Wael Bahia

Problem: Recurrent pregnancy loss (RPL), defined as two or more consecutive pregnancy losses before 20 weeks of gestation, affects 1%–5% of couples of reproductive age worldwide. Growing evidence indicates a role for the microbiome in reproductive health, particularly in unexplained RPL. Method of Study: Based on a review of literature from PubMed, EMBASE, and Web of Science databases from January 2020 to September 2025, this comprehensive overview explores the current understanding of the link between microbiome dysbiosis and RPL. Results: Microbiome dysbiosis, especially a reduction in Lactobacillus dominance and increased diversity, is strongly linked to RPL across multiple reproductive sites. RPL is associated with the loss of protective Lactobacillus crispatus and a higher presence of potentially harmful bacteria, including Gardnerella vaginalis and Atopobium vaginae. An altered gut microbiome, particularly with lipopolysaccharide‐producing gram‐negative bacteria, contributes to systemic inflammation and immune dysfunction by disrupting maternal‐fetal immune tolerance. The microbiome‐immune axis is essential for establishing maternal‐fetal tolerance, with dysbiosis promoting pro‐inflammatory Th1/Th17 responses while suppressing regulatory T cells. Multiple mechanisms connect microbiome dysbiosis to RPL, including local inflammation, systemic immune issues, disruption of maternal‐fetal immune tolerance, molecular mimicry, and autoimmunity. Conclusions: The microbiome is a promising new target for RPL treatment, with personalized microbial profiling and targeted therapies showing potential to improve pregnancy outcomes. Clinical implementation requires standardized protocols, larger randomized controlled trials, and validation of microbiome‐targeted interventions.

ASD etiology may be influenced by non-coding single-nucleotide polymorphisms (SNPs) in 3' untranslated regions (3'UTRs). These variations can affect post-transcriptional regulation by altering RNA structure and miRNA binding patterns. PTEN and TSC1, two key regulators of the PI3K/AKT/mTOR signaling pathway, are promising candidate genes for ASD. We performed a case-control study involving 108 individuals diagnosed with ASD and 184 healthy matched controls from the Tunisian population. Four specific 3'UTR SNPs (PTEN: rs701848, rs34140758; TSC1: rs739442, rs2809244) were analyzed through genotyping. Statistical associations were assessed using various genetic inheritance models, with multivariate logistic regression adjusting for gender, family psychiatric history, and parental age. A thorough bioinformatics approach was applied, which included miRNASNP-v3 for predicting altered miRNA binding sites, RNAhybrid for calculating the minimum free energy (MFE) of miRNA-mRNA duplexes, PhyloP and phastCons for evolutionary conservation analysis, and mfold for modeling RNA secondary structures. PTEN rs701848 showed a significant statistical association with ASD risk that remained significant after correction for multiple testing (corrected p < .0004), while TSC1 rs739442 showed a suggestive association (p = .009, corrected p = .036). Systematic functional assessment using multi-criteria evaluation identified rs701848 as having high predicted functional impact (extensive miRNA binding changes, including loss of neurodevelopmental miR-129 sites, high conservation (PhyloP = 2.24), and altered RNA stability), while rs739442 showed moderate impact, and rs2809244 minimal predicted functional significance. For mutant alleles, RNA structure modeling showed slight decreases in mRNA stability, which might affect transcript accessibility to regulatory factors. Evolutionary conservation analysis demonstrated that rs701848 and rs34140758 reside in highly conserved areas, whereas rs739442 and rs2809244 are in less evolutionarily conserved regions. By integrating population-genetics data with bioinformatics predictions, this study supports the relevance of non-coding SNPs as biological risk factors for ASD. It emphasizes their functional significance in modulating post-transcriptional gene regulation mechanisms.

Problem: This study analyzed the contribution of DRB1, DQB1, and DPB1 HLA class II alleles and resulting 3‐locus haplotypes to the overall risk of idiopathic recurrent pregnancy loss (RPL).Method of study: The study participants included 188 Tunisian participants comprising 84 women with established diagnoses of RPL, and 104 matched multiparous females as controls. Genotyping of HLA alleles was done by PCR‐SSP.Results: The allelic frequencies of DPB1, DRB1, and DQB1 were consistent with Hardy‐Weinberg equilibrium among control subjects. Among class II alleles, DRB1*04:01:01 (p = .03), DRB1*08:01:01 (p = .04), and DPB1*01:01:01 (p = .04) were positively associated, whereas DPB1*04:01:01 (p = .012) and DPB1*14:01:01 (p = .006) were negatively associated with risk of RPL. The association of DRB1 and DPB1 alleles with RPL was lost after correction for multiple comparisons. The prevalence of the seven determined DQB1 alleles was not significantly different among case and control groups. Higher prevalence of DRB1*07:01:01∼ DQB1*02:01:01∼DPB1*04:01:01 (0.0417 vs. 0.000; p = .049) coupled with lower prevalence of DRB1*13:01:01∼DQB1*06:01:01∼DPB1*02:01:01 (0.0167 vs. 0.1000; p = .014) haplotypes was noted in women with RPL, thus could be considered as an RPL at‐risk and RPL‐protective haplotype, respectively.Conclusion: The study establishes weak/no contribution of class II alleles and corresponding 3‐locus haplotypes to the altered risk of RPL among Tunisian women and does not confirm previous findings on other Arab‐speaking populations of an HLA association with RPL.

The growing prevalence of resistance to antibiotics potentially makes Escherichia coli and Staphylococcus aureus serious pathogens, necessitating the development of new antimicrobial agents. We extracted crude biosurfactants from a potential probiotic Bacillus spp. to control pathogenic bacteria associated with aerobic vaginal infection. Using nanotechnology formulations, we developed nanoemulsions based on biosurfactants at different concentrations (1% and 3.33%). The results showed that these nanoemulsions were stable, with a weighted index of 0.3, and demonstrated broad-spectrum antibacterial activity against Escherichia coli and Staphylococcus aureus, with MICs ranging between 1.25 and 4 mg/mL. Additionally, the nanoemulsions exhibited interesting antibiofilm effects. All strains became more sensitive to the antibiotics to which they were resistant because of various biosurfactant formulations combined with antibiotics. Lower concentrations of BNE1% and 3.33% were still more efficient than the crude biosurfactants. Our findings demonstrated that the biosurfactant had a strong antibiofilm effect against all tested pathogens. This antibacterial effect can be explained by their ability to alter cell physiology such as cell hydrophobicity and membrane disintegration. Thus, we can conclude that the use of nanotechnology formulations has improved this effect, and the nanoemulsions developed in this study can be used as a potential anti-infectious therapy against multidrug-resistant bacterial strains of clinical origin.

Recurrent pregnancy loss (RPL) is a major reproductive problem that affects approximately 5% of couples. The objective of this study was to assess vaginal flora dysbiosis in women suffering from unexplained RPL and to investigate the pathogenic properties of the microbiota associated with aerobic vaginitis (AV). The study included one hundred fifteen women, 65 with RPL and 50 controls. The diversity of vaginal microbiota isolated was evaluated by molecular sequencing. Then, pathogenic factors, such as acid-resistance, antibiotics susceptibility, and biofilm formation were evaluated. The prevalence of AV was five-fold higher in the RPL group than in the controls (64.6% vs. 12.0%). The most prevalent isolates in the case group were Enterococcus spp. (52%) and Staphylococcus spp. (26%). All bacterial strains tolerate low pH. The prevalence of multidrug resistance (MDR) among all bacteria was 47.7%. Of all strains, 91.0% were biofilm producers. The presence of MDR was found to be related to biofilm formation. The results provide evidence supporting an increased presence of dysbiosis of the vaginal flora, especially AV, in women with RPL in Tunisia. The viability of the AV-associated bacteria and their persistence in the genitals may be due to their ability to resist low pH and to produce a biofilm.

Background: The pathogenesis of recurrent pregnancy loss (RPL) is multifactorial and not completely elucidated. Dysregulated immunity was implicated with RPL, in which regulatory T cells (Tregs) are key. As Tregs development and function are regulated by forkhead box P3 (FOXP3) transcription factor, and as FOXP3 expression is genetically determined, a role for FOXP3 polymorphisms in RPL pathogenesis was suggested.Aim: To investigate the association of rs2294021, rs2232365, rs3761548, and rs141704699 FOXP3 variants with idiopathic RPL in Lebanese women.Methods: This retrospective case‐control study included 386 RPL cases and 398 age‐matched control women. Logistic odds ratios (OR) were estimated with 95% confidence interval after adjustment; a significance value of P<.05 was set.Results: Significantly lower rs22944021 and rs2232365 minor allele frequency (MAF) was found in patients with idiopathic RPL in comparison with the control group. Furthermore, statistically significantly lower frequency of heterozygous and homozygous rs2294021 and rs2232365 genotypes was seen in controls, while significantly lower rs3761548 heterozygous genotype frequencies were found in the patient group. Obesity, antihypertension treatment, smoking, positive RPL family history, abortion state, and infertility treatment correlated negatively with rs2294021, while rs2232365 negatively correlated with obesity, and rs3761548 negatively correlated with infertility treatment. Marked linkage disequilibrium (LD) was noted among FOXP3 SNPs, with TGCC and CGAC haplotypes being positive, while CAAC, CACC, and TGAC haplotypes being negatively associated with RPL risk. Except for CGAC, the association of these haplotypes with RPL persisted after adjustment.Conclusion: FOXP3 gene variants and haplotypes are associated with altered incidence of RPL, proposing the role of Treg in RPL pathogenesis.

Biosurfactants exhibit antioxidant, antibacterial, antifungal, and antiviral activities. They can be used as therapeutic agents and in the fight against infectious diseases. Moreover, the anti-adhesive properties against several pathogens point to the possibility that they might serve as an anti-adhesive coating agent for medical inserts and prevent nosocomial infections, without using synthetic substances. In this study, the antimicrobial, antibiofilm, cell surface hydrophobicity, and antioxidative activities of biosurfactant extracted from Bacillus sp., against four pathogenic strains of Staphylococcus spp. associated with vaginal infection, were studied. Our results have shown that the tested biosurfactant possesses a promising antioxidant potential, and an antibacterial potency against multidrug clinical isolates of Staphylococcus, with an inhibitory diameter ranging between 27 and 37 mm, and a bacterial growth inhibition at an MIC of 1 mg/ mL, obtained. The BioSa3 was highly effective on the biofilm formation of different tested pathogenic strains. Following their treatment by BioSa3, a significant decrease in bacterial attachment (p < 0.05) was justified by the reduction in the optical (from 0.709 to 0.111) following their treatment by BioSa3. The antibiofilm effect can be attributed to its ability to alter the membrane physiology of the tested pathogens to cause a significant decrease (p < 0.05) of over 50% of the surface hydrophobicity. Based on the obtained result of the bioactivities in the current study, BioSa3 is a good candidate in new therapeutics to better control multidrug-resistant bacteria and overcome bacterial biofilm-associated infections by protecting surfaces from microbial contamination.