Longitudinal profiling of human androgenotes through single-cell analysis unveils paternal gene expression dynamics in early embryo development.

STUDY QUESTION: How do transcriptomics vary in haploid human androgenote embryos at single cell level in the first four cell cycles of embryo development? SUMMARY ANSWER: Gene expression peaks at the fourth cell cycle, however some androcytes exhibit unique transcriptional behaviors. WHAT IS KNOWN ALREADY: The developmental potential of an embryo is determined by the competence of the oocyte and the sperm. However, studies of the contribution of the paternal genome using pure haploid androgenotes are very scarce. STUDY DESIGN, SIZE, DURATION: This study was performed analyzing the single-cell transcriptomic sequencing of 38 androcytes obtained from 10 androgenote bioconstructs previously produced in vitro (de Castro et al., 2023). These results were analyzed through different bioinformatics software such as g: Profiler, GSEA, Cytoscape, and Reactome. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single cell sequencing was used to obtain the transcriptomic profiles of the different androcytes. The results obtained were compared between the different cycles studied using the DESeq2 program and functional enrichment pathways using g: Profiler, Cytoscape, and Reactome. MAIN RESULTS AND THE ROLE OF CHANCE: A wave of paternally driven transcriptomic activation was found during the third-cell cycle, with 1128 upregulated and 225 downregulated genes and the fourth-cell cycle, with 1373 upregulated and 286 downregulated genes, compared to first-cell cycle androcytes. Differentially expressed routes related to cell differentiation, DNA-binding transcription, RNA biosynthesis and RNA polymerase II transcription regulatory complex, and cell death were found in the third and fourth with respect to the first-cell cycle. Conversely, in the fourth cell cycle, 153 downregulated and 332 upregulated genes were found compared with third cell cycle, associated with differentially expressed processes related to E-box binding and zinc finger protein 652 (ZNF652) transcription factor. Further, significant overexpression of LEUTX, PRAMEF1, DUXA, RFPL4A, TRIM43, and ZNF675 found in androgenotes, compared to biparental embryos, highlights the paternal contributions to zygote genome activation. LARGE SCALE DATA: All raw sequencing data are available through the Gene Expression Omnibus (GEO) under accessions number: GSE216501. LIMITATIONS, REASONS FOR CAUTION: Extrapolation of biological events from uniparental constructs to biparental embryos should be done with caution. Maternal and paternal genomes do not act independently of each other in a natural condition. The absence of one genome may affect gene transcription of the other. In this sense, the haploid condition of the bioconstructs could mask the transcriptomic patterns of the single cells. WIDER IMPLICATIONS OF THE FINDINGS: The results obtained demonstrated the level of involvement of the human paternal haploid genome in the early stages of embryo development as well as its evolution at the transcriptomic level, laying the groundwork for the use of these bioconstructs as reliable models to dispel doubts about the genetic role played by the paternal genome in the early cycles of embryo development. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Instituto de Salud Carlos III (ISCIII) through the project 'PI22/00924', co-funded by European Regional Development Fund (ERDF); 'A way to make Europe'. F.D. was supported by the Spanish Ministry of Economy and Competitiveness through the Miguel Servet program (CPII018/00002). M.J.E. was supported by Instituto de Salud Carlos III (PI19/00577 [M.J.E.]) and FI20/00086. P.dC. was supported by a predoctoral grant for training in research into health (PFIS PI19/00577) from the Instituto de Salud Carlos III. All authors declare having no conflict of interest with regard to this trial.

Inhibition of KIF20A by BKS0349 reduces endometriotic lesions in a xenograft mouse model.

Several studies have suggested a possible etiological association between ovarian endometriosis and ovarian cancer. Evidence has shown that KIF20A overexpression might confer a malignant phenotype to ovarian tumors by promoting proliferation and inhibiting apoptosis. However, no data about the role of KIF20A in endometriosis have been described. In this study, the human endometrium (n = 4) was transfected by mCherry adenovirus and intraperitoneally implanted in mice. Subsequently, mice were divided in three groups (n = 8/group) that were treated with Vehicle, BKS0349 (KIF20A-antagonist) or cabergoline (dopamine receptor agonist) for 21 days. mCherry-labeled endometriotic lesions were monitored over time using the IVIS Imaging System. Mice were sacrificed 72 h after the last administration; proliferation was evaluated by immunohistochemistry and apoptosis by TUNEL. CCND1 gene expression (G1 phase-related gene) was measured by qRT-PCR. A significant reduction in mCherry-fluorescent signal was observed in the BKS0349 group after treatment ended (D24) compared with D0 (P-value = 0.0313). Moreover, the mCherry signal on D24 showed a significant decrease in the BKS0349 group compared with controls (P-value = 0.0303), along with significant size reduction of endometriotic lesions observed in the BKS0349 group compared with control on D24 (P-value = 0.0006). Functional studies showed a significant reduction in proliferating cells in the BKS0349-treated group compared with controls (P-value = 0.0082). In addition, CCND1 expression was decreased in the BKS0349 group compared with control (P-value = 0.049) at D24 and a significant increase in apoptotic cells among endometriotic lesions in BKS0349-treated mice was observed compared with control (P-value = 0.0317). Based on these findings, we concluded that BKS0349 induces apoptosis and inhibits cell proliferation, reducing endometriotic lesion size and suggesting KIF20A inhibition by BKS0349 as a novel therapeutic treatment for endometriosis.

Phenotypic detection of clinical isolates of Haemophilus influenzae with altered penicillin-binding protein 3.

The aims of this study were to determine the correlation of mutations in the ftsI gene (coding for PBP3) of Haemophilus influenzae with aminopenicillin resistance and to evaluate the 2017 European Committee for Antibiotic Susceptibility Testing (EUCAST) guidelines for clinical categorization of ampicillin, amoxicillin, and amoxicillin-clavulanate for strains with mutated PBP3 conferring resistance (rPBP3). A panel of 91 H. influenzae isolates was genetically characterized by sequencing of the fstI gene. For all the studied isolates, a screening with benzylpenicillin 1U (BP1) was carried out and minimum inhibitory concentrations (MICs) of ampicillin, amoxicillin, and amoxicillin-clavulanate were tested and interpreted according to EUCAST recommendations. ftsI sequence analysis revealed a total of 14 different amino acid substitutions in PBP3. The substitution patterns most commonly observed were [D350N, M377I, A502V, N526K] among the bla-positive rPBP3 strains (37.5%) and [D350N, A502T, N526K] among the bla-negative rPBP3 strains (24.5%). Screening with BP1 was able to correctly categorize 100% of the bla-negative sPBP3 strains, 100% of the bla-positive strains, and 92% of the bla-negative rPBP3 ones. Only 29% of the bla-negative rPBP3 strains evaluated displayed ampicillin MICs above the current EUCAST resistant breakpoint defined at 1 µg/ml. The PBP3 substitution patterns of the strains evaluated are similar to the ones observed in previous Spanish and European studies. Although the screening with BP1 proved to be adequate in the detection of bla-negative rPBP3 strains, these cannot be reliably identified by current 2018 EUCAST breakpoints for ampicillin.

Non-molecular detection of carbapenemases in Enterobacteriaceae clinical isolates.

Infections caused by carbapenemase-producing bacteria are becoming a major clinical and public health concern. Detection of carbapenemase producing strains is often challenging, since susceptibility to carbapenems may vary significantly among carbapenemase producers. Some carbapenemases have shown to exhibit weak activity against carbapenems leading to minimum inhibitory concentrations of carbapenems below the breakpoint for defining clinical resistance and even below the proposed screening breakpoint. Thus, reliable and rapid detection of carbapenemase-activity is needed for an appropriate patient management and a rapid implementation of infection prevention and control measures. Over the last years, an increasing number of non-molecular assays for prompt detection of carbapenemase activity have been described and developed. However, none of the currently available phenotypic methods have proved to be full specific and sensitive. Selection of the appropriate methodological approach will depend on each situation. Factors to be considered include the epidemiological status, laboratory resources and availability of other confirmation tests. In this review, we provide an overview of the currently available non-molecular methods for detection of carbapenemase activity.

Mosaic results after preimplantation genetic testing for aneuploidy may be accompanied by changes in global gene expression.

Aneuploidy in preimplantation embryos is a major cause of human reproductive failure. Unlike uniformly aneuploid embryos, embryos diagnosed as diploid-aneuploid mosaics after preimplantation genetic testing for aneuploidy (PGT-A) can develop into healthy infants. However, the reason why these embryos achieve full reproductive competence needs further research. Current RNA sequencing techniques allow for the investigation of the human preimplantation transcriptome, providing new insights into the molecular mechanisms of embryo development. In this prospective study, using euploid embryo gene expression as a control, we compared the transcriptome profiles of inner cell mass and trophectoderm samples from blastocysts with different levels of chromosomal mosaicism. A total of 25 samples were analyzed from 14 blastocysts with previous PGT-A diagnosis, including five low-level mosaic embryos and four high-level mosaic embryos. Global gene expression profiles visualized in cluster heatmaps were correlated with the original PGT-A diagnosis. In addition, gene expression distance based on the number of differentially expressed genes increased with the mosaic level, compared to euploid controls. Pathways involving apoptosis, mitosis, protein degradation, metabolism, and mitochondrial energy production were among the most deregulated within mosaic embryos. Retrospective analysis of the duration of blastomere cell cycles in mosaic embryos revealed several mitotic delays compared to euploid controls, providing additional evidence of the mosaic status. Overall, these findings suggest that embryos with mosaic results are not simply a misdiagnosis by-product, but may also have a genuine molecular identity that is compatible with their reproductive potential.

Copper and lead exposures disturb reproductive features of primary endometrial stromal and epithelial cells.

This study investigates if Cu and Pb act as endocrine disruptors affecting endometrial cells. Primary EnSCs and EnECs were exposed to Cu (0, 50, 100 and 200 µM) or Pb (0, 30, 100 and 500 µM) and assessed for viability, decidualization, apoptosis and proliferation on EnSCs, and wound healing and adhesion capabilities on EnECs. Cu exposure decreased significantly cell viability in a dose-dependent manner. Cu and Pb negatively affected in vitro decidualization, showing a significant decrease in PRL secretion. HOXA10 and ERα mRNA levels significantly decreased in decidualized cells (dEnSCs) exposed to Cu. Cu and Pb decreased adhesion and regeneration capability in EnEC. This study reveals that Cu and Pb could negatively affect endometrial functionality, compromising the decidualization process and disrupting endometrial regeneration and embryo adhesion. Therefore, special care should be taken considering heavy metals exposure if pregnancy is being pursued.

Proteomic analysis of the human receptive versus non-receptive endometrium using differential in-gel electrophoresis and MALDI-MS unveils stathmin 1 and annexin A2 as differentially regulated.

BACKGROUND: The transcriptome of the endometrium throughout the menstrual cycle has been described in recent years. However, the proteomic of the window of implantation remains unknown. The aim of this study was to compare the proteome of the human endometrium in the pre-receptive phase versus the receptive phase by identifying and quantifying the proteins differentially expressed using differential in-gel electrophoresis (DIGE) and mass spectometry (MS). METHODS: Endometrial biopsies were collected at days 2 (pre-receptive) and 7 (receptive) after the urinary luteal hormone surge in the same menstrual cycle from eight fertile women (corresponding to days 16 and 21 of the menstrual cycle). Proteins were extracted and labeled with CyDye DIGE fluorofores and separated using two-dimensional gel electrophoresis. RESULTS: Image analysis using the DeCyder software followed by protein identification by matrix-assisted laser desorption/ionization-MS and database searching revealed 32 differentially expressed proteins, although only annexin A2 and stathmin 1 were consistently regulated in the two experiments performed. Validation and localization of annexin A2 and stathmin 1 were performed by western blot and immunohistochemistry. Annexin A2 and stathmin 1 were investigated using an endometrial refractoriness model. The results highlight the key potential of these proteins as possible targets for human endometrial receptivity and interception. CONCLUSION: This study shows that the human endometrium has a differential proteomic repertoire during the window of implantation. Consequently, we identified annexin A2 and stathmin 1 as differentially expressed molecules in the receptive endometrium.

CXCL10 and IL-6 induce chemotaxis in human trophoblast cell lines.

The investigation of trophoblast chemoattractive molecules in humans is of high interest for the reproductive field. Current evidence in ruminants demonstrates that CXCL10, formerly the interferon-gamma-inducible protein 10 (IP-10), is a potent chemotactic molecule implicated in the migration of trophoblast cells during early gestation. The aim of this work was to explore the existence of CXCL10/CXCR3 in the human model. Furthermore, chemotaxis assays were performed to demonstrate CXCL10 chemotactic activity in the human trophoblast cell lines JEG-3 and AC-1M88. Surprisingly, the conditioned media from epithelial endometrial cells (EEC) induced the highest trophoblast migration rate. Cytokine and chemokine membrane protein arrays were used to identify the secreted protein profile of EEC-conditioned media, and IL-6 was found to be the most abundant and CXCL13 the second most abundant molecule. Using a chemotaxis assay on AC-IM88, IL-6 antibody blocked the effect of EEC, indicating IL-6 to be an effective chemoattractive factor for trophoblast cells in the human model.

Evaluation of the carbapenem inactivation method (CIM) for detecting carbapenemase activity in enterobacteria.

OBJECTIVES: The objective of this study was to evaluate the accuracy of the CIM test in the detection of carbapenemase activity in 124 strains of Enterobacteriaceae. METHODS: A panel of 124 previously characterized Enterobacteriaceae was tested: 77 strains producing the following carbapenemase families: KPC (n = 14), GES (n = 22), NDM (n = 19), VIM (n = 4), IMP (n = 4) and OXA-48 (n = 14) and 47 non-carbapenemase producers. For the CIM method, an active susceptibility meropenem disc was exposed to a bacterial suspension of a test strain; when a carbapenemase is produced, the antibiotic is inactivated allowing uninhibited growth of an indicator strain after overnight incubation. A clear inhibition zone (≥20 mm) was considered indicative of no-carbapenemase activity. RESULTS: All KPC, NDM, VIM, IMP or OXA-48 producing strains were unequivocally detected with the CIM test. CIM false negative results were obtained with eleven Enterobacter cloacae producing GES-6. Two other E. cloacae not producing carbapenemase (one with SHV-12, one hyperproducing AmpC) were positive by the test. The sensitivity and specificity of the assay compared to those of molecular methods were 85.7% and 95.7%, respectively. CONCLUSIONS: The CIM method proved to be inexpensive and easy to interpret. It provided less than optimal results in the detection of GES-6 activity.

[Correlation between MALDI-TOF Vitek-MSTM system and conventional identification methods of gastrointestinal infection causing bacteria]. / Correlación entre el sistema de MALDI-TOF Vitek-MSTM y los métodos convencionales de identificación de bacterias causantes de infección gastrointestinal.

OBJECTIVE: Rapid identification of pathogens is essential for the diagnosis of gastrointestinal infections. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry has shown to be effective and fast for the identification of microorganisms. The objective of this study was to evaluate the correlation between Vitek-MSTM and conventional methods for bacterial identification causing gastrointestinal infection. METHODS: A total of 329 gastrointestinal pathogens were identified using Vitek-MSTM (v2 SARAMIS MS -ID, bioMérieux, Marcy-I´Étoile, France) and routine diagnostic methods simultaneously. In cases of discrepancy 16SrRNA gene sequencing was performed. RESULTS: The correlation between Vitek-MSTM and diagnostic methods was 100% except for Yersinia enterocolitica (94.1%), Helicobacter pylori (10%) and Aeromonas veronii (0 %). CONCLUSIONS: Vitek-MSTM is a quick and useful method for identification of enterophatogenic bacteria. It is necessary to improve the performance of the system for the identification of H. pylori and A. veronii.