MFN2 interacts with nuage-associated proteins and is essential for male germ cell development by controlling mRNA fate during spermatogenesis.

Mitochondria play a crucial role in spermatogenesis and are regulated by several mitochondrial fusion proteins. However, their functional importance associated with their structure formation and mRNA fate regulation during spermatogenesis remains unclear. Here, we show that mitofusin 2 (MFN2), a mitochondrial fusion protein, interacts with nuage-associated proteins (including MIWI, DDX4, TDRKH and GASZ) in mice. Conditional mutation of Mfn2 in postnatal germ cells results in male sterility due to germ cell developmental defects. Moreover, MFN2 interacts with MFN1, another mitochondrial fusion protein with a high-sequence similarity to MFN2, in testes to facilitate spermatogenesis. Simultaneous mutation of Mfn1 and Mfn2 in testes causes very severe infertile phenotypes. Importantly, we show that MFN2 is enriched in polysome fractions of testes and interacts with MSY2, a germ cell-specific DNA/RNA-binding protein, to control gamete-specific mRNA (such as Spata19) translational activity during spermatogenesis. Collectively, our findings demonstrate that MFN2 interacts with nuage-associated proteins and MSY2 to regulate male germ cell development by controlling several gamete-specific mRNA fates.

What human sperm RNA-Seq tells us about the microbiome.

PURPOSE: The study was designed to assess the capacity of human sperm RNA-seq data to gauge the diversity of the associated microbiome within the ejaculate. METHODS: Semen samples were collected, and semen parameters evaluated at time of collection. Sperm RNA was isolated and subjected to RNA-seq. Microbial composition was determined by aligning sequencing reads not mapped to the human genome to the NCBI RefSeq bacterial, viral and archaeal genomes following RNA-Seq. Analysis of microbial assignments utilized phyloseq and vegan. RESULTS: Microbial composition within each sample was characterized as a function of microbial associated RNAs. Bacteria known to be associated with the male reproductive tract were present at similar levels in all samples representing 11 genera from four phyla with one exception, an outlier. Shannon diversity index (p < 0.001) and beta diversity (unweighted UniFrac distances, p = 9.99e-4; beta dispersion, p = 0.006) indicated the outlier was significantly different from all other samples. The outlier sample exhibited a dramatic increase in Streptococcus. Multiple testing indicated two operational taxonomic units, S. agalactiae and S. dysgalactiae (p = 0.009), were present. CONCLUSION: These results provide a first look at the microbiome as a component of human sperm RNA sequencing that has sufficient sensitivity to identify contamination or potential pathogenic bacterial colonization at least among the known contributors.

A history of why fathers' RNA matters.

Having been debated for many years, the presence and role of spermatozoal RNAs is resolving, and their contribution to development is now appreciated. Data from different species continue show that sperm contain a complex suite of coding and noncoding RNAs that play a role in an individual's life course. Mature sperm RNAs provide a retrospective of spermatogenesis, with their presence and abundance reflecting sperm maturation, fertility potential, and the paternal contribution to the developmental path the offspring may follow.Sperm RNAs delivered upon fertilization provide some of the initial contacts with the oocyte, directly confront the maternal with the paternal contribution as a prelude to genome consolidation. Following syngamy, early embryo development may in part be modulated by paternal RNAs that can include epidydimal passengers. This provides a direct path to relay an experience and then initiate a paternal response to the environment to the oocyte and beyond. Their epigenetic impact is likely felt prior to embryonic genome activation when the population of sperm delivered transcripts markedly changes. Here, we review the insights gained from sperm RNAs over the years, the subtypes, and the caveats of the RNAs described. We discuss the role of sperm RNAs in fertilization and embryo development, and their possible mechanism(s) influencing offspring phenotype. Approaches to meet the future challenges as the study of sperm RNAs continues, include, elucidating the potential mechanisms underlying how paternal allostatic load, the constant adaptation of health to external conditions, may be relayed by sperm RNAs to affect future generations.

A comprehensive analysis of spermatozoal RNA elements in idiopathic infertile males undergoing fertility treatment.

Current approaches to diagnosing male infertility inadequately assess the complexity of the male gamete. Beyond the paternal haploid genome, spermatozoa also deliver coding and non-coding RNAs to the oocyte. While sperm-borne RNAs have demonstrated potential involvement in embryo development, the underlying mechanisms remain unclear. In this study, 47 sperm samples from normozoospermic males undergoing fertility treatment using donor oocytes were sequenced and analyzed to evaluate associations between sperm RNA elements (exon-sized sequences) and blastocyst progression. A total of 366 RNA elements (REs) were significantly associated with blastocyst rate (padj < 0.05), some of which were linked to genes related to critical developmental processes, including mitotic spindle formation and both ectoderm and mesoderm specification. Of note, 27 RE-associated RNAs are predicted targets of our previously reported list of developmentally significant miRNAs. Inverse RE-miRNA expression patterns were consistent with miRNA-mediated down-regulation. This study provides a comprehensive set of REs which differ by the patient's ability to produce blastocysts. This knowledge can be leveraged to improve clinical screening of male infertility and ultimately reduce time to pregnancy.

Hydroquinone impairs trophoblast migration and invasion via AHR-twist-IFITM1 axis.

INTRODUCTION: Embryo implantation is a tightly regulated process, critical for a successful pregnancy. After attachment of the blastocyst to the surface epithelium of the endometrium trophoblast migrate from the trophectoderm and invade into the stromal component of endometrium. Alterations on either process will lead to implantation failure or miscarriage. Volatile organic compounds (VOCs) such as benzene induce pregnancy complications, including preterm birth and miscarriages. The mechanism of this effect is unknown. The objective of this study was to elucidate the impact of benzene metabolite, Hydroquinone, on trophoblast function. We tested the hypothesis that Hydroquinone activates the Aryl hydrocarbon receptor (AhR) pathway modulating trophoblast migration and invasion. METHODS: First-trimester trophoblast cells (Sw.71) were treated with hydroquinone (6 and 25 µM). Trophoblast migration and invasion was evaluated using a 3D invasion/migration model. Gene expression was quantified by q-PCR and Western blot analysis. RESULTS: Hydroquinone impairs trophoblast migration and invasion. This loss is associated with the activation of the AhR pathway which reduced the expression of Twist1and IFITM1. IFITM1 overexpression can rescue impaired trophoblast migration. DISCUSSION: Our study highlights that hydroquinone treatment induces the activation of the AhR pathway in trophoblast cells, which impairs trophoblast invasion and migration. We postulate that activation of the AhR pathway in trophoblast suppress Twist1 and a subsequent IFITM1. Thus, the AhR-Twist1-IFITM1 axis represent a critical pathway involved in the regulation of trophoblast migration and it is sensitive to benzene exposure. These findings provide crucial insights into the molecular mechanisms underlying pregnancy complications induced by air pollution.

Trophectoderm non-coding RNAs reflect the higher metabolic and more invasive properties of young maternal age blastocysts.

Increasing female age is accompanied by a corresponding fall in her fertility. This decline is influenced by a variety of factors over an individual's life course including background genetics, local environment and diet. Studying both coding and non-coding RNAs of the embryo could aid our understanding of the causes and/or effects of the physiological processes accompanying the decline including the differential expression of sub-cellular biomarkers indicative of various diseases. The current study is a post-hoc analysis of the expression of trophectoderm RNA data derived from a previous high throughput study. Its main aim is to determine the characteristics and potential functionalities that characterize long non-coding RNAs. As reported previously, a maternal age-related component is potentially implicated in implantation success. Trophectoderm samples representing the full range of maternal reproductive ages were considered in relation to embryonic implantation potential, trophectoderm transcriptome dynamics and reproductive maternal age. The long non-coding RNA (lncRNA) biomarkers identified here are consistent with the activities of embryo-endometrial crosstalk, developmental competency and implantation and share common characteristics with markers of neoplasia/cancer invasion. Corresponding genes for expressed lncRNAs were more active in the blastocysts of younger women are associated with metabolic pathways including cholesterol biosynthesis and steroidogenesis.

The transcript integrity index (TII) provides a standard measure of sperm RNA.

Standardizing RNA quality is key to interpreting RNA-seq data as a compromised sample can mask the underlying biology. The challenge remains when evaluating RNA quality in samples with high RNA fragmentation. For example, programmed fragmentation and cytoplasmic expulsion, integral to sperm maturation, is a prime example of the complexities of interpreting RNA-seq data, given that fragmentation can be random and\or targeted. To meet this challenge, we developed an algorithm that accurately measures RNA quality in samples with high fragmentation, such as spermatozoa. The integrity of 1,000 previously identified abundant sperm transcripts were independently visualized and evaluated using the Transcript Integrity Index (TII) algorithm to identify intact transcripts. Full-length transcripts from visual and the TII algorithm were evaluated for testis preference in humans using the GTEx tissues database. Samples were then filtered by the Interquartile Range (IQR), identifying those in which the greatest number of transcripts failed to pass the visual or TII thresholds. Transcript lists were overlapped, forming the set of intact transcripts used as TII standards. Each sample was re-evaluated as a function of this TII set of intact transcripts, with poor quality samples identified as those failing in the largest number of transcripts. While ontologically enriched in roles related to spermatogenesis and/or fertilization, samples did not segregate based on birth outcome. The TII algorithm proved an effective means to identify samples of similar quality from sperm, a cell type enriched in biologically fragmented RNAs. The algorithm should facilitate other studies using samples compromised by high levels of RNA fragmentation, such as Formalin-Fixed Paraffin-Embedded samples. Requisite to assessing male health, TII provides a solution to the long-sought-after standard that identifies samples of similar quality.

COVID-19 and human reproduction: A pandemic that packs a serious punch.

The COVID-19 pandemic has led to a worldwide health emergency that has impacted 188 countries at last count. The rapid community transmission and relatively high mortality rates with COVID-19 in modern times are relatively unique features of this flu pandemic and have resulted in an unparalleled global health crisis. SARS-CoV-2, being a respiratory virus, mainly affects the lungs, but is capable of infecting other vital organs, such as brain, heart and kidney. Emerging evidence suggests that the virus also targets male and female reproductive organs that express its main receptor ACE2, although it is as yet unclear if this has any implications for human fertility. Furthermore, professional bodies have recommended discontinuing fertility services during the pandemic such that reproductive services have also been affected. Although increased safety measures have helped to mitigate the propagation of COVID-19 in a number of countries, it seems that there is no predictable timeline to containment of the virus, a goal likely to remain elusive until an effective vaccine becomes available  and widely distributed across the globe. In parallel, research on reproduction has been postponed for obvious reasons, while diagnostic tests that detect the virus or antibodies against it are of vital importance to support public health policies, such as social distancing and our obligation to wear masks in public spaces. This review aims to provide an overview of critical research and ethics issues that have been continuously emerging in the field of reproductive medicine as the COVID-19 pandemic tragically unfolds.Abbreviations: ACE2: angiotensin- converting enzyme 2; ART: Assisted reproductive technology; ASRM: American Society for Reproductive Medicine; CCR9: C-C Motif Chemokine Receptor 9; CDC: Centers for Disease Control and Prevention; COVID-19: Coronavirus disease 2019; Ct: Cycle threshold; CXCR6: C-X-C Motif Chemokine Receptor 6; ELISA: enzyme-linked immunosorbent assay; ESHRE: European Society of Human Reproduction and Embryology; ET: Embryo transfer; FSH: Follicle Stimulating Hormone; FFPE: formalin fixed paraffin embedded; FYCO1: FYVE And Coiled-Coil Domain Autophagy Adaptor 1; IFFS: International Federation of Fertility Societies; IUI: Intrauterine insemination; IVF: In vitro fertilization; LH: Luteinizing Hormone; LZTFL1: Leucine Zipper Transcription Factor Like 1; MAR: medically assisted reproduction services; MERS: Middle East Respiratory syndrome; NGS: Next Generation Sequencing; ORF: Open Reading Frame; PPE: personal protective equipment; RE: RNA Element; REDa: RNA Element Discovery algorithm; RT-PCR: Reverse=trascriptase transcriptase-polymerase chain reaction; SARS: Severe acute respiratory syndrome; SARS-CoV-2: Severe Acute Respiratory Syndrome Coronavirus 2; SLC6A20: Solute Carrier Family 6 Member 20; SMS: Single Molecule Sequencing; T: Testosterone; TMPRSS2: transmembrane serine protease 2; WHO: World Health Organization; XCR1: X-C Motif Chemokine Receptor.

The effects of aging on molecular modulators of human embryo implantation.

Advancing age has a negative impact on female fertility. As implantation rates decline during the normal maternal life course, age-related, embryonic factors are altered and our inability to monitor these factors in an unbiased genome-wide manner in vivo has severely limited our understanding of early human embryo development and implantation. Our high-throughput methodology uses trophectoderm samples representing the full spectrum of maternal reproductive ages with embryo implantation potential examined in relation to trophectoderm transcriptome dynamics and reproductive maternal age. Potential embryo-endometrial interactions were tested using trophectoderm sampled from young women, with the receptive uterine environment representing the most 'fertile' environment for successful embryo implantation. Potential roles for extracellular exosomes, embryonic metabolism and regulation of apoptosis were revealed. These biomarkers are consistent with embryo-endometrial crosstalk/developmental competency, serving as a mediator for successful implantation. Our data opens the door to developing a diagnostic test for predicting implantation success in women undergoing fertility treatment.

Human chromatin remodeler cofactor, RNA interactor, eraser and writer sperm RNAs responding to obesity.

In the United States almost 33% of adults are considered obese (BMI > 30 kg/m2). Both animal models and to a lesser extent human studies, have associated BMI, a measure of obesity, with alterations in sperm DNA methylation and RNAs. Sperm RNAs from the Assessment of Multiple Gestations from Ovarian Stimulation trial, were isolated and sequenced. A Generalized Linear Model identified 487 BMI associated human sperm RNA elements (short exon-sized sequences). They partitioned into four patterns; a continual increase with BMI, increase once obese (BMI>30 kg/m2); a steady decrease with BMI; and decrease once overweight (BMI 25 - 30 kg/m2). Gene Ontology revealed a unique relationship between BMI and transcripts associated with chromosome organization, adipogenesis, cellular stress and obesity-related inflammation. Coregulatory networks linked by Chromatin remodeler cofactors, RNA interactors, Erasers and Writers (CREWs) were uncovered to reveal a hierarchical epigenetic response pathway.