We performed whole exome sequencing to identify an unknown genetic cause of azoospermia and male infertility in a large Pakistani family. Three infertile males were subjected to semen analysis, hormone testing, testicular histology, ultrasonography, karyotyping, Y-chromosome microdeletion and CFTR testing. The clinical testing suggested a diagnosis of obstructive azoospermia (OA). To identify the cause, we performed whole exome sequencing (WES) for 2 infertile brothers and 2 fertile family members. For segregation analysis and variant confirmation, we performed Sanger sequencing. WES data analysis of the family revealed segregated variants in 3 candidate genes. We considered novel nonsense variant c.2440C > T(p.Arg814*) in X-linked gene ADGRG2 as biologically most plausible. It is predicted to truncate the protein by 204 amino acids (aa) at a key transmembrane domain. Adgrg2-knockout male mice show sperm loss due to obstructive fluid stasis, while ADGRG2 mutations cause OA in the infertile male patients. Our analysis of testicular histology reveals secondary severe reduction of spermatogenesis, consistent with human and knockout mouse phenotypes. The ADGRG2 nonsense mutation is absent in the largest population databases, ExAC and gnomAD. Analysis of the novel nonsense mutation in extended family members confirmed co-segregation of the mutation with OA in all affected males. The likely pathogenic nature of the mutation is supported by its truncation effect on the transmembrane domain and distinctive ultrasound results. The study demonstrates effectiveness of WES in discovering a genetic cause of azoospermia.

Purpose

Male infertility is a complex health condition. To our knowledge there are no molecular biomarkers of male infertility. Sperm RNA is a potential biomarker for detecting sperm abnormalities and viability at infertility clinics. However, RNA use is hindered by its inconsistent quantity, quality, multiple cell types in semen and condensed sperm structure.

Materials and methods

We tested the usefulness of high quality RNA isolated from mature sperm and whole semen by our protocol, which reduces RNA degradation by maintaining semen and protocol components at 37 C and decreasing processing time. We isolated RNA from 83 whole semen samples, 18 samples of motile sperm prepared by the swim-up protocol and 18 of sperm prepared by the standard Percoll gradient method.

Results

Electrophoretic and spectral analysis of RNA revealed high quality 18S and 28S rRNAs in 71 of 83 whole semen samples (86%) and 15 of 18 mature sperm swim-up samples (83%). However, high quality RNA was isolated from only 7 of 18 Percoll gradient sperm samples (39%). Interestingly quantitative reverse transcriptase-polymerase chain reaction analysis of 4 somatic and 10 germ cell markers showed that whole semen and swim-up samples had similar RNA profiles. RNA sequencing revealed that most encoded proteins were involved in mature sperm function, regulation of DNA replication, transcription, translation, cell cycle and embryo development.

Conclusions

We believe that semen and sperm specific RNAs are highly informative biomarkers for germ cell stages and somatic cell contribution. Therefore, these RNAs could be valuable diagnostic indicators of sperm survival, fertilization and early embryogenesis, and could serve as a predictor of the in vitro fertilization prognosis.

Background

The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men.

Methods

We performed array comparative genomic hybridization testing in blood samples obtained from 15 patients with azoospermia, and we performed mutation screening by means of direct Sanger sequencing of the testis-expressed 11 gene (TEX11) open reading frame in blood and semen samples obtained from 289 patients with azoospermia and 384 controls.

Results

We identified a 99-kb hemizygous loss on chromosome Xq13.2 that involved three TEX11 exons. This loss, which was identical in 2 patients with azoospermia, predicts a deletion of 79 amino acids within the meiosis-specific sporulation domain SPO22. Our subsequent mutation screening showed five novel TEX11 mutations: three splicing mutations and two missense mutations. These mutations, which occurred in 7 of 289 men with azoospermia (2.4%), were absent in 384 controls with normal sperm concentrations (P=0.003). Notably, five of those TEX11 mutations were detected in 33 patients (15%) with azoospermia who received a diagnosis of azoospermia with meiotic arrest. Meiotic arrest in these patients resembled the phenotype of Tex11-deficient male mice. Immunohistochemical analysis showed specific cytoplasmic TEX11 expression in late spermatocytes, as well as in round and elongated spermatids, in normal human testes. In contrast, testes of patients who had azoospermia with TEX11 mutations had meiotic arrest and lacked TEX11 expression.

Conclusions

In our study, hemizygous TEX11 mutations were a common cause of meiotic arrest and azoospermia in infertile men. (Funded by the National Institutes of Health and others.).

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.