Cardiac developmental toxicity and transcriptome analyses of zebrafish (Danio rerio) embryos exposed to Mancozeb.

Mancozeb (MZ), an antibacterial pesticide, has been linked to reproductive toxicity, neurotoxicity, and endocrine disruption. However, whether MZ has cardiactoxicity is unclear. In this study, the cardiotoxic effects of exposure to environment-related MZ concentrations ranging from 1.88 µM to 7.52 µM were evaluated at the larval stage of zebrafish. Transcriptome sequencing predicted the mechanism of MZ-induced cardiac developmental toxicity in zebrafish by enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Consistent with morphological changes, the osm, pfkfb3, foxh1, stc1, and nrarpb genes may effect normal development of zebrafish heart by activating NOTCH signaling pathways, resulting in pericardial edema, myocardial fibrosis, and congestion in the heart area. Moreover, differential gene expression analysis indicated that cyp-related genes (cyp1c2 and cyp3c3) were significantly upregulated after MZ treatment, which may be related to apoptosis of myocardial cells. These results were verified by real-time quantitative RT-qPCR and acridine orange staining. Our findings suggest that MZ-mediated cardiotoxic development of zebrafish larvae may be related to the activation of Notch and apoptosis-related signaling pathways.

Fertility-Preserving Treatment in Young Women With Grade 1 Presumed Stage IA Endometrial Adenocarcinoma: A Meta-Analysis.

OBJECTIVES: The aim of this study was to investigate the different efficacies of various fertility-preserving therapies for grade 1 presumed stage IA endometrial cancer. METHODS: We searched the major online databases (PubMed, MEDLINE, Cochrane Library, Web of Science, and Ovid) and retrieved all the research on fertility-preserving treatment for young, grade 1 presumed stage IA endometrial adenocarcinoma patients since January 2000. We used the systemic evaluation of the Cochrane Collaboration to select the literature and merge the data we collected using R3.2.2 software (R Development Core Team, Auckland, New Zealand). By comparing the remission, recurrence, and pregnancy rates, we evaluated the efficiency of 3 existing fertility-preserving treatments indirectly: a) taking oral progestin only therapy, b) hysteroscopic resection followed by progestin therapy, and c) intrauterine progestin therapy: levonorgestrel-releasing intrauterine system combined with gonadotropin-releasing hormone agonist/progestin therapy. RESULTS: Twenty-eight studies met the selection criteria. A total of 619 cases were included in this study. The group that took oral progestin only (456 patients) achieved a complete remission rate (CRR), recurrence rate (ReR), and pregnancy rate (PregR) of 76.3%, (95% confidence interval [CI], 70.7%-81.1%); 30.7% (95% CI, 21.0%-42.4%); and 52.1% (95% CI, 41.2%-66.0%), respectively. The hysteroscopic resection followed by progestin therapy group (73 patients) achieved a CRR, ReR, and PregR of 95.3% (95% CI, 87.8%-100%); 14.1% (95% CI, 7.1%-26.1%); and 47.8% (95% CI, 33.0%-69.5%), respectively. The intrauterine progestin therapy group (90 patients) achieved a CRR, ReR, and PregR of 72.9% (95% CI, 60.4%-82.5%); 11.0% (95% CI, 5.1%-22.0%); and 56.0% (95% CI, 37.3%-73.1%), respectively. CONCLUSIONS: The existing results show that patients who received hysteroscopic resection followed by progestin therapy achieved the highest CRR. Patients who received oral progestin only might be more likely to recur and have more systemic adverse effects. Recent intrauterine progestin therapy such as levonorgestrel-releasing intrauterine system combined with gonadotropin-release hormone receptor agonist/progestin have a satisfactory PregR and low ReR rate. Considering the inherent limitations of the studies we included, further well-designed, randomized controlled trials are necessary to confirm and update this analysis.

Seminal plasma S100A8/A9 as a potential biomarker of genital tract inflammation.

ABSTRACT: Infections and inflammatory reactions in the male genital tract are the leading causes of male infertility with a prevalence of 6%-10%, primarily affecting testicular and epididymal function and ultimately compromising sperm quality. However, most infertile patients with genital infection/inflammation are asymptomatic and easily overlooked. Traditional indicators, including white blood cells, elastase, and other components in semen, can reflect inflammation of the genital tract, but there is still a lack of a uniform standard method of detection. Therefore, it is necessary to explore reliable markers in semen that reflect the inflammatory status of the genital tract. Using the experimental autoimmune orchitis (EAO) model to simulate noninfectious chronic orchitis, we successfully collected ejaculated seminal fluid from EAO rats using optimized electrical stimulation devices. Proteomic analysis was performed using isobaric tags for relative and absolute quantification (iTRAQ). Compared to the control group, 55 upregulated and 105 downregulated proteins were identified in seminal plasma samples from the EAO group. In a preliminary screening, the inflammation-related protein S100A8/A9 was upregulated. We further verified that S100A8/A9 was increased in seminal plasma and highly expressed in testicular macrophages of the EAO model. In patients with oligoasthenospermia and genital tract infections, we also found that S100A8/A9 levels were remarkably increased in seminal plasma and testicular macrophages. S100A8/A9 in semen may be a potential biomarker for chronic genital inflammation. Our study provides a new potential biomarker for early diagnosis and further understanding of male infertility caused by genital inflammation.

Molecular mechanism of ethylparaben on zebrafish embryo cardiotoxicity based on transcriptome analyses.

Ethylparaben (EP), one of the parabens, a ubiquitous food and cosmetic preservatives, has caused widespread concern due to its health risks. Recently, studies have found that parabens exposure during pregnancy is negatively correlated with fetal and early childhood development. However, studies about EP on embryo development are few. In this study, the cardiotoxicity effects of EP concentrations ranging from 0 to 20 mg/L on zebrafish embryo development were explored. Results showed that EP exposure induce abnormal cardiac function and morphology, mainly manifested as pericardial effusion and abnormal heart rate in early-stage development of zebrafish embryos. Through transcriptome sequencing followed by Gene Ontology enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, we further confirmed that EP exposure ultimately leads to cardiac morphologic abnormalities via the following three mechanisms: 1. Disruption of the retinoic acid signaling pathway related to original cardiac catheter development; 2. Inhibition of gene expression related to myocardial contraction; 3. Orientation development disturbance of heart tube. Moreover, O-Dianisidine staining, whole-mount in situ hybridization at 30 and 48 hours post fertilization (hpf) and hematoxylin-eosin staining results all confirmed the decreased heart's return blood volume, misoriented heart tubes toward either the right or the middle side, and heart loop defects. For the first time, we explored the mechanism by which EP exposure causes abnormal heart development in zebrafish embryos, laying the foundation for further revealing of the EP toxicity on embryonic development.

Obesity Causes Abrupt Changes in the Testicular Microbiota and Sperm Motility of Zebrafish.

Background: Obesity is a recognized risk factor for low fertility and is becoming increasingly prevalent in many countries around the world. Obesity changes intestinal microbiota composition, causes inflammation of various organs, and also reduces sperm quality. Several microorganisms are present in the testis. However, whether obesity affects the changes of testicular microbiota and whether these changes are related to reduced fertility in obese men remain to be elucidated. Methods: In the present study, a zebrafish obesity model was established by feeding with egg yolk powder. Sperm motility was measured by the Computer Assisted Sperm Analysis system, testicular microbial communities was assessed via 16s RNA sequencing, the immune response in zebrafish testis was quantified by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and the testicular tissue structure was detected by electron microscopy and hematoxylin-eosin staining. Results: Compared with the control group, zebrafish sperm motility was dramatically reduced, the expression of testicular proinflammatory cytokines in the testes was upregulated, and the blood-testis barrier structure was disrupted in the obese group. In addition, testicular microbiome composition was clearly altered in the obese group. Conclusion: Obesity alters testicular microbiota composition, and the reason behind the decreased sperm motility in obese zebrafish may be related to changes in the testicular microbial communities.

Severe Acute Respiratory Syndrome Coronavirus 2 and Male Reproduction: Relationship, Explanations, and Clinical Remedies.

Coronavirus disease 2019 (COVID-2019) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been an ongoing pandemic and worldwide public health emergency, having drawn a lot of attention around the world. The pathogenesis of COVID-19 is characterized by infecting angiotensin-converting enzyme 2 (ACE2)-expressing cells, including testis-specific cells, namely, Leydig, Sertoli, and spermatogenic cells, which are closely related to male reproduction. This leads to aberrant hyperactivation of the immune system generating damage to the infected organs. An impairment in testicular function through uncontrolled immune responses alerts more attention to male infertility. Meanwhile, the recent clinical data indicate that the infection of the human testis with SARS-CoV-2 may impair male germ cell development, leading to germ cell loss and higher immune cell infiltration. In this review, we investigated the evidence of male reproductive dysfunction associated with the infection with SARS-CoV-2 and its possible immunological explanations and clinical remedies.

Evaluation of 3,4,4,9-trichlorocarbanilide to zebrafish developmental toxicity based on transcriptomics analysis.

Triclocarban (TCC), considered an endocrine-disrupting, persistent, and bioaccumulating organic matter, has attracted a great deal of attention for its pollution and health risks. However, studies on its toxicological mechanism, especially for embryo development are limited. This article explores the cardiac developmental toxicity induced in zebrafish embryos after exposure to different TCC concentrations. First, liquid chromatography-tandem mass spectrometry was used in detecting TCC in embryos in vivo after exposure to various TCC. Results showed that embryonic TCC content reached 9.23 ng after exposure to 300 µg/L TCC, the heart rates of the embryos markedly decreased, heart abnormalities significantly increased. In addition, obvious pericardial effusion was observed in the larvae. Through transcriptome sequencing, 200 differential gene expression (DGE) patterns were detected in the TCC (300 µg/L) experimental and control groups. The results of GO function analysis and KEGG pathway of DGE showed that aryl hydrocarbon receptor (AhR) activation and cyp-related genes (cyp1a, cyp1b1 and cyp1c) were significantly up-regulated. these affected the normal development of zebrafish embryonic heart, tissue edema, and hemorrhage. TCC exhibited strong cardiac teratogenic effects and developmental toxicity, which is partly related to AhR activation. Transcriptome-based results are helpful in precisely determining the risk of TCC exposure. The potential mechanism between TCC and AhR should be further investigated.

tmbim4 protects against triclocarban-induced embryonic toxicity in zebrafish by regulating autophagy and apoptosis.

Triclocarban (TCC), an antibacterial agent widely used in personal care products, can affect embryonic development. However, the specific molecular mechanism of TCC-induced embryonic developmental damage remains unclear. In this study, TCC exposure was found to increase the expression of tmbim4 gene in zebrafish embryos. The tmbim4 mutant embryos are more susceptible to TCC exposure than wild-type (WT) embryos, with tmbim4 overexpression reducing TCC-induced embryonic death in the former. Exposure of tmbim4 mutant larvae to 400 µg/L TCC substantially increased apoptosis in the hindbrain and eyes. RNA-sequencing of WT and tmbim4 mutant larvae indicated that knockout of the tmbim4 gene in zebrafish affects the autophagy pathway. Abnormalities in autophagy can increase apoptosis and TCC exposure caused abnormal accumulation of autophagosomes in the hindbrain of tmbim4 mutant zebrafish embryos. Pretreatment of TCC-exposed tmbim4 mutant zebrafish embryos with autophagosome formation inhibitors, substantially reduced the mortality of embryos and apoptosis levels. These results indicate that defects in the tmbim4 gene can reduce zebrafish embryo resistance to TCC. Additionally, apoptosis induced by abnormal accumulation of autophagosomes is involved in this process.

Folic acid deficiency damages male reproduction via endoplasmic reticulum stress-associated PERK pathway induced by Caveolin-1 in mice.

Folic acid is critical to maintaining normal male reproductive function. Endoplasmic reticulum (ER) stress plays a crucial role in folic acid deficiency. Studies have shown that Caveolin-1 (Cav-1) is involved in ER stress, but the specific mechanism in male reproduction is still unclear. This study aimed to investigate the effects of folic acid deficiency on spermatogenesis and elucidate the underlying mechanisms. C57BL/6 mice fed with folic acid deficiency induced diet(0.3 mg/kg) were used. A significant decrease in the sperm concentration in the folic acid deficiency group was observed. Meanwhile, folic acid deficiency decreased Cav-1 expression in the testis tissue and increased endoplasmic reticulum stress-related PERK, eIF2α, ATF4, CHOP gene expression. Our results suggest that folic acid deficiency can affect male reproduction through the Cav-1-PERK-eIFα-ATF4-CHOP pathway.Abbreviations: ATF4: activating transcription factor 4; Ca2+: calcium ion; Cav-1: Caveolin-1; CCK-8: cell counting kit-8; CHOP: CCAAT-enhancer-binding protein homologous protein; DNA: Deoxyribonucleic acid; DSB: double strand breakage; eIF2α: eukaryotic Initiation Factor 2 alpha; ER: endoplasmic reticulum; FD: folic acid deficiency; FITC: fluorescein isothiocyanate; HE: hematoxylin and eosin; H3K4me3: histone H3 lysine 4 trimethylation; PERK: protein kinase RNA-like endoplasmic reticulum kinase; PI: propidium iodide; RT-qPCR: quantitative reverse transcription PCR; TUNEL: TdT mediated dUTP Nick End Labeling.

Prokineticin 2 via Calcium-Sensing Receptor Activated NLRP3 Inflammasome Pathway in the Testicular Macrophages of Uropathogenic Escherichia coli-Induced Orchitis.

Reproductive tract infections contribute to the development of testicular inflammatory lesions, leading to male infertility. Previous research shows that the activation of the NLRP3 inflammasome in orchitis promotes the secretion and maturation of IL-1ß and, thus, decreases male fertility. The calcium-sensing receptor (CaSR) is closely related to the secretion of proinflammatory cytokines. An increase in the CaSR level promotes the assembly and activation of the NLRP3 inflammasome. However, the role of CaSRs in orchitis is unknown. We first constructed a uropathogenic Escherichia Coli (UPEC) rat orchitis model and then detected the expression of CaSR and NLRP3 inflammatory pathway proteins in testicular macrophages (TM) through RT-PCR and WB, calcium levels in TM through flow cytometry, and proinflammatory factor IL-1ß through ELISA. In addition, testosterone levels in the serum samples were detected using liquid chromatography-mass spectrometry (LC-MS). Here, we show that CaSR upregulation after infection in TM in a rat model of UPEC induces the activation of the NLRP3 inflammasome pathway and thereby enhances IL-1ß secretion and reduces the testosterone level in the blood. Moreover, CaSR inhibitors can alleviate inflammatory impairment. After UPEC challenge in vitro, CaSR promoted NLRP3 expression and released IL-1ß cleaved from TM into the supernatant. Overall, elevated CaSR levels in TM in testes with UPEC-induced orchitis may impair testosterone synthesis through the activation of the NLRP3 pathway and PK2 is an upstream regulatory protein of CaSR. Our research further shows the underlying mechanisms of inflammation-related male infertility and provides anti-inflammatory therapeutic targets for male infertility.

Effect of paternal age on offspring birth defects: a systematic review and meta-analysis.

OBJECTIVE: This systematic review and meta-analysis was aimed at determining whether paternal age is a risk factor for offspring birth defects. RESULTS: A total of 38 and 11 studies were included in the systematic review and meta-analysis, respectively. Compared with reference, fathers aged 25 to 29, young fathers (< 20 years) could increase the risk of urogenital abnormalities (OR: 1.50, 95 % CI: 1.03-2.19) and chromosome disorders (OR: 1.38, 95 % CI: 1.12-1.52) in their offsprings; old fathers (≥ 40 years) could increase the risk of cardiovascular abnormalities (OR: 1.10, 95 % CI: 1.01-1.20), facial deformities (OR: 1.08, 95 % CI: 1.00-1.17), urogenital abnormalities (OR: 1.28, 95 % CI: 1.07-1.52), and chromosome disorders (OR: 1.30, 95 % CI: 1.12-1.52). CONCLUSIONS: Our study indicated that paternal age is associated with a moderate increase in the incidence of urogenital and cardiovascular abnormalities, facial deformities, and chromosome disorders. METHODS: PubMed, Web of Science, the Cochrane Library, and Embase were searched for relevant literatures from 1960 to February 2020. The systematic review follows PRISMA guidelines. Relevant meta-analyses were performed.