Complete Sertoli Cell-only Syndrome Research Articles

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome

BackgroundThe most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), which refers to the lack of all spermatogenic cells in the testes. The genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells.MethodsWhole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS, and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human-induced pluripotent stem cells (hiPSCs) into germ cell-like cells.ResultsThe homozygous loss-of-function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking all functional domains, and no PIWIL2 expression was detected in the patient’s testes. The patient and PIWIL2−/− hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses suggested the inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2−/− group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs.ConclusionsOur study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments.

Does detection of DDX4 mRNA in cell-free seminal plasma represents a reliable noninvasive germ cell marker in patients with nonobstructive azoospermia?

This study aimed to investigate the potential application of DDX4 gene expression in cell-free seminal mRNA as a noninvasive biomarker for the identification of the presence of germ cells in men with nonobstructive azoospermia and to correlate this factor with testicular biopsy. Male reproductive organ-specific genes were chosen: DDX4, which is a germ cell-specific gene and transglutaminase 4, which is a prostate-specific gene that was used as a control gene. Thirty-nine azoospermic males and twenty-eight normospermic fertile males (serving as a control group) participated in the study. Histopathological examination of testicular biopsies categorised azoospermic males into 20.5% with maturation arrest, 17.9% with incomplete Sertoli cell-only syndrome and 61.5% with complete Sertoli cell-only syndrome. Using real-time polymerase chain reaction, positivity for DDX4 gene was detected in 17 of 39 males with NOA which was due to maturation arrest in 35.3% (n=6/17) of cases, due to incomplete Sertoli cell only in 23.5% (n=4/17) and due to complete Sertoli cell only in 41.2% (n=7/17). The study recommends joint utilisation of molecular transcripts as noninvasive biomarkers and histopathological examination of testicular biopsies in management of cases with azoospermia of the nonobstructive type.

P450‐aromatase activity and expression in human testicular tissues with severe spermatogenic failure

There is evidence that impaired spermatogenesis is associated with an imbalance in the oestradiol/testosterone ratio and with Leydig cell (LC) dysfunction. In testis, P450-aromatase, encoded by CYP19, is responsible for the conversion of testosterone to oestradiol. The aims of this study were to quantify CYP19 mRNA expression, aromatase activity and protein localization, and to measure the oestradiol to testosterone ratio in testicular tissues of men with spermatogenic impairment. Twenty-four men with complete Sertoli cell-only syndrome (SCOS), 14 with focal SCOS, 14 with maturation arrest (MA), 8 with mixed atrophy and 30 controls with normal spermatogenesis were subjected to testicular biopsy. All subjects underwent a physical examination, cytogenetic and serum hormonal studies. Testicular CYP19 mRNA was quantified using real time RT-PCR. Testicular aromatase activity was measured using the (3)H(2)0 assay and protein expression was evaluated using immunohistochemistry. In cases, serum testosterone and oestradiol were normal, but the testosterone/LH ratio was lower compared with controls (p < 0.05). Aromatase was localized in the Leydig, Sertoli and germ cells of all tissues, although stronger intensity was observed in LC. Aromatase mRNA and activity were not altered in cases and correlated positively with LC number (r = 0.516 and r = 0.369; p < 0.008). The intratesticular oestradiol/testosterone ratio was elevated (p = 0.005) in complete SCOS patients compared with controls. In conclusion, testicular aromatase seems to be normal in most subjects with impaired spermatogenesis. However, an altered intratesticular oestradiol/testosterone ratio in some patients with complete SCOS suggests that aromatase is increased, which might contribute to Leydig cell dysfunction.

ESX1 gene expression as a robust marker of residual spermatogenesis in azoospermic men

It would be of value to identify ongoing spermatogenesis molecular markers which can predict successful sperm recovery in patients with non-obstructive azoospermia undergoing conventional or microsurgical testicular sperm extraction (TESE/microTESE). ESX1 is an X-linked homeobox gene expressed in testis, placenta, brain and lung in humans and specifically in pre- and post-meiotic germ cells of the testis in mice. We investigated the sequence, expression (by RT-PCR) and epigenetic status (by promoter pyrosequencing) of ESX1 in testicular tissue samples, obtained from 81 azoospermic subjects in the context of surgical sperm extraction, to check a possible association between ESX1 alterations and impaired spermatogenesis, as determined by histological analysis. The ESX1 transcript was detected in 100% of cases diagnosed as obstructive azoospermia (33), hypospermatogenesis (18) and incomplete maturation arrest (MA) (2), and sperm recovery was also successful in 100% of these cases. ESX1 mRNA was also detected in 5 of 6 patients with incomplete Sertoli cell-only syndrome, in 4 of 6 subjects with complete MA but in only 3 of 16 cases of complete Sertoli cell-only syndrome (cSCOS), whereas sperm recovery was successful in 4 of 6, 2 of 6 and 5 of 16 of these patients, respectively. In cases of focal spermatogenesis, ESX1 expression and sperm retrieval were concordant in 14 of 19 (74%) cases subjected to TESE, but in only 3 of 11 (27%) men who underwent microTESE. With TESE, but not with microTESE, both samples originated from adjacent testicular areas. The pyrosequencing of the ESX1 CpG island revealed methylation levels that were significantly lower in ESX1 expressors when compared with non-expressors. ESX1 emerges as a potentially reliable spermatogenesis molecular marker, whose clinical value as a predictor of successful sperm retrieval warrants further studies.

Microsurgical TESE versus conventional TESE for ICSI in non-obstructive azoospermia: a randomized controlled study

In a population of non-obstructive azoospermia patients, the efficacy of microsurgical testicular sperm extraction (microTESE) and conventional TESE was evaluated in a randomized controlled study on 138 testicles, classified and paired in a 48-square table according to the different classes of the following three variables: patient plasma FSH concentration, orchidometry and testicular histology. Sperm retrieval was positive in 21/22 testicles with hypospermatogenesis (11/11, 10/11; microTESE, TESE respectively), in 12/14 with maturation arrest (6/7, 6/7), in 16/22 with incomplete Sertoli cell-only syndrome (8/11, 8/11), and in 16/80 with complete Sertoli cell-only syndrome (11/40, 5/40). Sperm recovery was positive in 5/24 patients with FSH concentration > or = 3 x maximum value of normal range (N) (4/12, 1/12), in 17/40 patients with 2N < or = FSH < 3N (9/20, 8/20), in 30/48 patients with N < FSH < 2N (17/24, 13/24), and in 13/26 patients with FSH = N (6/13, 7/13). Regarding orchidometry, sperm recovery was positive in 11/18 testicles with volume (V) > or = 12 ml (6/9, 5/9), in 27/56 testicles with 8 ml < or = V < 12 ml (15/28, 12/28), and in 27/64 testicles with V < 8 ml (15/32, 12/32). FSH value and the surgical procedure were the two variables significantly (P < 0.05) predicting positive sperm retrieval.

Effect of TGF-β/Smad signaling on sertoli cell and possible mechanism related to complete sertoli cell-only syndrome

The roles of TGF-beta and the interaction between TGF-beta and EGFR signaling are critical in Sertoli cell, though the knowledge about them is limited. RT-PCR was used to characterize the status of TGF-beta signaling in clinical testicular specimens with complete Sertoli cell-only syndrome (SCOS). The mouse Sertoli cell TM4 was used to investigate the interaction between TGF-beta and EGFR signaling by using mitogenic assay, luciferase assay, and western blot, while TM3 (mouse leydig cell), 3T3 (mouse embryo fibroblasts), and B82 (mouse lung fibroblasts) were selected as control. The RT-PCR assay indicated that the expression levels of TbetaRII and Smad2 in SCOS testes were upregulated compared to that in the normal controls. In the in vitro experiment, the TGF-beta1 downregulated cellular proliferation of TM3 and B82 cell (P < 0.05), but it did not changed the proliferation of TM4 and 3T3 cells (P > 0.05). On contrast, TGF-beta1 only increased the TGF response elements p3TP-lux activity significantly (P < 0.05) in Sertoli cell TM4. Also, the Western blot assay shows an obvious increase of Smad2 in TM4, 3T3, and TM3 cells after TGF-beta1 treatment while the EGFR expression level was significantly increased in TM4 cells only. In conclusion, the TGF-beta pathway and the cross-link between TGF-beta and EGFR signaling may play an important role on the dysfunction of Sertoli cells which induce germ stem cells' disappearance in SCOS.

Is there a role for the nuclear export factor 2 gene in male infertility?

To determine the presence of mutations in the NXF2 gene of patients with nonobstructive azoospermia. Molecular analysis of male infertility. University genetic laboratory and reproductive clinic. Sixty-five patients with Sertoli cell-only syndrome (SCOS) and 20 control men. Polymerase chain reaction, sequencing analysis, RNA extraction, and reverse transcription polymerase chain reaction. Expression of NXF2 messenger RNA and analysis of the NXF2 gene for the presence of mutations and polymorphisms. Messenger RNA derived from the NXF2 gene could be amplified from normal human testicular tissue. Sequencing analysis showed the presence of two polymorphisms in the NXF2 gene. A first alteration, c.1779C>T, was observed in one man who had complete SCOS. Although it is located near an intron-exon boundary, this change probably does not affect splicing. The second alteration, c.1857A>G, was detected in 22 patients with complete SCOS and in 13 patients with incomplete SCOS. Also, 15 of 20 men with normal spermatogenesis had this alteration. Neither of these alterations causes a change at the amino acid level. No mutations were detected in the NXF2 gene, from which we concluded that there is no need to screen for mutations in the NXF2 gene in a routine IVF program.

Quantification of DDX3Y, RBMY1, DAZ and TSPY mRNAs in testes of patients with severe impairment of spermatogenesis

Y chromosome microdeletion is the most important genetic cause of impairment of spermatogenesis. Nevertheless, a significant proportion of patients with spermatogenic failure do not have this condition. This study investigated the expression level of AZF genes, DDX3Y (DBY), RBMY1, DAZ and TSPY in testicular tissues of 42 subjects with impaired spermatogenesis compared with 33 with normal spermatogenesis. Histopathological evaluation was performed in all subjects and tissues were classified according to Johnsen Score. Transcript amounts were determined by quantitative-competitive RT-PCR. Patients with complete Sertoli cell-only syndrome (SCOS) did not exhibit RBMY1, DAZ and TSPY gene expression, however, we detected very low expression of DDX3Y transcript. Tissue samples with focal SCOS showed significantly decreased expression of all genes (P < 0.001). Maturation arrest and hypospermatogenesis tissues expressed significantly low levels of DDX3Y testicular transcript (P < 0.001), while the mRNA levels of the other genes were similar to that in tissues from the normal spermatogenesis group. Negative or diminished gene expression of DDX3Y, RBMY1, DAZ and TSPY in tissues samples with SCOS or focal SCOS reflects the absence or the lower number of germ cells, respectively. The finding that the testicular transcript of DDX3Y is significantly decreased in patients with severe spermatogenenic failure, especially in those presenting maturation arrest, suggests an important role of DDX3Y during spermatogenesis.

High prevalence of testicular cancer in azoospermic men without spermatogenesis

An increased risk of testicular cancer in men with infertility and poor semen quality has been reported. Our aim was to investigate the prevalence of testicular nodules and cancer in azoospermic subjects with different spermatogenetic patterns. A total of 1443 consecutive infertile men were investigated, out of which 145 (10.0%) were found to be azoospermic. By using clinical examination and testicular ultrasound, 11 out of the 145 patients showed testicular nodules (2.8-26 mm). To obtain spermatozoa for assisted reproduction, 97 subjects required testicular sperm extraction (TESE) and biopsy, including the 11 patients with nodules. They were divided into two groups according to biopsy results: Group A (n = 38) with complete Sertoli cell-only syndrome (SCOS) and Group B (n = 59) with varying spermatogenetic patterns. Ten nodules were found in Group A and one in Group B. In azoospermic men, the overall prevalence of nodules was 7.5%. In complete SCOS, the prevalence of nodules and cancer was 10/38 (26.3%) and 4/38 (10.5%), respectively. Amongst the cancers, one embryonal carcinoma, one seminoma and two in-situ carcinomas were found. The prevalence of testicular nodules and cancer in azoospermic men with complete SCOS is very high. In these subjects, the role of clinical evaluation, ultrasound and biopsy should be emphasized.

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.

Intracytoplasmic injection of round and elongated spermatids from azoospermic patients: results and review.

Microinjection is established as the method of choice in the treatment of severe male factor infertility as well as in azoospermic patients. Recent studies have shown that fertilization and cleavage can be achieved by injection of ejaculated as well as testicular elongated spermatids into oocytes. Here we report on the two first pregnancies worldwide resulting from elongated spermatid injection from frozen-thawed testicular tissue. Four patients with complete Sertoli cell-only syndrome (SCOS) and two with spermatogenetic maturation arrest were included in our microinjection programme. Tissues from open testicular biopsies were cryopreserved until the time of follicle puncture. A total of 67 oocytes were harvested. In the two patients with maturation arrest, cryopreserved elongated spermatids were successfully injected, while in two of the other four SCOS patients only cryopreserved round spermatids were available to be injected into the oocytes. Out of 18 injected oocytes, 10 were fertilized in the first group, while nine out of 49 injected oocytes showed fertilization and cleavage in the second group. Two clinical pregnancies were achieved with elongated spermatids from frozen-thawed testicular tissue, while no pregnancy was established in the case of round spermatids. This study confirms that fertilization, cleavage and pregnancy can be successfully achieved in cases with spermatogenetic maturation arrest by injecting cryopreserved elongated spermatids into oocytes. The literature on pregnancies following spermatid injection, as well as the problems using this technique and possible risks, are discussed.

Does the gonadotrophic axis play a role in the pathogenesis of Sertoli-cell-only syndrome?

Infertile men with Sertoli-cell-only syndrome (SCO) have highly elevated serum FSH immunoactivity related to the degree of histological damage. The activity that serum FSH exerts at the target site depends on its glycosylation pattern and FSH receptor (FSHR) function. Either could be impaired, leading to failure of spermatogenesis. The aim of the present investigation was to study bioactivity and the glycosylation pattern of serum FSH and the occurrence of mutations in the FSH receptor in infertile patients with SCO compared to normal men. Blood was taken from 19 patients with bilateral testicular focal or complete SCO and eight normozoospermic controls. FSH bioactivity in serum was measured using an in-vitro FSH bioassay based on recombinant rat FSHR. The glycosylation pattern of serum FSH was determined by concanavalin A chromatography. Inhibin B was determined in serum using a recently available assay. Genomic DNA extracted from blood lymphocytes was amplified by PCR using primers specific for the FSHR and screened by single-stranded conformation polymorphism gel electrophoresis. Men with SCO showed significantly higher FSH in-vitro bioactivity (34.9 +/- 5.0 IU/l) than controls (9.6 +/- 0.8 IU/l: p < 0.01), as well as significantly elevated FSH immunoactivity (14.9 +/- 1.7 IU/l) compared to controls (3.1 +/- 0.5; p < 0.01). Immunoactivity of serum FSH was correlated with in-vitro bioactivity (r = 0.9; p < 0.001) and was related to the degree of testicular damage (proportion of SCO-tubules) (ANOVA: p < 0.001) and total testicular volume (r = -0.76; p < 0.01). An inverse relationship between serum FSH and inhibin B levels (r = -0.93; p < 0.001) was found. In the serum of SCO patients a slight increase in less glycosylated FSH isoforms was found (6.7 +/- 0.6% versus 3.6 +/- 0.3%; p < 0.05). No mutations of the FSHR were observed in SCO patients. We conclude that the spermatogenic failure observed in infertile patients with SCO histology and elevated FSH serum levels can be explained neither by a change in FSH bioactivity nor by mutations in the FSHR. The slight change in the FSH glycosylation pattern is probably related to higher hormonal secretion rates in SCO patients. The inverse relationship between serum FSH and inhibin B points to an intact endocrine testicular-pituitary circuit responsible for the compensatory increase of FSH in SCO.