenital bilateral absence of the vas deferens. CONCLUSIONS: The results of this study demonstrate the presence of NOS activity in human semen and support a role of nitric oxide in sperm function in vivo. Our results also suggest that the seminal vesicles may be an important source of NOS activity in semen.

Histologic effects of testicular sperm extraction on the testicle in men with nonobstructive azoospermia.

OBJECTIVES: Testicular sperm extraction (TESE) is a therapeutic technique that has revolutionized the treatment of severe male infertility presenting as nonobstructive azoospermia. However, the procedure is not without side effects, involving at least a transient effect on spermatogenesis. The purpose of this study was to demonstrate the histologic effects of TESE on the testicle. METHODS: Testicular biopsy specimens were analyzed from 7 patients with nonobstructive azoospermia who each underwent two consecutive TESE procedures. We evaluated two biopsies at the same site on the testicle so that we could examine the histologic effects of the first TESE procedure with the second biopsy specimen. First, a quantitative evaluation of seminiferous tubular volume was performed with a 121-point grid over multiple fields of the testicular specimen slides. The second step of the analysis involved a comparison of the number of germ cells per tubule in each set of specimens. Both Student's t test and Wilcoxon matched pairs tests were used for analysis. RESULTS: In the first set of TESE specimens, tubules comprised 33,158 of 63,525 grid points, or 52.2% of the specimen area. This decreased to 28,637 points, or 45.1%, in the second set of specimens. This decrease in seminiferous tubular volume and corresponding increase in interstitial tissue was statistically significant (P <0.00042). Our data also showed a 5.5% decrease in the number of germ cells per 91 tubules in each data set (from 3222 to 2887, P = 0.25), which suggests a trend toward a lower number of germ cells per tubule. CONCLUSIONS: These findings support our clinical observation that TESE causes a decrease in seminiferous tubular volume within the testicular parenchyma adjacent to the biopsy site. This reflects a potentially adverse local effect of TESE on the testis that may have important clinical consequences for men with nonobstructive azoospermia.

Evidence of a treatable endocrinopathy in infertile men.

PURPOSE: We establish whether a subset of infertile men have decreased serum testosterone-to-estradiol ratios and whether this condition can be corrected with an oral aromatase inhibitor. MATERIALS AND METHODS: The serum testosterone-to-estradiol ratios of 63 men with severe male factor infertility or hypergonadotropic hypogonadism (mean follicle-stimulating hormone 21.2 +/- 1.8) were compared with those of an age matched, fertile, control reference group. Of the 63 men 43 were azoospermic with biopsy proved severe male infertility and 20 were oligospermic. The men with the lowest ratios (less than 20th percentile) were treated with 50 to 100 mg of the aromatase inhibitor testolactone orally twice daily. Testosterone-to-estradiol ratios and semen analyses were evaluated during testolactone therapy. RESULTS: Men with severe male infertility had significantly lower testosterone (328 versus 543 ng/dl, p <0.01) and higher estradiol (58.4 versus 43.5 ng/l, p = 0.01) than fertile control reference subjects, resulting in a decreased testosterone-to-estradiol ratio (x10(-1) = 6.9 +/- 0.6 versus 14.5 +/- 1.2, respectively, p <0.01). Of the 45 men treated with testolactone a correction of these abnormalities was seen and ratios (x10(-1)) increased into the normal range (5.0 +/- 0.3 to 12.7 +/- 1.2, p <0.01). Semen analyses were considered evaluable only in men with sperm in the ejaculate before aromatase inhibitor treatment. Semen analyses before and during testolactone treatment revealed significant increases in sperm concentration (16.1 to 28.9 million sperm per ml, p = 0.03) and motility (27.1% to 45.3%, p <0.01) in 12 oligospermic men. CONCLUSIONS: We identified an endocrinopathy in men with severe male factor infertility that is characterized by a decreased serum testosterone-to-estradiol ratio. This ratio can be corrected by aromatase inhibition, resulting in a significant improvement in semen parameters in oligospermic patients.

Acquired hypogonadotropic hypogonadism presenting as decreased seminal volume.

A 32-year-old man with decreased ejaculatory volume was found to have acquired hypogonadotropic hypogonadism. Initial evaluation demonstrated castrate levels of testosterone with low serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels. Semen analysis revealed a volume of 0.35 cc and severe oligospermia. Administration of gonadotropin-releasing hormone (GnRH) did not effect an increase in LH or FSH, indicating a pituitary defect. Magnetic resonance imaging revealed a partially empty sella turcica. Treatment with human chorionic gonadotropin (hCG) alone resulted in normalization of testosterone levels, sperm concentration, and semen volume, as well as the successful conception and delivery of a healthy baby girl. The findings from this case demonstrate the importance of considering low serum testosterone levels in the evaluation of low semen volume, as well as the role of hCG alone as an infertility treatment for acquired hypogonadotropic hypogonadism.

Use of electively cryopreserved microsurgically aspirated epididymal sperm with IVF and intracytoplasmic sperm injection for obstructive azoospermia.

OBJECTIVE: To investigate the efficacy of using intentionally cryopreserved epididymal sperm in selected cases of obstructive azoospermia. DESIGN: A retrospective, nonrandomized study. SETTING: Academic research environment. PATIENTS: One hundred forty-one couples undergoing first-time IVF/ICSI using either fresh or cryopreserved epididymal sperm. INTERVENTIONS: The epididymides were microsurgically aspirated. MAIN OUTCOME MEASURES: Clinical pregnancy rates. RESULTS: Motile sperm were obtained from all men. For the fresh group, the mean total sperm aspirated was 99 x 10(6) with 5.5 vials frozen per patient after ICSI and 82 x 10(6) with 4.7 vials frozen per patient in the cryopreserved group. No statistically significant difference in oocyte fertilization rate or number of embryos transferred was noted between groups. Of 108 patients using freshly aspirated sperm, 72 (66.7%) achieved clinical pregnancy. Of 33 patients in the group using cryopreserved sperm, 20 (60.6%) achieved clinical pregnancy (P=0.47). CONCLUSIONS: In selected ideal cases of unreconstructable azoospermia, elective open microsurgical epididymal sperm aspiration with cryopreservation yields pregnancy rates similar to that employing fresh sperm. The advantages of this method are: (1) Use of cryopreserved sperm obviates the logistics problems associated with the use of fresh sperm. (2) Abundant high-quality sperm can be cryopreserved in a single procedure for all future attempts at IVF/ICSI. Rarely, viable sperm will not be present after thawing, and fresh retrieval will be necessary.

Xenogeneic transplantation of human spermatogonia.

In the last 3 years, several studies have shown that xenogeneic transplantation of rodent spermatogonia is feasible. The treatment of infertile patients with spermatogenic arrest using the injection of immature germ cells has yielded only poor results. We attempted to establish a complete spermatogenetic line in the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) transplanted with germ cells from azoospermic men. Spermatogenic cells were obtained from testicular biopsy specimens of men (average age of 34.3 +/- 9 years) undergoing infertility treatment because of obstructive and non-obstructive azoospermia. Testicular tissue was digested with collagenase to promote separation of individual spermatogenic cells. The germ cells were injected into mouse testicular seminiferous tubules using a microneedle (40 microm inner diameter) on a 10 ml syringe. To assess the penetration of the cell suspension into the tubules, trypan blue was used as an indicator. Mice were maintained for 50 to 150 days to allow time for germ cell colonisation and development prior to them being killed. Testes were then fixed for histological examination and approximately 100 cross-sectioned tubules were examined for human spermatogenic cells. A total of 26 testicular cell samples, 16 frozen and 10 fresh, were obtained from 24 men. The origin of the azoospermia was obstructive (OA) in 16 patients and non-obstructive (NOA) in 8 patients. The concentration of spermatogenic cells in the OA group was 6.6 x 10(6) cells/ml, and 1.3 x 10(6) cells/ml in the NOA group (p < 0.01). The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogenia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes. A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection. However, sections from the remaining testes examined up to 150 days after injection showed tubules lined with Sertoli cells and xenogeneic germ cells were not found. The reason why the two strains of mouse used as recipients did not allow the implantation of human germ cells is probably due to interspecies specificity involving non-compatible cell adhesion molecules and/or immunological rejection.

Use of frozen-thawed testicular sperm for intracytoplasmic sperm injection.

OBJECTIVE: To determine the feasibility of using frozen-thawed testicular spermatozoa for intracytoplasmic sperm injection. DESIGN: Prospective clinical study. SETTING: A university hospital. PATIENT(S): One hundred seventy-five azoospermic men participating in a routine intracytoplasmic sperm injection program. INTERVENTION(S): The men underwent testicular biopsy for cryopreservation of tissue to be used in consecutive intracytoplasmic sperm injection treatment cycles. Their female partners underwent controlled ovarian hyperstimulation for conventional IVF treatment. MAIN OUTCOME MEASURE(S): Fertilization and pregnancy rates. RESULT(S): In 77% of the patients, spermatozoa could be harvested from the testis by an open testicular biopsy technique and used for intracytoplasmic sperm injection after freezing and thawing of testicular tissue. Histopathologic evaluation revealed a Sertoli cell-only pattern in 21%, maturation arrest in 60%, and hypospermatogenesis in 19% of the patients. In 2. 9% of the patients, carcinoma in situ or a germ cell tumor was detected. In all patients, viable spermatozoa could be visualized after the tissue samples were thawed. One hundred thirty-five intracytoplasmic sperm injection treatment cycles were performed, with a fertilization rate of 45% and a clinical pregnancy rate of 30% per oocyte retrieved. CONCLUSION(S): The use of frozen-thawed testicular tissue allows ovarian stimulation of the female partner to be timed and avoids cancellation of ovum pick-up when spermatozoa cannot be retrieved.