The genetic basis of nonobstructive azoospermia is unknown in the majority of infertile men.
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.
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.
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.).
Structural aberrations involving more than two breakpoints on two or more chromosomes are known as complex chromosomal rearrangements (CCRs). They can reduce fertility through gametogenesis arrest ...developed due to disrupted chromosomal pairing in the pachytene stage. We present a familial case of two infertile brothers (with azoospermia and cryptozoospermia) and their mother, carriers of an exceptional type of CCR involving chromosomes 1 and 7 and three breakpoints. The aim was to identify whether meiotic disruption was caused by CCR and/or genomic mutations. Additionally, we performed a literature survey for male CCR carriers with reproductive failures. The characterization of the CCR chromosomes and potential genomic aberrations was performed using: G-banding using trypsin and Giemsa staining (GTG banding), fluorescent in situ hybridization (FISH) (including multicolor FISH (mFISH) and bacterial artificial chromosome (BAC)-FISH), and genome-wide array comparative genomic hybridization (aCGH). The CCR description was established as: der(1)(1qter->1q42.3::1p21->1q42.3::7p14.3->7pter), der(7)(1pter->1p2 1::7p14.3->7qter). aCGH revealed three rare genes variants:
,
, and
, which were ruled out due to unlikely biological functions. The aCGH analysis of three breakpoint CCR regions did not reveal copy number variations (CNVs) with biologically plausible genes. Synaptonemal complex evaluation (brother-1; spermatocytes II/oligobiopsy; the silver staining technique) showed incomplete conjugation of the chromosomes. Associations between CCR and the sex chromosomes (by FISH) were not found. A meiotic segregation pattern (brother-2; ejaculated spermatozoa; FISH) revealed 29.21% genetically normal/balanced spermatozoa. The aCGH analysis could not detect smaller intergenic CNVs of few kb or smaller (indels of single exons or few nucleotides). Since chromosomal aberrations frequently do not affect the phenotype of the carrier, in contrast to the negative influence on spermatogenesis, there is an obvious need for genomic sequencing to investigate the point mutations that may be responsible for the differences between the azoospermic and cryptozoospermic phenotypes observed in a family. Progeny from the same parents provide a unique opportunity to discover a novel genomic background of male infertility.
Reproductive genetics and the aging male Yatsenko, Alexander N.; Turek, Paul J.
Journal of assisted reproduction and genetics,
06/2018, Letnik:
35, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Purpose
To examine current evidence of the known effects of advanced paternal age on sperm genetic and epigenetic changes and associated birth defects and diseases in offspring.
Methods
Review of ...published PubMed literature.
Results
Advanced paternal age (> 40 years) is associated with accumulated damage to sperm DNA and mitotic and meiotic quality control mechanisms (mismatch repair) during spermatogenesis. This in turn causes well-delineated abnormalities in sperm chromosomes, both numerical and structural, and increased sperm DNA fragmentation (3%/year of age) and single gene mutations (relative risk, RR 10). An increase in related abnormalities in offspring has also been described, including miscarriage (RR 2) and fetal loss (RR 2). There is also a significant increase in rare, single gene disorders (RR 1.3 to 12) and congenital anomalies (RR 1.2) in offspring. Current research also suggests that autism, schizophrenia, and other forms of “psychiatric morbidity” are more likely in offspring (RR 1.5 to 5.7) with advanced paternal age. Genetic defects related to faulty sperm quality control leading to single gene mutations and epigenetic alterations in several genetic pathways have been implicated as root causes.
Conclusions
Advanced paternal age is associated with increased genetic and epigenetic risk to offspring. However, the precise age at which risk develops and the magnitude of the risk are poorly understood or may have gradual effects. Currently, there are no clinical screenings or diagnostic panels that target disorders associated with advanced paternal age. Concerned couples and care providers should pursue or recommend genetic counseling and prenatal testing regarding specific disorders.
Abstract
Infertility is a problem that affects approximately 15% of couples, and male infertility is responsible for 40–50% of these cases. The cause of male infertility is still poorly diagnosed and ...treated. One of the prominent causes of male infertility is disturbed spermatogenesis, which can lead to nonobstructive azoospermia (NOA). Whole-genome sequencing (WGS) allows us to identify novel rare variants in potentially NOA-associated genes, among others, in the
ESX1
gene. The aim of this study was to activate the
ESX1
gene using CRISPRa technology in human germ cells (testicular seminoma cells—TCam-2). Successful activation of the
ESX1
gene in TCam-2 cells using the CRISPRa system was achieved, and the expression level of the
ESX1
gene was significantly higher in modified TCam-2 cells than in WT cells or the negative control with nontargeted gRNA (
p
< 0.01). Using RNA-seq, a network of over 50 genes potentially regulated by the
ESX1
gene was determined. Finally, 6 genes,
NANOG, CXCR4, RPS6KA5, CCND1, PDE1C,
and
LINC00662
, participating in cell proliferation and differentiation were verified in azoospermic patients with and without a mutation in the
ESX1
gene as well as in men with normal spermatogenesis, where inverse correlations in the expression levels of the observed genes were noted.
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.
In this work, we have compared photosynthetic characteristics of photosystem II (PSII) in
Tradescantia
leaves of two contrasting ecotypes grown under the low light (LL) and high light (HL) regimes ...during their entire growth period. Plants of the same genus,
T. fluminensis
(shade-tolerant) and
T. sillamontana
(sun-resistant), were cultivated at 50–125 µmol photons m
−2
s
−1
(LL) or at 875–1000 µmol photons m
−2
s
−1
(HL). Analyses of intrinsic PSII efficiency was based on measurements of fast chlorophyll (Chl)
a
fluorescence kinetics (the OJIP test). The fluorescence parameters
F
v
/
F
m
(variable fluorescence) and
F
0
(the initial level of fluorescence) in dark-adapted leaves were used to quantify the photochemical properties of PSII. Plants of different ecotypes showed different sustainability with respect to changes in the environmental light intensity and temperature treatment. The sun-resistant species
T. sillamontana
revealed the tolerance to variations in irradiation intensity, demonstrating constancy of maximum quantum efficiency of PSII upon variations of the growth light. In contrast to
T. sillamontana
, facultative shade species
T. fluminensis
demonstrated variability of PSII photochemical activity, depending on the growth light intensity. The susceptibility of
T. fluminensis
to solar stress was documented by a decrease in
F
v
/
F
m
and a rise of
F
0
during the long-term exposition of
T. fluminensis
to HL, indicating the loss of photochemical activity of PSII. The short-term (10 min) heat treatment of leaf cuttings caused inactivation of PSII. The temperature-dependent heating effects were different in
T. fluminensis
and
T. sillamontana
. Sun-resistant plants
T. sillamontana
acclimated to LL and HL displayed the same plots of
F
v
/
F
m
versus the treatment temperature (
t
), demonstrating a decrease in
F
v
/
F
m
at
t
≥ 45 °C. The leaves of shadow-tolerant species
T. fluminensis
grown under the LL and HL conditions revealed different sensitivities to heat treatment. Plants grown under the solar stress conditions (HL) demonstrated a gradual decline of
F
v
/
F
m
at lower heating temperatures (
t
≥ 25 °C), indicating the “fragility” of their PSII as compared to
T. fluminensis
grown at LL. Different responses of sun and shadow species of
Tradescantia
to growth light and heat treatment are discussed in the context of their biochemical and ecophysiological properties.
Purpose The causes of male infertility are heterogeneous but more than 50% of cases have a genetic basis. Specific genetic defects have been identified in less than 20% of infertile males and, thus, ...most causes remain to be elucidated. The most common cytogenetic defects associated with nonobstructive azoospermia are numerical and structural chromosome abnormalities, including Klinefelter syndrome (47,XXY) and Y chromosome microdeletions. To refine the incidence and nature of chromosomal aberrations in males with infertility we reviewed cytogenetic results in 668 infertile men with oligozoospermia and azoospermia. Materials and Methods High resolution Giemsa banding chromosome analysis and/or fluorescence in situ hybridization were done in 668 infertile males referred for routine cytogenetic analysis between January 2004 and March 2009. Results The overall incidence of chromosomal abnormalities was about 8.2%. Of the 55 patients with abnormal cytogenetic findings sex chromosome aneuploidies were observed in 29 (53%), including Klinefelter syndrome in 27 (49%). Structural chromosome abnormalities involving autosomes (29%) and sex chromosomes (18%) were detected in 26 infertile men. Abnormal cytogenetic findings were observed in 35 of 264 patients (13.3%) with azoospermia and 19 of 365 (5.2%) with oligozoospermia. Conclusions Structural chromosomal defects and low level sex chromosome mosaicism are common in oligozoospermia cases. Extensive cytogenetic assessment and fluorescence in situ hybridization may improve the detection rate in males with oligozoospermia. These findings highlight the need for efficient genetic testing in infertile men so that couples may make informed decisions on assisted reproductive technologies to achieve parenthood.
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.
Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of ...diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes
,
,
,
and
by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the
gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.
Background
Genetic causes that lead to spermatogenetic failure in patients with nonobstructive azoospermia (NOA) have not been yet completely established.
Objective
To identify low‐frequency ...NOA‐associated single nucleotide variants (SNVs) using whole‐genome sequencing (WGS).
Materials and methods
Men with various types of NOA (n = 39), including samples that had been previously tested with whole‐exome sequencing (WES; n = 6) and did not result in diagnostic conclusions. Variants were annotated using the Ensembl Variant Effect Predictor, utilizing frequencies from GnomAD and other databases to provide clinically relevant information (ClinVar), conservation scores (phyloP), and effect predictions (i.e., MutationTaster). Structural protein modeling was also performed.
Results
Using WGS, we revealed potential NOA‐associated SNVs, such as: TKTL1, IGSF1, ZFPM2, VCX3A (novel disease causing variants), ESX1, TEX13A, TEX14, DNAH1, FANCM, QRICH2, FSIP2, USP9Y, PMFBP1, MEI1, PIWIL1, WDR66, ZFX, KCND1, KIAA1210, DHRSX, ZMYM3, FAM47C, FANCB, FAM50B (genes previously known to be associated with infertility) and ALG13, BEND2, BRWD3, DDX53, TAF4, FAM47B, FAM9B, FAM9C, MAGEB6, MAP3K15, RBMXL3, SSX3 and FMR1NB genes, which may be involved in spermatogenesis.
Discussion and conclusion
In this study, we identified novel potential candidate NOA‐associated genes in 29 individuals out of 39 azoospermic males. Note that in 5 out of 6 patients subjected previously to WES analysis, which did not disclose potentially causative variants, the WGS analysis was successful with NOA‐associated gene findings.
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