Innovative all-in-one exome sequencing strategy for diagnostic genetic testing in male infertility: Validation and 10-month experience.

Background: Current guidelines indicate that patients with extreme oligozoospermia or azoospermia should be tested for chromosomal imbalances, azoospermia factor (AZF) deletions and/or CFTR variants. For other sperm abnormalities, no genetic diagnostics are recommended.

Objectives: To determine whether exome sequencing (ES) with combined copy number variant (CNV) and single nucleotide variant (SNV) analysis is a reliable first-tier method to replace current methods (validation study), and to evaluate the diagnostic yield after 10 months of implementation (evaluation study).

Inherited defects of piRNA biogenesis cause transposon de-repression, impaired spermatogenesis, and human male infertility.

piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm.

Improved phenotypic classification of male infertility to promote discovery of genetic causes.

An increasing number of genes are being described in the context of non-syndromic male infertility. Linking the underlying genetic causes of non-syndromic male infertility with clinical data from patients is important to establish new genotype-phenotype correlations. This process can be facilitated by using universal nomenclature, but no standardized vocabulary is available in the field of non-syndromic male infertility.

A multicentre double-blinded randomized controlled trial on the efficacy of laser-assisted hatching in patients with repeated implantation failure undergoing IVF or ICSI.

Study Question: Does assisted hatching increase the cumulative live birth rate in subfertile couples with repeated implantation failure?

Summary Answer: This study showed no evidence of effect for assisted hatching as an add-on in subfertile couples with repeated implantation failure.

What Is Known Already: The efficacy of assisted hatching, with regard to the live birth rate has not been convincingly demonstrated in randomized trials nor meta-analyses. It is suggested though that especially poor prognosis women, e.

The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans.

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons.

Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male Infertility.

Male infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported.

Transient reduction of DNA methylation at the onset of meiosis in male mice.

Background: Meiosis is a specialized germ cell cycle that generates haploid gametes. In the initial stage of meiosis, meiotic prophase I (MPI), homologous chromosomes pair and recombine. Extensive changes in chromatin in MPI raise an important question concerning the contribution of epigenetic mechanisms such as DNA methylation to meiosis.

Silencing markers are retained on pericentric heterochromatin during murine primordial germ cell development.

Background: In the nuclei of most mammalian cells, pericentric heterochromatin is characterized by DNA methylation, histone modifications such as H3K9me3 and H4K20me3, and specific binding proteins like heterochromatin-binding protein 1 isoforms (HP1 isoforms). Maintenance of this specialized chromatin structure is of great importance for genome integrity and for the controlled repression of the repetitive elements within the pericentric DNA sequence. Here we have studied histone modifications at pericentric heterochromatin during primordial germ cell (PGC) development using different fixation conditions and fluorescent immunohistochemical and immunocytochemical protocols.

Incomplete meiotic sex chromosome inactivation in the domestic dog.

Background: In mammalian meiotic prophase, homologous chromosome recognition is aided by formation and repair of programmed DNA double-strand breaks (DSBs). Subsequently, stable associations form through homologous chromosome synapsis. In male mouse meiosis, the largely heterologous X and Y chromosomes synapse only in their short pseudoautosomal regions (PARs), and DSBs persist along the unsynapsed non-homologous arms of these sex chromosomes.

Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications.

The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos.

Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice.

Pachytene piRNAs are a class of Piwi-interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon-derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor-domain proteins and processing intermediates of pachytene piRNA primary transcripts.

A role for retrotransposon LINE-1 in fetal oocyte attrition in mice.

Fetal oocyte attrition (FOA) is a conserved but poorly understood process of elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth. We now implicate retrotransposons LINE-1 (L1), activated during epigenetic reprogramming of the embryonic germline, in FOA in mice. We show that wild-type fetal oocytes possess differential nuclear levels of L1ORF1p, an L1-encoded protein essential for L1 ribonucleoprotein particle (L1RNP) formation and L1 retrotransposition.

Differential expression and sex chromosome association of CHD3/4 and CHD5 during spermatogenesis.

ATP-dependent nucleosome remodelers of the CHD family play important roles in chromatin regulation during development and differentiation. The ubiquitously expressed CHD3 and CHD4 proteins are essential for stem cell function and serve to orchestrate gene expression in different developmental settings. By contrast, the closely related CHD5 is predominantly expressed in neural tissue and its role is believed to be restricted to neural differentiation.

Optimized flow cytometry isolation of murine spermatocytes.

Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis, and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment, and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages.

Intracellular detection of cytosine incorporation in genomic DNA by using 5-ethynyl-2'-deoxycytidine.

5-Ethynyl-2'-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo(-) and DNA polymerase β. Incorporation of 5-ethynyl-2'-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC.

Bodies of evidence - compartmentalization of the piRNA pathway in mouse fetal prospermatogonia.

Epigenetic reprogramming of embryonic mouse germ cells involves DNA demethylation of the genome that is accompanied by derepression of transposable elements (TEs). Threatening the genome's integrity, TE activation is efficiently countered by the concerted action of de novo DNA methylation and PIWI-interacting small RNAs (piRNAs). Recent studies have closely examined the subcellular localization of various piRNA pathway proteins in fetal prospermatogonia of wild-type and piRNA pathway mutant mice.

Defending the genome in tudor style.

Repression of transposable elements is crucial for the survival of germ cells. In this issue of Developmental Cell, Shoji et al. provide evidence that a Tudor domain protein TDRD9 partners with MIWI2 to specifically silence LINE-1 transposons in the fetal germline of male mice.

Synaptonemal complex length variation in wild-type male mice.

Meiosis yields haploid gametes following two successive divisions of a germ cell in the absence of intervening DNA replication. Balanced segregation of homologous chromosomes in Meiosis I is aided by a proteinaceous structure, the synaptonemal complex (SC). The objective of this study was to determine total average autosomal SC lengths in spermatocytes in three commonly used mouse strains (129S4/SvJae, C57BL/6J, and BALB/c).

Cytoplasmic compartmentalization of the fetal piRNA pathway in mice.

Derepression of transposable elements (TEs) in the course of epigenetic reprogramming of the mouse embryonic germline necessitates the existence of a robust defense that is comprised of PIWI/piRNA pathway and de novo DNA methylation machinery. To gain further insight into biogenesis and function of piRNAs, we studied the intracellular localization of piRNA pathway components and used the combination of genetic, molecular, and cell biological approaches to examine the performance of the piRNA pathway in germ cells of mice lacking Maelstrom (MAEL), an evolutionarily conserved protein implicated in transposon silencing in fruit flies and mice. Here we show that principal components of the fetal piRNA pathway, MILI and MIWI2 proteins, localize to two distinct types of germinal cytoplasmic granules and exhibit differential association with components of the mRNA degradation/translational repression machinery.

Transient relaxation of transposon silencing at the onset of mammalian meiosis.

Postmigratory mouse primordial germ cells (PGCs) undergo extensive epigenetic remodeling that includes DNA methylation (DM) reprogramming of imprinted genes and, surprisingly, of transposable elements (TEs). Given the danger posed by TEs to the integrity of the germline, even a brief derepression of TEs is counterintuitive and puzzling. In the male fetal gonocytes, a sophisticated repressive mechanism that uses DM and TE-targeting piRNAs has evolved to stably silence TEs.

Mouse maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis.

Tight control of transposon activity is essential for the integrity of the germline. Recently, a germ-cell-specific organelle, nuage, was proposed to play a role in transposon repression. To test this hypothesis, we disrupted a murine homolog of a Drosophila nuage protein Maelstrom.