Identification and Functional Investigation of Novel Heterozygous HELQ Mutations in Patients with Sertoli Cell-only Syndrome.

Background: Male infertility is a major health concern in couples of childbearing ages. Nonobstructive azoospermia (NOA) is an extreme form of male infertility that affects ∼1% of adult men, and the etiology remains unknown in most cases. Sertoli cell-only syndrome (SCOS) is the most severe type of NOA. Aims: To explore novel human candidate variants that cause SCOS. Methods: (1) Whole exome sequencing (WES) of 20 men with SCOS, (2) Sanger sequencing of the HELQ gene in an additional 163 men with SCOS, (3) in vitro functional assays, and (4) in vivo studies. Results: WES of 20 patients with SCOS led to the identification of two heterozygous missense mutations (M1 and M2) in two unrelated Chinese patients with infertility. Using subsequent Sanger sequencing covering all the coding regions of the HELQ gene for 163 additional SCOS cases, we identified four additional heterozygous mutations (M3-M6) in unrelated patients. In vitro functional analyses revealed that two of these mutations (M5, c.2538T > G and M6, c.2945G > T) might affect the function of the HELQ protein. Two heterozygous mutant mouse models with mutations similar to those of two patients (M5 and M6) did not show any considerable spermatogenic defects. Conclusion: Assuming that the mouse models accurately reflect the impact of the mutations, heterozygous HELQ variants alone did not lead to the development of the SCOS phenotype in mice. However, we cannot rule out the risk variants in Chinese or other human populations, and a larger dataset is needed to confirm the association between HELQ mutations with SCOS.

Sertoli Cells Only Syndrome - Case Report.

Exclusive Sertoli Cell Syndrome (ESCS) is a rare condition that has male infertility as its main consequence. It is one of the most serious forms of non-obstructive azoospermia, with a poor reproductive prognosis. In some cases, however, such as the type II of the syndrome, sperm can be recovered through testicular puncture and subsequent ICSI, with a 13% success rate. This article aims to report the case of an azoospermic 35-year-old patient, with no other significant changes in complementary exams. After percutaneous puncture of the epididymis and biopsy with no sperm, we diagnosed ESCS, and indicated IVF with donor semen.

Pituitary hyperplasia with Sertoli cell-only and 47,XYY syndromes: an uncommon triad.

We report the case history of a 32-year-old man with no phenotypical abnormalities who presented with infertility. Semen analysis revealed azoospermia and testicular biopsy confirmed Sertoli cell-only (SCO) syndrome. Karyotyping revealed 47,XYY and pituitary hyperplasia was found on MRI pituitary. In our patient, 47,XYY karyotype is likely to have given rise to SCO syndrome that in turn resulted in pituitary hyperplasia. The patient was evaluated by various members of the multidisciplinary team including the pituitary surgeon, endocrinologist and andrologist. The patient's partner successfully delivered a healthy baby via in vitro fertilisation with donor sperm. This triad of diagnoses (SCO syndrome, 47,XYY karyotype and pituitary hyperplasia) has not been reported previously. SCO syndrome should be considered in the presence of azoospermia, elevated follicle-stimulating hormone, low inhibin-B and normal testosterone levels. Our case report also highlights the importance of excluding genetic causes of infertility even when the patient has no phenotypical abnormalities.

Testicular sperm extraction in men with sertoli cell-only testicular histology - 1680 cases.

OBJECTIVE: To study the outcomes of testicular sperm extraction (TESE) among men with pure Sertoli cell-only histology identified during diagnostic testicular biopsy. METHODS: This retrospective cohort study involved 1680 cases of patients with nonobstructive azoospermia (NOA) diagnosed with pure Sertoli cell-only histology who underwent testicular biopsy with TESE in a reference center in Brazil by a single surgeon. Sperm retrieval rates (SSR) were the main outcome measure. RESULTS: Overall, 14.83% of patients with Sertoli cell-only had sperm retrieved with TESE in quantity that allowed the performance of ICSI. No differences were observed in SSR based on testis volume (<15 mL vs. <15 mL) or serum FSH level. CONCLUSIONS: Patients with Sertoli cell-only histology can be counseled that they have some likelihood of sperm retrieval with TESE. Based on the findings, patients to be submitted to testicular biopsy for histologic analysis may be concomitantly prepared for ICSI with TESE in case sperm is available.

Noonan syndrome males display Sertoli cell-specific primary testicular insufficiency.

Context Abnormalities in the hypothalamo-pituitary-gonadal axis have long been reported in Noonan syndrome (NS) males with only few data available in prepubertal children. Objective The aim of this study was to describe the gonadal function of NS males from childhood to adulthood. Design It is a retrospective chart review. Patients and methods A total of 37 males with a genetically confirmed diagnosis of NS were included. Clinical and genetic features, as well as serum hormone levels (LH, FSH, testosterone, anti-Müllerian hormone (AMH), and inhibin B) were analysed. Results Of the 37 patients, 16 (43%) children had entered puberty at a median age of 13.5 years (range: 11.4-15.0 years); age at pubertal onset was negatively correlated with BMI SDS (r = -0.541; P = 0.022). In pubertal boys, testosterone levels were normal suggesting a normal Leydig cell function. In contrast, NS patients had significant lower levels of AMH (mean SDS: -0.6 ± 1.1; P = 0.003) and inhibin B (mean SDS: -1.1 ± 1.2; P < 0.001) compared with the general population, suggesting a Sertoli cell dysfunction. Lower AMH and inhibin B levels were found in NS-PTPN11 patients, whereas these markers did not differ from healthy children in SOS1 patients. No difference was found between cryptorchid and non-cryptorchid patients for AMH and inhibin B levels (P = 0.43 and 0.62 respectively). Four NS-PTPN11 patients had a severe primary hypogonadism with azoospermia/cryptozoospermia. Conclusions NS males display Sertoli cell-specific primary testicular insufficiency, whereas Leydig cell function seems to be unaffected.

MicroRNAs in Sertoli cells: implications for spermatogenesis and fertility.

In recent decades, infertility has been considered a major widespread public health issue of very high concern. Currently, almost 50% of infertility cases are due to male factors, including semen disorders, obstructions, cryptorchidism, varicocele and testicular failures, which can occur due to malfunctions in both somatic and germ cells. In this context, besides other approaches, different miRNAs have been used as biomarkers for the diagnosis of male infertility, with different pathologic conditions such as Sertoli cell-only syndrome, mixed atrophy, and germ cell arrest. However, most studies related to male fertility do not point out the functions and cell targets of the described miRNAs. Initial investigations using experimental assays in murine and porcine models were performed, providing the first evidence of the influence of miRNAs on Sertoli cell function including, for instance, proliferation, maturation and hormone responses of these cells. The aim of this mini-review is therefore to summarize our present knowledge of this relevant subject and to highlight the importance of future investigations concerning the miRNA influence in the control of Sertoli cells, spermatogenesis and male fertility.

Testicular histology may predict the successful sperm retrieval in patients with non-obstructive azoospermia undergoing conventional TESE: a diagnostic accuracy study.

PURPOSE: The present study sought to determine the diagnostic accuracy of FSH level, testicular volume, and testicular histology in predicting the successful sperm retrieval (SSR) in a large cohort of patients with non-obstructive azoospermia undergoing conventional testicular sperm extraction (TESE). METHODS: We retrospectively evaluated 356 patients with non-obstructive azoospermia between June 2004 and July 2009. Binary logistic regression was used to evaluate the diagnostic accuracy of our predicting model, identifying sperm retrieval rate as binary dependent variable. The predictive accuracy of all variables individually evaluated was quantified with area under curve (AUC) estimates derived from receiver operating characteristic (ROC) curve. RESULTS: The mean patients' age was 36.8 years. Testicular sperm were retrieved in 158 out of 356 patients (44.3 %). Histological diagnosis of Sertoli cell only syndrome (SCO) was obtained in 216 patients (60.6 %), while 55 patients (15.4 %) had maturation arrest (MA) and 85 (23.8 %) had hypospermatogenesis (HYPO). The binary logistic regression model was statistically significant (χ 2 = 96.792, p < 0.0001) and correctly classified 72.8 % of cases with 46.8 % sensitivity and 93.4 % specificity, positive predictive value (PPV) 85.06 %, negative predictive value (NPV) 68.7 %, +likelihood ratio (LR) 7.13, and -LR 0.57. Only testicular histology was significant to the model, while FSH and testicular volume were not. Sperm retrieval rate (SRR) was significantly higher in patients with HYPO compared to patients with SCO or MA (88.2 vs 30.5 and 30.9 %, respectively, p < 0.0001) CONCLUSIONS: This study demonstrates that including testicular histology in a model for predicting sperm retrieval increases its diagnostic accuracy. As histology is not available prior to TESE, this model applies only to patients with previous testicular surgery.

Discrimination and characterization of Sertoli cell-only syndrome in non-obstructive azoospermia using cell-free seminal DDX4.

Cell-free seminal mRNA (cfs-mRNA) contains testis-specific transcripts from bilateral testes. This study determined the presence of DEAD box polypeptide 4 (DDX4) in cfs-mRNA to identify and characterize the incidence of Sertoli cell-only (SCO) syndrome in men with non-obstructive azoospermia (NOA). DDX4 cfs-mRNA was determined in 315 men with NOA, and compared with testicular samples obtained by microdissection from 19 NOA patients. Karyotype and azoospermia factor microdeletion analysis were performed, and clinical features were evaluated. Negative DDX4 cfs-mRNA suggestive of SCO was found in 13.7% of NOA patients, with a similar incidence in NOA men with known genetic causes and those without known genetic causes. DDX4 cfs-mRNA was absent in 44% of SCO cases diagnosed by testicular histopathology, but present in all patients presenting with maturation arrest or hypospermatogenesis. Furthermore, 84.2% of NOA men with DDX4 cfs-positive mRNA had a DDX4-positive testicular sample. In NOA men without genetic causes, SCO patients discriminated by negative DDX4 cfs-mRNA showed different clinical features when compared with non-SCO cases. These results suggest that the evaluation of DDX4 cfs-mRNA is more accurate than testicular histopathology in discriminating SCO, and also permits the identification of a specific group of NOA men with distinct clinical features.

Diagnostic value of fine needle aspiration cytology in testicular disorders of red deer (Cervus elaphus): a case report.

We used fine needle aspiration cytology (FNAC) to diagnose Sertoli cell-only pattern and hypospermatogenesis in an Iberian red deer (Cervus elaphus hispanicus). Cytologic diagnosis was confirmed by histology and epididymal sperm analysis. We conclude that FNAC can be an important diagnostic tool in testicular diseases of wildlife.

A novel application of cell-free seminal mRNA: non-invasive identification of the presence of germ cells or complete obstruction in men with azoospermia.

BACKGROUND: Cell-free seminal mRNA (cfs-mRNA) exists in human ejaculate at high concentrations and with high stability, and contains many tissue-specific transcripts secreted from the male reproductive system. Owing to the sensitivity of RNA technology, cfs-mRNAs are ideal candidates for non-invasive biomarkers of physiopathological conditions. This study applied cfs-mRNA in identifying the presence of either germ cells or complete obstruction in men with azoospermia. METHODS: RT-PCR was performed to amplify the germ cell-specific (DDX4), seminal vesicle-specific (SEMG1) and prostate-specific (TGM4) mRNAs from cfs-mRNAs, which were isolated from the seminal plasma of men with non-obstructive azoospermia (NOA) or obstructive azoospermia (OA). The 39 patients with NOA, diagnosed by testicular biopsy, included 8 men with maturation arrest (MA), 3 men with incomplete sertoli cell only (iSCO) syndrome and 28 men with complete SCO (cSCO). The 29 patients with OA, confirmed by the presence of sperm in the testis or epididymis, included 8 men with congenital bilateral absence of the vas deferens (CBAVD) and 21 men with non-CBAVD. Healthy individuals and vasectomized men were enrolled as controls. RESULTS: TGM4 was detected in all participants. Consistent with their diagnosis, DDX4 was detected in all patients with MA or iSCO but was absent in most cases of cSCO (n = 21, 75.0%) or non-CBAVD (n = 18, 85.7%), and in all men with vasectomy or CBAVD. The presence of DDX4 in the other seven men with cSCO and three patients with non-CBAVD suggests the presence of germ cells in the testis, and incomplete obstruction, respectively. SEMG1 was undetectable in three patients with CBAVD with bilateral absence of the seminal vesicles, and in two non-CBAVD cases with low ejaculate volume. CONCLUSIONS: These results suggest that, with high sensitivity and representativity, cfs-mRNA could be non-invasive biomarkers for identifying the presence of germ cells or complete obstruction in azoospermia.

Recognising the Sertoli-cell-only (SCO) syndrome: a case study.

Total Sertoli-cell-only (SCO) syndrome is often confused with a focal SCO picture, in which testicular illness caused damage to seminiferous tubules and compromised the Sertoli cell range of maturation and functions, but from which still some spermatozoa can be retrieved for assisted reproductive techniques. Here, a possibly new SCO syndrome phenotype is reported exhibiting complete lack of germ cells despite normal architecture of the seminiferous tubules with presence of mature Sertoli cells and normal Leydig cells in the intertubular tissue. Sertoli cells are immunonegative for the prepubertal differentiation markers cytokeratin-18, anti-Muellerian hormone and M2A antigen, but reveal a positive signal for the gap junctional protein connexin 43 known to be expressed in Sertoli cells with an adult type of differentiation. The complete lack of germ cells in combination with fully differentiated adult-type Sertoli cells in this case is in contradiction with known SCO subtypes and with the current hypothesis of reciprocal regulation of Sertoli and germ cell differentiation.

Azoospermia and Sertoli-cell-only syndrome: hypoxia in the sperm production site due to impairment in venous drainage of male reproductive system.

Sertoli-cell-only (SCO) syndrome, or germ cell aplasia, is diagnosed on testicular biopsy when germ cells are seen to be absent without histological impairment of Sertoli or Leydig cells. It is considered a situation of irreversible infertility. Recent studies have shown that varicocele, a bilateral disease, causes hypoxia in the testicular microcirculation. Destruction of one-way valves in the internal spermatic veins (ISV) elevates hydrostatic pressure in the testicular venules, exceeding the pressure in the arteriolar system. The positive pressure gradient between arterial and venous system is reversed, causing hypoxia in the sperm production site. Sperm production deteriorates gradually, progressing to azoospermia. Our prediction was that, if genetic problems are excluded, SCO may be the final stage of longstanding hypoxia which deteriorates sperm production in a progressive process over time. This would indicate that SCO is not always an independent disease entity, but may represent deterioration of the testicular parenchyma beyond azoospermia. Our prediction is confirmed by histology of the seminiferous tubules demonstrating that SCO is associated with extensive degenerative ischaemic changes and destruction of the normal architecture of the sperm production site. Adequate treatment of bilateral varicocele by microsurgery or by selective sclerotherapy of the ISV resumes, at least partially, the flow of oxygenated blood to the sperm production site and restored sperm production in 4 out of 10 patients. Based on our findings the following statements can be made: (i) SCO may be related in part of the cases to persistent, longstanding testicular parenchymal hypoxia; (ii) germ cells may still exist in other areas of the testicular parenchyma; and (iii) if genetic problems are excluded, adequate correction of the hypoxia may restore very limited sperm production in some patients.

Usefulness of testicular fine needle aspiration cytology in cases of infertility.

Testicular fine needle aspiration cytology (FNAC) is an important investigation in management of male infertility, especially to differentiate between obstructive and non obstructive causes of azoospermia. It is less invasive and associated with no or minimal complications. Nowadays when assisted fertilization techniques are being practiced, fibrosis after biopsy may further hamper in sperm extraction for intra cytoplasmic sperm injection (ICSI). Present study describes a detailed analysis of aspiration cytology in 546 cases and also compared 48 cases of testicular biopsies with cytology. The cytological diagnoses correlated well with histological diagnoses and helped in management of infertility. FNAC can help in management of surgical and medical causes of infertility and can save unnecessary expensive investigations in cases of sertoli cell only syndrome and atrophic patterns. FNAC in combination with semen analysis and serum follicle stimulating hormone levels are of great help in management of male infertility.