Vertical transmission of maternal DNA through extracellular vesicles associates with altered embryo bioenergetics during the periconception period.

The transmission of DNA through extracellular vesicles (EVs) represents a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. We aimed to investigate the potential for vertical DNA transmission within maternal endometrial EVs to the pre-implantation embryo and describe any effect on embryo bioenergetics. We discovered that the human endometrium secretes all three general subtypes of EV - apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs) - into the human endometrial fluid (EF) within the uterine cavity. EVs become uniformly secreted into the EF during the menstrual cycle, with the proportion of different EV populations remaining constant; however, MVs contain significantly higher levels of mitochondrial (mt)DNA than ABs or EXOs. During the window of implantation, MVs contain an eleven-fold higher level of mtDNA when compared to cells-of-origin within the receptive endometrium, which possesses a lower mtDNA content and displays the upregulated expression of mitophagy-related genes. Furthermore, we demonstrate the internalization of EV-derived nuclear-encoded (n)DNA/mtDNA by trophoblast cells of murine embryos, which associates with a reduction in mitochondrial respiration and ATP production. These findings suggest that the maternal endometrium suffers a reduction in mtDNA content during the preconceptional period, that nDNA/mtDNA become packaged into secreted EVs that the embryo uptakes, and that the transfer of DNA to the embryo within EVs occurs alongside the modulation of bioenergetics during implantation.

Oral immunization with heat-inactivated Mycobacterium bovis reduces local parasite dissemination and hepatic granuloma development in mice infected with Leishmania amazonensis.

Aiming to explore whether oral immunization with heat-inactivated Mycobacterium bovis (HIMB) protects mice against Leishmania infection, 18 female BALB/c mice were randomly assigned to the immunized group, that received oral HIMB, or the control group, and were infected by inoculation of 10,000 Leishmania amazonensis promastigotes in the footpad. Spleen culture was positive in 55.55% of immunized mice and in 100% of control mice (p = 0.082). The number of immunolabeled amastigotes number in the popliteal lymph node was lower in the immunized group (p = 0.009). The immunized group presented fewer mature granulomas in the liver (p = 0.005) and more Lys + macrophages (p = 0.002) and fewer CD3+ T lymphocytes (p < 0.001) per hepatic granuloma. We conclude that immunization with HIMB via the oral route limited local parasite dissemination and hepatic granuloma development in mice challenged with Leishmania amazonensis through stimulation of macrophages, which is compatible with trained immunity.

The human periconceptional maternal-embryonic space in health and disease.

Pregnancy is established during the periconceptional period as a continuum beginning with blastocyst attachment to the endometrial epithelial surface followed by embryo invasion and placenta formation. This period sets the foundation for the child and mother's health during pregnancy. Emerging evidence indicates that prevention of downstream pathologies in both the embryo/newborn and pregnant mother may be possible at this stage. In this review, we discuss current advances in the periconceptional space, including the preimplantation human embryo and maternal endometrium. We also discuss the role of the maternal decidua, the periconceptional maternal-embryonic interface, the dialogue between these elements, and the importance of the endometrial microbiome in the implantation process and pregnancy. Finally, we discuss the myometrium in the periconceptional space and review its role in determining pregnancy health.

Vestibulotoxicity in Patients Undergoing Cisplatin-Based Cancer Treatment: A Phase IIIB Randomized Controlled Clinical Trial.

INTRODUCTION: This study aimed to evaluate the incidence of balance disorders and the efficacy of dexamethasone in protecting patients undergoing cisplatin-based cancer treatment against vestibulototoxicity. METHODS: This study was a randomized controlled phase IIIB clinical trial. The subjects participating in the clinical trial were patients with a neoplastic disease whose treatment protocol included cisplatin. The average dose of cisplatin was 444.87 mg (SD 235.2 mg). Treatment consisted of intratympanically administering dexamethasone via a passive diffusion device called Microwick (8 mg/24 h dose) from the start of treatment with cisplatin to 3 weeks after the last cycle. Patients were administered the medication to one ear, and the contralateral ear was used as the control. The treated ears were randomly chosen using a computer system (randomization). Vestibular system was evaluated by video head impulse test before each cisplatin cycle. RESULTS: Thirty-four patients were recruited over a 2-year period at a reference tertiary hospital, of whom 11 were excluded. Forty-six ears were analyzed (23 treated and 23 control ears). Vestibular analysis presented no changes in the mean increase in the vestibulo-ocular response in all patients evaluated, both in treated and control ears. Both 8.69% infection complications during treatment and 34.8% permanent perforation at 6 months were detected after device removal. CONCLUSION: Ototoxicity related to cisplatin-based treatment does not affect the vestibular system. Long-term high-dose intratympanic dexamethasone treatment is safe for the vestibular system.

Effect of heat-inactivated Mycobacterium avium subspecies paratuberculosis (MAP) vaccine on the lesions and immunopathology developed in target tissues of naturally MAP-infected goats.

Paratuberculosis (PTB) is a disease caused by Mycobacterium avium subspecies paratuberculosis (MAP), which affects a broad range of hosts, including domestic and wild animals. PTB is a chronic granulomatous enteritis and lymphadenitis that compromises animal welfare and causes economic losses. The objective of this study was to evaluate the effect of a commercial heat-inactivated MAP vaccine on lesions and immunopathology developed in the target tissues of goats naturally infected with MAP. Lesions compatible with PTB in the intestine and regional lymph nodes (LNs), as well as local immune response to MAP, were evaluated and compared in Gudair®-vaccinated (n = 14) and unvaccinated (n = 11) goats from a MAP-infected farm. The percentage of animals with multifocal granulomatous lesions in the jejunal (p = 0.05) and ileocecal (p = 0.02) LNs was higher in unvaccinated animals, while a lesion score reduction of 50% was found in the LNs of vaccinated animals. Unvaccinated animals showed increased numbers and wider distribution of macrophages (MΦs, CD68 +) in histiocytic infiltrate (p = 0.0003), associated with increased numbers of mycobacteria. Increased inducible nitric oxide synthase (iNOS) expression was also reported in these animals, while M2 MΦs (CD163 +) were scarce in both groups. Vaccinated animals showed an increase in CD3 + lymphocytes, although differences in interferon gamma (IFNγ) were negligible. These results support the hypothesis that heat-inactivated MAP vaccination could reduce the severity of PTB lesions and mycobacterial load in target tissues in vaccinated adult goats.

The Endometrial Microbiome and Its Impact on Human Conception.

Changes in the female genital tract microbiome are consistently correlated to gynecological and obstetrical pathologies, and tract dysbiosis can impact reproductive outcomes during fertility treatment. Nonetheless, a consensus regarding the physiological microbiome core inside the uterine cavity has not been reached due to a myriad of study limitations, such as sample size and experimental design variations, and the influence of endometrial bacterial communities on human reproduction remains debated. Understanding the healthy endometrial microbiota and how changes in its composition affect fertility would potentially allow personalized treatment through microbiome management during assisted reproductive therapies, ultimately leading to improvement of clinical outcomes. Here, we review current knowledge regarding the uterine microbiota and how it relates to human conception.

Endometrial microbiota composition is associated with reproductive outcome in infertile patients.

BACKGROUND: Previous evidence indicates associations between the female reproductive tract microbiome composition and reproductive outcome in infertile patients undergoing assisted reproduction. We aimed to determine whether the endometrial microbiota composition is associated with reproductive outcomes of live birth, biochemical pregnancy, clinical miscarriage or no pregnancy. METHODS: Here, we present a multicentre prospective observational study using 16S rRNA gene sequencing to analyse endometrial fluid and biopsy samples before embryo transfer in a cohort of 342 infertile patients asymptomatic for infection undergoing assisted reproductive treatments. RESULTS: A dysbiotic endometrial microbiota profile composed of Atopobium, Bifidobacterium, Chryseobacterium, Gardnerella, Haemophilus, Klebsiella, Neisseria, Staphylococcus and Streptococcus was associated with unsuccessful outcomes. In contrast, Lactobacillus was consistently enriched in patients with live birth outcomes. CONCLUSIONS: Our findings indicate that endometrial microbiota composition before embryo transfer is a useful biomarker to predict reproductive outcome, offering an opportunity to further improve diagnosis and treatment strategies. Video Abstract.

Evaluating the Efficacy of Intratympanic Dexamethasone in Protecting Against Irreversible Hearing Loss in Patients on Cisplatin-Based Cancer Treatment: A Randomized Controlled Phase IIIB Clinical Trial.

OBJECTIVE: To determine the efficacy of long-term high-dose intratympanic dexamethasone in protecting the hearing capacity of cancer patients undergoing cisplatin-based ototoxic treatment. DESIGN: A randomized controlled phase IIIB clinical trial to evaluate the efficacy of dexamethasone in protecting against hearing loss in patients undergoing cisplatin treatment. The subjects participating in the clinical trial were patients with a neoplastic disease whose treatment protocol included cisplatin. The average dose of cisplatin was 444.87 mg (SD 235.2 mg). Treatment consisted of intratympanically administering dexamethasone via a passive diffusion device called Microwick (8 mg/24 h dose) from the start of treatment with cisplatin to 3 weeks after the last cycle. Patients were administered the medication to one ear, and the contralateral ear was used as the control. The treated ears were randomly chosen using a computer system (randomization). The hearing threshold was evaluated using pure tone audiometry before each cisplatin cycle. RESULTS: Thirty-four patients were recruited over a 2-year period at a reference tertiary hospital, of whom 11 were excluded. Forty-six ears were analyzed (23 treated and 23 control ears). When treatment was completed, the audiometric analysis showed a higher hearing threshold in the study group than in the control group. Differences were statistically significant at frequencies of 500, 1000, and 6000 Hz: 4.9 dB (1.1 to 8.7), 5.5 dB (0.8 to 10.3), and 16 dB (3.2 to 28.7), respectively, (p < 0.05, 95% confidence interval), but were not clinically significant according to the ASHA hearing loss criteria. Both 8.69% infection complications during treatment and 34.8% permanent perforation at 6 mo were detected after device removal. CONCLUSIONS: Long-term high-dose intratympanic dexamethasone treatment did not prevent cisplatin-induced hearing loss.

Equine infection with Leishmania spp. in Costa Rica: Study of five cases.

BACKGROUND: Cutaneous forms of leishmaniosis due to Leishmania braziliensis have been reported in horses in the New World. Domestic animals play a role in the transmission of the disease. In Costa Rica, human cases of L. braziliensis, L. panamensis and L. infantum have been reported. OBJECTIVES: The present report describes five cases of equine cutaneous leishmaniosis in Costa Rica. The aetiological diagnosis was based on the presence of the parasite within the lesions. METHODS: Skin biopsies were used to perform histopathological analyses of the lesions. Immunohistochemistry was used to detect the presence of the Leishmania spp. antigens in tissue sections. Laser-capture micro-dissection and quantitative real-time PCR techniques were carried out to detect the pathogen nucleic acid within the microscopic lesions. RESULTS: Histopathological analyses showed a granulomatous inflammation within the dermis, with multi-nucleated giant cells, macrophages, lymphocytes and few neutrophils and eosinophils. We detected the parasite by immunohistochemistry, using a rabbit polyclonal antibody raised against Leishmania spp. However, we could not identify Leishmania spp. by quantitative real-time PCR in formalin-fixed paraffin-embedded tissues, using specific primers for the conserved region in the minicircle of the Leishmania DNA kinetoplast. CONCLUSIONS: Our results emphasise the importance of Leishmania spp. not only as a causative agent of equine cutaneous disease in the New World, but also as a possible emerging pathogen. Leishmaniosis is one of the most prevalent parasitic public health problems worldwide, and equines may have a role in the epidemiology of the disease.

A Concise Synthesis of a BODIPY-Labeled Tetrasaccharide Related to the Antitumor PI-88.

A convergent synthetic route to a tetrasaccharide related to PI-88, which allows the incorporation of a fluorescent BODIPY-label at the reducing-end, has been developed. The strategy, which features the use of 1,2-methyl orthoesters (MeOEs) as glycosyl donors, illustrates the usefulness of suitably-designed BODIPY dyes as glycosyl labels in synthetic strategies towards fluorescently-tagged oligosaccharides.

Mitochondria selective trackers for long-term imaging based on readily accessible neutral BODIPYs.

We report the design of a new model based on a small neutral 8-aryl-3-formylBODIPY and its suitability to develop privileged highly bright and photostable fluorescent probes for selective and, more importantly, covalent staining of mitochondria.

First Lanthanide Complex for De Novo Phasing in Native Protein Crystallography at 1 Å Radiation.

Phasing agents enabling de novo protein structure determination at ca. 1 Å, the wavelength corresponding to the maximum intensity of the synchrotron facilities applied in biomacromolecular crystallography, have been long sought-after. The first phasing agent designed for solving native protein structures at 0.97934 Å is described herein. The agent consists of a neutral ytterbium(III)-caged complex that exhibits higher anomalous signals at shorter wavelengths when compared to the best, currently applied lanthanide-based phasing agents, all of them based on gadolinium or terbium. As a proof of principle, the complex allows determining the 3D structure of a 36 kDa protein without setting the incident beam wavelength at the metal absorption edge, the strategy followed to date to gain the strongest anomalous signal even at the expense of crystallographic resolution. The agent becomes nondisruptive to the diffraction quality of the marked crystals and allows determining accurate phases, both leading to high-quality electron-density maps that enable the full tracing of the protein structure only with one agent unit bound to the protein. The high phasing power, efficient binding to the protein, low metal-macromolecule ratio, and easy handling support the developed Yb(III) complex as the best phasing agent for X-ray crystallography of a complex biomacromolecule without using modified analogues.

Abscisic Acid-Triggered Persulfidation of the Cys Protease ATG4 Mediates Regulation of Autophagy by Sulfide.

Hydrogen sulfide is a signaling molecule that regulates essential processes in plants, such as autophagy. In Arabidopsis (Arabidopsis thaliana), hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species via an unknown mechanism. Comparative and quantitative proteomic analysis was used to detect abscisic acid-triggered persulfidation that reveals a main role in the control of autophagy mediated by the autophagy-related (ATG) Cys protease AtATG4a. This protease undergoes specific persulfidation of Cys170 that is a part of the characteristic catalytic Cys-His-Asp triad of Cys proteases. Regulation of the ATG4 activity by persulfidation was tested in a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Sulfide significantly and reversibly inactivates AtATG4a. The biological significance of the reversible inhibition of the ATG4 by sulfide is supported by the results obtained in Arabidopsis leaves under basal and autophagy-activating conditions. A significant increase in the overall ATG4 proteolytic activity in Arabidopsis was detected under nitrogen starvation and osmotic stress and can be inhibited by sulfide. Therefore, the data strongly suggest that the negative regulation of autophagy by sulfide is mediated by specific persulfidation of the ATG4 protease.

Single-cell transcriptomic atlas of the human endometrium during the menstrual cycle.

In a human menstrual cycle the endometrium undergoes remodeling, shedding and regeneration, all of which are driven by substantial gene expression changes in the underlying cellular hierarchy. Despite its importance in human fertility and regenerative biology, our understanding of this unique type of tissue homeostasis remains rudimentary. We characterized the transcriptomic transformation of human endometrium at single-cell resolution across the menstrual cycle, resolving cellular heterogeneity in multiple dimensions. We profiled the behavior of seven endometrial cell types, including a previously uncharacterized ciliated cell type, during four major phases of endometrial transformation, and found characteristic signatures for each cell type and phase. We discovered that the human window of implantation opens with an abrupt and discontinuous transcriptomic activation in the epithelia, accompanied with a widespread decidualization feature in the stromal fibroblasts. Our study provides a high-resolution molecular and cellular characterization of human endometrial transformation across the menstrual cycle, providing insights into this essential physiological process.

Menstruation: science and society.

Women's health concerns are generally underrepresented in basic and translational research, but reproductive health in particular has been hampered by a lack of understanding of basic uterine and menstrual physiology. Menstrual health is an integral part of overall health because between menarche and menopause, most women menstruate. Yet for tens of millions of women around the world, menstruation regularly and often catastrophically disrupts their physical, mental, and social well-being. Enhancing our understanding of the underlying phenomena involved in menstruation, abnormal uterine bleeding, and other menstruation-related disorders will move us closer to the goal of personalized care. Furthermore, a deeper mechanistic understanding of menstruation-a fast, scarless healing process in healthy individuals-will likely yield insights into a myriad of other diseases involving regulation of vascular function locally and systemically. We also recognize that many women now delay pregnancy and that there is an increasing desire for fertility and uterine preservation. In September 2018, the Gynecologic Health and Disease Branch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a 2-day meeting, "Menstruation: Science and Society" with an aim to "identify gaps and opportunities in menstruation science and to raise awareness of the need for more research in this field." Experts in fields ranging from the evolutionary role of menstruation to basic endometrial biology (including omic analysis of the endometrium, stem cells and tissue engineering of the endometrium, endometrial microbiome, and abnormal uterine bleeding and fibroids) and translational medicine (imaging and sampling modalities, patient-focused analysis of menstrual disorders including abnormal uterine bleeding, smart technologies or applications and mobile health platforms) to societal challenges in health literacy and dissemination frameworks across different economic and cultural landscapes shared current state-of-the-art and future vision, incorporating the patient voice at the launch of the meeting. Here, we provide an enhanced meeting report with extensive up-to-date (as of submission) context, capturing the spectrum from how the basic processes of menstruation commence in response to progesterone withdrawal, through the role of tissue-resident and circulating stem and progenitor cells in monthly regeneration-and current gaps in knowledge on how dysregulation leads to abnormal uterine bleeding and other menstruation-related disorders such as adenomyosis, endometriosis, and fibroids-to the clinical challenges in diagnostics, treatment, and patient and societal education. We conclude with an overview of how the global agenda concerning menstruation, and specifically menstrual health and hygiene, are gaining momentum, ranging from increasing investment in addressing menstruation-related barriers facing girls in schools in low- to middle-income countries to the more recent "menstrual equity" and "period poverty" movements spreading across high-income countries.

The first glimpse of the endometrial microbiota in early pregnancy.

Investigation of the microbial community in the female reproductive tract with the use of sequencing techniques has revealed that endometrial samples obtained through a transvaginal catheter are dominated by Lactobacillus species. Dysbiotic changes in the endometrial microbiota may be associated with implantation failure or early spontaneous abortion in patients who undergo assisted reproductive technology treatment. Whether or not there is an endometrial microbiota in early pregnancy is unknown. Herein we describe, the human endometrial microbiota in a patient who subsequently had an 8th week spontaneous clinical miscarriage with euploid embryos in the next cycle and, for the first time, during a successful pregnancy in which the endometrial fluid was sampled at 4 weeks of gestation. The microbial profile found on the endometrial sample before the spontaneous abortion had higher bacterial diversity and lower Lactobacillus abundance than the endometrial fluid from the healthy pregnancy. Functional metagenomics detected different Lactobacillus species between the 2 samples. Lactobacillus crispatus was present in the endometrium before the spontaneous abortion, as were other bacteria involved in dysbiosis, which had an unstable functional pattern characterized by transposases and insertion elements. Lactobacillus iners was the most prevalent microbe found in the endometrium during early pregnancy; its presence was associated with defense mechanisms and basal functions. These novel observations prompt future investigations to understand the potential implications of microbiology on healthy and pathologic human pregnancy.

Identification and Characterization of Extracellular Vesicles and Its DNA Cargo Secreted During Murine Embryo Development.

Extracellular vesicles (EVs) are known to transport DNA, but their implications in embryonic implantation are unknown. The aim of this study was to investigate EVs production and secretion by preimplantation embryos and assess their DNA cargo. Murine oocytes and embryos were obtained from six- to eight-week-old females, cultured until E4.5 and analyzed using transmission electron microscopy to examine EVs production. EVs were isolated from E4.5-day conditioned media and quantified by nanoparticle tracking analysis, characterized by immunogold, and their DNA cargo sequenced. Multivesicular bodies were observed in murine oocytes and preimplantation embryos together with the secretion of EVs to the blastocoel cavity and blastocyst spent medium. Embryo-derived EVs showed variable electron-densities and sizes (20-500 nm) and total concentrations of 1.74 × 107 ± 2.60 × 106 particles/mL. Embryo secreted EVs were positive for CD63 and ARF6. DNA cargo sequencing demonstrated no differences in DNA between apoptotic bodies or smaller EVs, although they showed significant gene enrichment compared to control medium. The analysis of sequences uniquely mapping the murine genome revealed that DNA contained in EVs showed higher representation of embryo genome than vesicle-free DNA. Murine blastocysts secrete EVs containing genome-wide sequences of DNA to the medium, reinforcing the relevance of studying these vesicles and their cargo in the preimplantation moment, where secreted DNA may help the assessment of the embryo previous to implantation.

Endometrial Liquid Biopsy Provides a miRNA Roadmap of the Secretory Phase of the Human Endometrium.

CONTEXT: Endometrial liquid biopsy (ELB) is a minimally invasive alternative for research and diagnosis in endometrial biology. OBJECTIVE: We sought to establish an endometrial micro ribonucleic acid (miRNA) roadmap based on ELB during the secretory phase of the menstrual cycle in both natural and hormonal replacement therapy (HRT) cycles. DESIGN: Human ELB samples (n = 58) were obtained from healthy ovum donors undergoing a natural and an HRT cycle consecutively. miRNA profiles were identified using next-generation sequencing (NGS). For functional analysis, messenger ribonucleic acid targets were chosen among those reported in the endometrial receptivity analysis. RESULTS: The human endometrial secretory phase is characterized by a dynamic miRNA secretion pattern that varies from the prereceptive to the receptive stages. No differences in miRNA profiles were found among natural versus HRT cycles in the same women, reinforcing the similarities in functional and clinical outcomes in natural versus medicated cycles. Bioinformatic analysis revealed 62 validated interactions and 81 predicted interactions of miRNAs differentially expressed in the HRT cycle. Annotation of these genes linked them to 51 different pathways involved in endometrial receptivity. CONCLUSION: This NGS-based study describes the miRNA signature in human ELB during the secretory phase of natural and HRT cycles. A consistent endometrial miRNA signature was observed in the acquisition of endometrial receptivity. Interestingly, no significant differences in miRNA expression were found in natural versus HRT cycles reinforcing the functional clinical similarities between both approaches.