Germ cell transplantation into mouse testes procedure.

OBJECTIVE: To illustrate the step-by-step protocol followed to assay germ cell transplantation into the seminiferous epithelium of mouse testes. DESIGN: Video presentation of an animal model for research in reproductive and regenerative medicine. SETTING: Research laboratory. ANIMAL(S): Male nude mice (NU-Foxn1(nu)). INTERVENTION(S): Mice were chemically sterilized with alkylant compounds (busulfan) followed by gonadal microsurgery to inject donor germ cells. MAIN OUTCOME MEASURE(S): Donor cells should be labeled with reporter genes, such as green fluorescent protein (GFP), lactose operon (LacZ), or alternatively design an effective strategy with specific antibodies to track them within the recipient testes. Sperm detection in the ejaculate can also be used as a read out. However, in this case detection of the donor genotype in the sperm is mandatory to elucidate their origin. RESULT(S): In the present study we describe the complete protocol for germ cell transplant by efferent duct injection, including the preparation of recipient mice, surgery for the germ cell transplant, and analysis of recipient testes. The main strength of this technique is that it constitutes the gold standard for a functional test of the germ cell potential as only spermatogonial stem cells are able to properly colonize the seminal lumen. Both fresh and frozen/thawed testicular cells are suitable for this technique as donor germ cells. Also, enrichment of living spermatogonial stem cells, previous to the transplant, seems to improve the efficiency of colonization. For proper colonization of germ cells, the niche should be available and thus mouse strains that lack endogenous spermatogenesis such as W/W(v) mutant mice are usually used. In the case of nonmatched donor cells, seminiferous epithelium of immune-suppressed recipient mice should be germ cell depleted before the transplant. One limitation of this technique is that the procedure can take up to 3 months. Also, in contrast to the full recovery of spermatogenesis in mouse-to-mouse transplants, xenotransplantation of germ cells from phylogenetically distant species, such as humans into mouse recipients, results in colonization of donor cells and spermatogonial expansion, but fail in their spermatogenic progression due to evolutive incompatibilities with the recipient niche. Xenografting of pieces of donor testis tissue under the skin of mouse hosts is an alternative approach that is currently being investigated to try to solve this limitation. CONCLUSION(S): Transplantation of spermatogonial stem cells into the seminal lumen of mouse testes is a functional assay that defines this cellular subpopulation by its ability to colonize it. This technique can be used as a model to elucidate the insights of spermatogonial stem cells, to produce transgenic animals by genetically manipulating donor cells before transplantation, but also it has potential applications in fertility preservation in cattle and humans as it is feasible in large animals, as recent reports have demonstrated with rhesus monkeys, that recovered spermatogenesis after allogenic transplantation, and even from human cadaver testes. Therefore spermatogonial stem cells isolated from prepuberal boys, who are treated with alkylant chemotherapy, could be returned to their testis to regenerate spermatogenesis in the future.

Cell-free circulating plasma hTERT mRNA is a useful marker for prostate cancer diagnosis and is associated with poor prognosis tumor characteristics.

BACKGROUND: Serum prostate-specific antigen (PSA) is the most widely used marker for diagnosing prostate cancer (PCa). It lacks specificity and predictive value, resulting in inaccurate diagnoses and overtreatment of the disease. The aim of this study was to assess the usefulness of plasma telomerase reverse transcriptase (hTERT) mRNA as a diagnostic and prognostic tool for PCa and its association with clinicopathological parameters of tumors. PRINCIPAL FINDINGS: Plasma hTERT mRNA levels were determined by qRT-PCR in 105 consecutive patients with elevated PSA levels and in 68 healthy volunteers. The diagnostic accuracy, the efficacy as a prognostic factor of biochemical recurrence and the association with tumor clinicopathological parameters of plasma hTERT mRNA and serum PSA tests were determined using univariate and multivariate analyses. The results show that plasma hTERT mRNA is a non-invasive biomarker for PCa diagnosis that shows higher sensitivity (85% vs. 83%), specificity (90% vs. 47%), positive predictive value (83% vs. 56%), and negative predictive value (92% vs. 77%) than serum PSA. Plasma hTERT mRNA is significantly associated with poor prognosis tumor clinicopathological parameters and is a significant independent predictor of PCa (p<0.0001). Univariate analysis identified plasma hTERT mRNA (but not serum PSA) as a significant prognostic factor of biochemical recurrence. Plasma hTERT mRNA Kaplan-Meier curves confirmed the significant differences between groups and patients with higher levels than the cut-off value showed diminished recurrence-free survival (p=0.004), whereas no differences were observed with serum PSA (p=0.38). Multivariate analysis indicated that plasma hTERT mRNA (but not serum PSA) and stage were significantly associated with biochemical recurrence. CONCLUSIONS: Overall, these findings indicate that hTERT mRNA is a useful non-invasive tumor marker for the molecular diagnosis of PCa, affording a greater diagnostic and prognostic accuracy than the PSA assay and may be of relevance in the follow-up of the disease.

Plasma hTERT mRNA discriminates between clinically localized and locally advanced disease and is a predictor of recurrence in prostate cancer patients.

INTRODUCTION: Since the introduction of prostate-specific antigen (PSA) testing, new prostate cancer (PCa) patients are diagnosed earlier and most have localized and locally advanced disease. Current diagnosis methods lack specificity and sensitivity, leading to overdiagnosis and overtreatment of patients with low-risk organ-confined localized disease. Therefore, new non-invasive molecular tools are needed to discriminate between localized and locally advanced disease. METHODS: Plasma telomerase reverse transcriptase (hTERT) mRNA levels were determined by qRT-PCR in 49 patients with localized and locally advanced PCa. Diagnostic accuracy and efficacy as a prognostic factor of biochemical recurrence of plasma hTERT mRNA were determined using univariate and multivariate analyses and compared with conventional tumor markers. RESULTS: Patients with locally advanced disease had significantly (p < 0.05) higher plasma hTERT mRNA and serum PSA levels than those with localized disease. Plasma hTERT mRNA test showed lower sensitivity (83% vs. 100%), higher specificity (73% vs. 43%), AUC ROC curve (0.911 vs. 0.757), and positive likelihood ratios (6.17 vs. 1.76) than the PSA assay in discriminating between localized and locally advanced disease. At multivariate analysis, plasma hTERT mRNA levels and age but not PSA showed a positive trend (p = 0.05) in the risk of locally advanced PCa. On univariate analysis, plasma hTERT mRNA and serum PSA were identified as significant prognostic factors of biochemical recurrence. Using ROC curves and the appropriate cutoff, both tests showed high sensitivity (85%) and specificity (72%). Kaplan-Meier curves confirmed the significant differences between the groups and patients with higher levels than the cutoff value showed diminished recurrence-free survival (p < 0.05). At multivariate analysis, Gleason score and PSA were the strongest factors associated with biochemical recurrence (p < 0.05), whereas hTERT mRNA did not reach statistical significance, although a positive trend was observed (p = 0.09). CONCLUSION: Plasma hTERT mRNA quantification can be both a useful non-invasive tumor marker for discriminating between localized and locally advanced PCa, as well as a prognostic factor of recurrence at the molecular level.

Impact of benign prostatic hyperplasia surgery on erectile function.

AIM: We evaluate the prevalence of erectile dysfunction (ED) prior to surgery for benign prostatic hyperplasia (BPH) and changes produced after surgical intervention. PATIENTS AND METHODS: This prospective study included 128 patients treated surgically for BPH. The prevalence of ED was determined before and after surgery according to the International Index of Erectile Function (IIEF). The influence of different clinical variables on erectile function (EF) improvement or deterioration after surgery was determined using uni- and multivariate analyses. RESULTS: Mean IIEF score before surgery was 20.5 +/- 7.6. Overall, ED was absent in 32% of patients, mild in 42%, moderate in 13.3%, and severe in 12.5%. Mean IIEF score following surgery was 21.5 +/- 7.4 (p = n.s.). After surgery EF improved in 26.6% (34/128) of patients and worsened in 18.8% (24/128) (p < 0.05). Analysing the subset of patients with presurgical ED, 39% reported improvement and 21.1% reported worsening of EF postoperatively. None of the variables analyzed showed a significant relationship with improvement or worsening of EF. Only age was related to worsening EF in the subgroup of non-ED patients. CONCLUSIONS: There is a high prevalence of ED amongst candidates for BPH surgery. Although the risk of worsening EF exists postsurgically, an important percentage of ED patients will improve.

Emotional changes in men treated with sildenafil citrate for erectile dysfunction: a double-blind, placebo-controlled clinical trial.

INTRODUCTION: Erectile dysfunction (ED) has been associated with several comorbidities and can cause significant loss of quality of life and self-esteem. AIM: In men with ED, to use the validated Self-Esteem and Relationship (SEAR) questionnaire to evaluate changes in self-esteem associated with sildenafil treatment of ED and to assess changes dependent on concomitant comorbid conditions. METHODS: This was a 14-week, international, randomized, parallel-group, double-blind, flexible-dose (25, 50, or 100 mg), placebo-controlled study of sildenafil in men aged >or=18 years with a clinical diagnosis of ED (score

First report of the absence of viral load in testicular sperm samples obtained from men with hepatitis C and HIV after washing and their subsequent use.

Human immunodeficiency virus and hepatitis C infections are sexually transmitted diseases that require sperm samples to be pretreated to eliminate the viral presence before their safe use in assisted reproduction treatments. In this report we describe our experience with sperm washing protocols applied to sperm cells from testicular biopsies as well as the results obtained in subsequent assisted reproduction treatments on seropositive males that are also azoospermic.

Relationship between the Saint Louis University ADAM questionnaire and sexual hormonal levels in a male outpatient population over 50 years of age.

OBJECTIVES: To determine the relationship between the ADAM questionnaire and the sexual hormonal levels in a male population older than 50 yr, and to know the predictive capacity of this questionnaire with regard to biochemical hypogonadism in the ageing male. METHODS: A prospective study was carried out on 230 Spanish men. Patients were evaluated by clinical history. The ADAM questionnaire and the Yesavage's Geriatric Depression Scale were completed by each patient. Blood tests were performed including total testosterone, SHBG, free testosterone (FT), dehidroepiandrosterone sulfate (DHEA-S), androstenedione, 17-beta-estradiol, FSH, LH, and prolactin. The relationship between positive ADAM questionnaire and age, clinical and sociodemographic backgrounds, and hormone levels was analysed by means of uni- and multivariate tests. The capacity of the ADAM questionnaire to predict biochemical hypogonadism was determined with a chi-square test. RESULTS: ADAM questionnaire (excluding men with positive Yesavage's Scale) was positive in 140 patients (67.9%). With respect to clinical backgrounds, diabetes mellitus and age had a significant relationship with an ADAM-positive questionnaire. With respect to hormones, FT and DHEA-S levels were significantly lower when the ADAM questionnaire was positive. In the multivariate analysis, age, FT, and diabetes were independently related to an ADAM-positive questionnaire. Prevalence of biochemical hypogonadism (FT<0.228 nmol/l) was 24.6%. The ADAM test had a sensitivity of 84.0% and a specificity of 36.6% to detect biochemical hypogonadism. CONCLUSIONS: FT is inversely related to the ADAM-positive questionnaire, independently of age. The ADAM questionnaire is a valid test to detect hypogonadism but has low specificity.

Percentage of cancer in prostate biopsies as prognostic factor for staging and postoperative biochemical failure after radical prostatectomy.

OBJECTIVE: To assess if the percentage of cancer in prostate needle biopsies provides independent prognostic information for predicting pathological stage and/or biochemical relapse after radical prostatectomy. METHODS: One hundred and forty prostate cancer patients who underwent radical prostatectomy were evaluated. Preoperative parameters analyzed were patient age, PSA, clinical stage, and the information obtained from sextant biopsies (Gleason score, maximum percentage of cancer in a core, percentage of tissue with cancer in all biopsies and the number of cores positive for cancer). Univariate and multivariate analyses (logistic regression) for the dependent variables (prostate cancer, organ-confined and biochemical relapse) were performed. RESULTS: The tumor was organ-confined in 73.6% of patients. In those patients studied for disease progression (n = 126), no biochemical recurrence was observed in 76.2%. In the multivariate analysis for organ-confined disease, the total percentage of biopsy tissue with cancer, the preoperative PSA level, the Gleason score and the clinical stage were the most accurate predictive factors of pathological stage. The multivariate analysis for the study of biochemical failure indicated that only the total percentage of biopsy tissue with cancer, the preoperative PSA level and the Gleason score were independent predictive factors. According to the logistic regression analysis for disease recurrence, 3 risk groups could be identified: low risk (less than 10% probability of disease progression), intermediate risk (30%) and high risk (more than 70%). CONCLUSIONS: The percentage of cancer in prostate biopsy provides independent prognostic information for predicting pathological stage and the risk of biochemical failure after radical prostatectomy.

Testosterone levels in men with erectile dysfunction.

OBJECTIVE: To investigate the frequency of hypogonadism in men with erectile dysfunction (ED) and to assess which factors are related with low testosterone levels. PATIENTS AND METHODS: In all, 165 men with ED were assessed; the evaluation included: hormonal profiles, serum total and free testosterone (using Vermeulen's formula) levels, and self-reported questionnaires on erectile function and desire domains of the International Index of Erectile Function. The frequency of hypogonadism was established using total and free testosterone levels as diagnostic criteria. The factors that might influence testosterone levels were evaluated by univariate and multivariate statistical analysis, and a logistic regression was used to determine which factors can predict free testosterone levels below normal limits (biochemical hypogonadism). RESULTS: Using the total testosterone levels, 4.8% of the men were hypogonadal, whereas when using the free testosterone levels, 17.6% were hypogonadal. In the univariate analyses, not smoking and hypertension were associated with lower total and free testosterone levels. Ageing, absence of nocturnal erections and a lower erectile function score were only associated with lower free testosterone serum levels. There was no association between total and free testosterone levels and desire. In the multivariate analysis, only total testosterone levels were related to hypertension, while free testosterone levels were related to age and nocturnal erections. For biochemical hypogonadism, simple logistic regression analysis selected age, erectile function score and aetiological diagnosis of ED as predictors. In the multivariate analysis only the erectile function score had significant independent prognostic value. CONCLUSIONS: The frequency of hypogonadism is higher when free testosterone levels are used for diagnosis. The total and free testosterone levels were not related to the level of sexual desire in men with ED. The free testosterone levels could be related to the quality and frequency of nocturnal erections, and when ED is more severe, it is more probable that free testosterone levels are below the 'normal' limit.

Prognostic value of DNA ploidy and nuclear morphometry in prostate cancer treated with androgen deprivation.

OBJECTIVES: To assess the prognostic value of flow cytometry and nuclear morphometry in prostate cancer after androgen deprivation treatment. METHODS: A total of 127 patients with a prostate cancer diagnosis who had undergone androgen suppression were retrospectively studied. The DNA content by flow cytometry and nuclear morphometry was studied from biopsy specimens. In the patients with Stage M0, two multivariate analyses by the Cox proportional regression model were performed to determine whether the experimental variables (DNA content and nuclear area) added independent information to the classic prognostic factors (Gleason score and stage). Using the statistical analysis results, risk groups were created. RESULTS: T and M categories, Gleason score, DNA ploidy, and mean nuclear area proved to have prognostic value in the univariate analysis. For the group of patients free of metastasis (M0), it was possible to create low, intermediate, and high-risk groups using stage and Gleason score with statistically significant differences in survival. Multivariate analysis, combining the classic and experimental variables, selected Gleason score and DNA content as prognostic independent factors. Also, risk groups with statistically significant differences in survival were created. However, the net result of combining both kinds of factors was at least as valuable as the combination of stage and Gleason score in predicting survival. CONCLUSIONS: The determination of DNA ploidy and mean nuclear area do not add enough independent information to improve the predictive value to justify their use in this group of patients treated with hormonal therapy.