Ali A. Dabaja

Possible germ cell-Sertoli cell interactions are critical for establishing appropriate expression levels for the Sertoli cell-specific MicroRNA, miR-202-5p, in human testis

BackgroundTo examine human microRNA expression in fertile men and subsequently to compare expression patterns of miRNAs in fertile and infertile men, specifically men with Sertoli Cell Only (SCO) histopathology.MethodsTesticular tissues from men with azoospermia and SCO, as well as those of men with normal spermatogenesis, were analyzed. MicroRNA was isolated using the miRCURY™ RNA Purification Kit. A miRCURY LNA™ Universal RT system was used for detection of microRNA by quantitative real-time PCR. MicroRNA localization was performed by in situ hybridizations (ISH) on formalin-fixed paraffin embedded (FFPE) tissue utilizing miRCURY LNA™ microRNA ISH technology. Statistical analysis was performed by GenEx V5.0.ResultsMicroRNA expression was determined for 13 normal fertile men and 5 men with the confirmed diagnosis of diffuse SCO. MiR-202-5p expression was reduced by 17-fold (Pu2009<u20090.00001) in tissue from SCO men compared to normal. MiR-34c-5p was reduced by 346-fold (Pu2009<u20090.00001), miR-10b was reduced 18-fold (Pu2009<u20090.00001), miR-191 was reduced 20-fold (Pu2009=u20090.001) and miR-126 was reduced 40-fold (Pu2009<u20090.00001)) in tissues from SCO compared to normal fertile men. Using ISH, miR-202-5p was localized to Sertoli cells of men with normal spermatogenesis, but not in the Sertoli cells of men with SCO.ConclusionNumber of miRNAs are differentially expressed in normal fertile men compared to men with SCO. MicroRNA-202-5p is localized to Sertoli cells and its expression dramatically differs between fertile men and men whose germ cells are depleted, suggesting a novel interaction for regulating microRNA expression between the somatic and germ cell components of the seminiferous epithelium.AbstractObjectifsEvaluer l’expression des microARN chez des hommes féconds puis comparer les profils d’expression de ces miRNAs chez des hommes féconds et des inféconds qui présentent plus particulièrement un syndrome de Sertoli seules (SCO) à l’histologie testiculaire.Matériel et MéthodesOnt été analysés des tissues testiculaires d’hommes avec azoospermie et SCO ainsi que ceux d’hommes avec spermatogenèse normale. Les miRNAs ont été isolés avec la trousse de Purification miRCURY™ RNA. Le système miRCURY LNA™ Universal RT a été utilisé pour la détection quantitative de miARNs par PCR en temps réel. La localisation des miARNs a été réalisée par hybridation in situ (HIS) sur des tissus fixés au formol et inclus en paraffine en utilisant la technologie miRCURY LNA™ microRNA ISH. Les analyses statistiques ont été faites avec GenEx V5.0.RésultatsL’expression des microARNs a été faite chez 13 hommes féconds et 5 hommes avec un diagnostic confirmé de SCO diffus. L’expression de miR-202-5p est réduite d’un facteur 17 (Pu2009<u20090.00001) dans le tissu des hommes SCO par rapport au tissu des hommes à spermatogenèse normale. L’expression de miR-34c-5p est réduite d’un facteur 346 (Pu2009<u20090.00001), celle de miR-10b d’un facteur 18 (Pu2009<u20090.00001), celle de miR-191 d’un facteur 20 (Pu2009=u20090.001) et celle de miR-126 d’un facteur 40 (Pu2009<u20090.00001) dans les tissus des hommes SCO comparés à ceux des hommes à spermatogenèse normale. MiR-202-5p a été localisé par HIS dans les cellules de Sertoli des hommes à spermatogenèse normale, mais pas dans les cellules de Sertoli des hommes SCO.ConclusionsNombre de miARNs sont exprimés différentiellement chez les hommes féconds par rapport aux hommes SCO. MicroARN-202-5p est localisé dans les cellules de Sertoli et son expression diffère de façon marquée entre les hommes féconds et ceux dont les cellules germinales sont absentes; ceci suggère une nouvelle interaction – entre les cellules somatiques et germinales constitutives de l’épithélium séminifère – impliquée dans la régulation de l’expression des microARNs.

Medical treatment of male infertility

The majority of male infertility is idiopathic. However, there are multiple known causes of male infertility, and some of these causes can be treated medically with high success rates. In cases of idiopathic or genetic causes of male infertility, medical management is typically empirical; in most instances medical therapy represents off-label use that is not specifically approved by the FDA. Understanding the hypothalamic-pituitary-gonadal (HPG) axis and the effect of estrogen excess is critical for the assessment and treatment of male infertility. The use of certain medical treatment has been associated with an increase in sperm production or motility, and primarily focuses on optimizing testosterone (T) production from the Leydig cells, increasing follicle-stimulating hormone (FSH) levels to stimulate Sertoli cells and spermatogenesis, and normalizing the T to estrogen ratio.

Varicocele and Hypogonadism

Accumulating evidence suggests that varicocele, long associated with male infertility, is also a risk factor for low testosterone levels. The exact pathophysiology of the negative effects of varicocele on testicular function is not well understood, but theories include venous stasis, increased testicular temperature, oxidative stress, and resulting toxic environment. While prior studies report conflicting effects of non-microsurgical varicocelectomy on testosterone level, recent literature demonstrates that microsurgical varicocelectomy improves testosterone levels in men with varicocele and low testosterone preoperatively.

When is a varicocele repair indicated: the dilemma of hypogonadism and erectile dysfunction?

In the past, the indications for varicocelectomy are primarily for infertility with abnormal semen parameters, testicular hypotrophy/atrophy in adolescents, and/or pain. The surgical treatment of varicocele for hypogonadism is controversial and debated. Recently, multiple reports in the literature have suggested that varicocele is associated with hypogonadism and varicocele repair can increase testosterone levels. Men with hypogonadal symptoms should have at least two serum testosterone levels. Microsurgical varicocelectomy may be beneficial for men with clinically palpable varicoceles with documented hypogonadism. In this review, we summarize the most recent literature linking varicocele to hypogonadism and sexual dysfunction and the impact of repair on serum testosterone levels. We performed a search of the published English literature. The key words used were varicocele and hypogonadism and varicocele surgery and testosterone. We included published studies after 1998. We, also, evaluated the effect of surgery on the changes in the serum testosterone level regardless of the indication for the varicocele repair.

Ubiquitin Specific Protease 26 (USP26) Expression Analysis in Human Testicular and Extragonadal Tissues Indicates Diverse Action of USP26 in Cell Differentiation and Tumorigenesis

Ubiquitin specific protease 26 (USP26), a deubiquitinating enzyme, is highly expressed early during murine spermatogenesis, in round spermatids, and at the blood-testis barrier. USP26 has also been recognized as a regulator of androgen receptor (AR) hormone-induced action involved in spermatogenesis and steroid production in in vitro studies. Prior mutation screening of USP26 demonstrated an association with human male infertility and low testosterone production, but protein localization and expression in the human testis has not been characterized previously. USP26 expression analysis of mRNA and protein was completed using murine and human testis tissue and human tissue arrays. USP26 and AR mRNA levels in human testis were quantitated using multiplex qRT-PCR. Immunofluorescence colocalization studies were performed with formalin-fixed/paraffin-embedded and frozen tissues using primary and secondary antibodies to detect USP26 and AR protein expression. Human microarray dot blots were used to identify protein expression in extra-gonadal tissues. For the first time, expression of USP26 and colocalization of USP26 with androgen receptor in human testis has been confirmed predominantly in Leydig cell nuclei, with less in Leydig cell cytoplasm, spermatogonia, primary spermatocytes, round spermatids, and Sertoli cells. USP26 likely affects regulatory proteins of early spermatogenesis, including androgen receptor with additional activity in round spermatids. This X-linked gene is not testis-specific, with USP26 mRNA and protein expression identified in multiple other human organ tissues (benign and malignant) including androgen-dependent tissues such as breast (myoepithelial cells and secretory luminal cells) and thyroid tissue (follicular cells). USP26/AR expression and interaction in spermatogenesis and androgen-dependent cancer warrants additional study and may prove useful in diagnosis and management of male infertility.

The effect of hypogonadism and testosterone‐enhancing therapy on alkaline phosphatase and bone mineral density

To evaluate the relationship of testosterone‐enhancing therapy on alkaline phosphatase (AP) in relation to bone mineral density (BMD) in hypogonadal men.

When to ask male adolescents to provide semen sample for fertility preservation

Background Fertility preservation in adolescents undergoing sterilizing radiation and/or chemotherapy is the standard of care in oncology. The opportunity for patients to provide a semen sample by ejaculation is a critical issue in adolescent fertility preservation. Methods Fifty males with no medical or sexual developmental abnormalities were evaluated. The subjects were screened for evidence of orgasmic, erectile, and ejaculatory dysfunction. A detailed sexual development history was obtained under an Institutional Review Board (IRB)-approved protocol. Results Fifty males, aged 18-65 years (mean 39±16.03 years) volunteered to be part of this study. The mean reported age for the onset of puberty was 12.39 years (95% CI, 11.99-12.80 years), 13.59 years (95% CI, 13.05-14.12 years) for the first ejaculation, 12.56 years (95% CI, 11.80-13.32 years) for the start of masturbation, and 17.26 years (95% CI, 16.18-18.33 years) for the first experienced intercourse. Seventy-five percent of the cohort reached puberty by the age of 13.33, experienced masturbation by 14.5, first ejaculated by the age of 14.83, and had intercourse at age of 19.15 years. The first experienced ejaculation fell 1.5 years after the onset of puberty in 80% present of the cohort, and 84% starts masturbation 1.5 years after the onset of puberty. The mean response between the younger and the older subject was not statistical significance. Conclusions It is appropriate to consider a request for semen specimens by masturbation from teenagers at one year and six months after the onset of puberty; the onset age of puberty plus 1.5 years is an important predictor of ejaculation and sample collection for cryopreservation.

A critical analysis of candidacy for penile revascularization.

INTRODUCTIONnPenile revascularization (PR) is a potentially curative procedure for young men with isolated arteriogenic erectile dysfunction. Standard preoperative evaluation is erectile hemodynamics (HDX) using duplex Doppler penile ultrasound (DUS) and/or cavernosometry (DIC) and assessment of cavernosal arterial anatomy by selective internal pudendal arteriography (SIPA).nnnAIMnThe aim of this study was to review our experience with men who sought a second opinion from us regarding their candidacy for PR.nnnMETHODnStudy population consisted of men (i) who presented to us for a second opinion regarding PR; (ii) who had DUS/DIC and SIPA; and (iii) had been advised by outside surgeon to undergo PR. Review of the HDX study and SIPA was conducted. Discrepancies between these studies resulted in repeating the DIC in men with normal SIPA or repeating the SIPA in men with normal HDX studies.nnnMAIN OUTCOME MEASURESnDiscrepancies between HDX and SIPA and the results of repeat HDX or SIPA were the main outcome measures.nnnRESULTnForty-five patients participated in the study; mean age was 33 years with 4% ≥50 years old. Median vascular risk factor number was 1 (ranged 0-3). A credible trauma history was present in 11%. Thirty-three percent had prior DIC and 49% of patients had a significant discrepancy between HDX study and SIPA, including all patients seen by a community urologist. Thirty-eight percent had a discrepancy between side of abnormality on HDX and SIPA where both studies were abnormal (group A). Seven percent had abnormal HDX and normal SIPA (group B). Four percent had a normal HDX study with an abnormal SIPA (group C). Repeat DIC (nu2009=u200920) was conducted in groups Au2009+u2009B and was normal in 70% of cases. Repeat SIPA (nu2009=u20092) was conducted in group C and was normal in both patients.nnnCONCLUSIONnAlmost one half of patients had a significant discrepancy between HDX and SIPA. Of these, 73% had normal repeat studies, making them no longer candidates for penile revascularization.

Bulbocavernosus muscle area measurement: a novel method to assess androgenic activity

Serum testosterone does not correlate with androgen tissue activity, and it is critical to optimize tools to evaluate such activity in males. Ultrasound measurement of bulbocavernosus muscle (BCM) was used to assess the relationship between the number of CAG repeats (CAGn) in the androgen receptor (AR) and the BCM size; the changes in the number of CAGn over age were also evaluated. Transperineal ultrasound measurement of the BCM was also performed. AR CAGn were determined by high performance liquid chromatography, and morning hormone levels were determined using immunoassays. Forty-eight men had CAG repeat analysis. Twenty-five were <30 years of age, mean 23.7 years (s.d. = 3.24) and 23 were >45 years of age, mean 53 years (s.d. = 5.58). The median CAGn was 21 (13–29). BCM area was greater when the number of CAGn were <18 as compared to the number of CAGn >24 (P = 0.04). There was a linear correlation between the number of CAGn and the BCM area R2 = 16% (P = 0.01). In the 45 to 65-years-old group, a much stronger negative correlation (R2 = 29%, P = 0.01) was noticed. In the 19 to 29-years-old group, no such correlation was found (R2 = 4%, P = 0.36). In older men, the number of CAGn increased with age (R2 = 32%, P = 0.01). The number of CAGn in the AR correlates with the area of the BCM. Ultrasound assessment of the BCM is an effective surrogate to evaluate end-organ activity of androgens. The number of CAGn may increase with age.

Microsurgical Hydrocelectomy: Rationale and Technique

Introduction: Hydrocelectomy may be complicated by hematoma, infection, testicular atrophy, epididymal injury and recurrence. Microsurgical hydrocelectomy allows for better visualization of epididymal tubules and testicular vasculature. We investigated the clinical outcomes of microsurgical hydrocelectomy. Methods: We retrospectively reviewed the charts of all men who underwent microsurgical hydrocelectomy from 1999 to 2013. All operations were performed through a scrotal incision. The spermatic vessels, vas deferens and epididymis were identified under an operating microscope at 6× to 25× magnification. The hydrocele sac was excised under the microscope. Assessment of operative outcomes included postoperative physical examination, need for subsequent fertility treatment and recurrence. Pathology reports were reviewed and descriptive analysis was performed. Results: Mean ± SD patient age was 44.4 ± 17.4 years. Mean hydrocele volume was 275.5 ± 260 ml. The etiology was prior inguinal hernia repair in 21% of patients, nonmicrosurgical varicocelectomy in 16%, recurrent hydroceles in 14%, epididymitis or orchitis in 7%, trauma or scrotal surgery in 7% and an idiopathic cause in 35%. Surgery was performed on 70 units (12 bilateral) in a total of 58 men. There was a 12% postoperative complication rate and all complications were Clavien grade I. No recurrence developed during a mean followup of 68.8 months. At hydrocelectomy concomitant surgical procedures were done in 60% of cases, of which 50% were spermatocelectomy. In 69 hydrocele sac pathology specimens no epididymal tubules were identified. In an older man in whom fertility was not an issue part of the epididymis was intentionally transected with the sac. Conclusions: Microsurgical hydrocelectomy minimizes the risk of injury to the epididymis, vas deferens or testicular blood supply as well as the risk of infection or hematoma.