pH-triggered polymeric nanoparticles in gel for preventing vaginal transmission of HIV and unintended pregnancy.

Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/ AIDS) and unplanned pregnancy affect female reproductive health globally. A single product providing a dual purpose of HIV prophylaxis and contraception may improve adherence to the therapy. Thus, we formulated a female-centric multipurpose prevention technology (MPT) comprising of nanoparticle loaded vaginal gel formulation acting as a contraceptive and microbicide. Eudragit® S100 nanoparticles of Atazanavir sulphate (ATZ; antiviral) and Fluoxetine hydrochloride (FLX; repurposed spermicide) were prepared for pH dependent drug release and loaded in carrageenan and HPMC K200M gel. The particle size of ATZ and FLX nanoparticles was 396.7 ± 20.64 nm and 226.5 ± 2.08 nm respectively. The in vitro release of the gel formulation in simulated seminal fluid (pH 7.6) showed 96.16% and 95.98% release of ATZ and FLX respectively at the end of 8 h. The in vitro anti-HIV and spermicidal activity of the formulation was above 80% for low drug concentrations. In vivo studies on murine model showed no signs of inflammation or vaginal epithelial injury. Curcumin based imaging confirmed the retention of the formulation in the reproductive tract of mice with minimal leakage. Nanoparticles in gel enabled non-invasive and localised delivery with minimal side effects and can be an effective prophylactic therapy.

Mapping the vaginal microbiota variations in women from a community clinic in Mumbai, India.

PURPOSE: The vaginal microbiome contributes significantly to women's reproductive health and fluctuates due to various physiological and pathological factors. The study's objective is to map the vaginal microbiome of non-pregnant women and evaluate variations based on various potential factors influencing vaginal milieu. METHODS: Fifty-two sexually active, non-pregnant women between 18 and 45 years were recruited from a community clinic and clinical history was recorded. Vaginal swabs were collected to assess the vaginal microbiome by sequencing the V3-V4 region of the 16S rRNA using the Illumina HiSeq platform, followed by data analysis with QIIME 2. Vaginal milieu was assessed by Nugent score and profiling cytokines in the cervico-vaginal lavage. RESULTS: Lactobacillus iners (34.3%) were the most abundant species in all women. Significant changes in abundance of genera (Lactobacillus, Prevotella and Anaerococcus), expression of pro-inflammatory cytokine IFN-γ and changes in alpha and beta diversity was observed in women having asymptomatic bacterial vaginosis (BV). Differences in beta diversity were seen between healthy women and women exhibiting presence of Candida spp. Variations in the abundance of genera (Lactobacillus, Bifidobacterium, Porphyromonas) were observed in women who had delivery less than twelve months back, probably as more of these women (50%, 53.7%) had higher abnormal Nugent score. CONCLUSION: Lactobacillus iners was the most prevalent vaginal species in women from a Mumbai community clinic. Maximum variations in the vaginal microbiome characterized by a perturbation of the Lactobacillus predominant vaginal microbiota are seen in those women who have asymptomatic BV and childbirth within last twelve months.

The dynamics of SARS-CoV-2 infection in unvaccinated and vaccinated populations in Mumbai, India, between 28 December 2020 and 30 August 2021.

The emergence and evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants that could compromise vaccine efficacy (VE) with re-infections in immunized individuals have necessitated continuous surveillance of VE. Here, the occurrence and dynamics of SARS-CoV-2 infections in the context of vaccination during the second wave of infection in Mumbai were evaluated. RT-PCR cycle threshold (Ct) values of the open reading frame (ORF)/envelope (E)/nucleocapsid (N) genes obtained from a total of 42415 samples, comprising unvaccinated (96.88%) and vaccinated cases (3.12%) were analyzed between December 28, 2020, and August 30, 2021. A lower incidence of SARS-CoV-2 infection in fully vaccinated cases (5.07%) compared to partially vaccinated cases (6.5%) and unvaccinated cases (13.453%) was recorded. VE was significant after the first dose of vaccination (ORF gene p-value = 0.003429, and E/N gene p-value = 0.000866). Furthermore, VE was observed to be significant when the post-immunization (first dose) period was stratified to within 30 days (ORF gene p-value = 0.0094 and E/N gene p-value = 0.0023) and to 60 days following the second dose of vaccination (ORF gene p-value = 0.0238). Also, significantly higher efficacy was observed within individuals receiving two doses compared to a single dose (ORF gene p-value = 0.0132 and E/N gene p-value = 0.0387). The emergence of breakthrough infections was also evident (odds ratio= 0.34; 95% confidence interval= 0.27-0.43). Interestingly, viral loads trended towards being higher in some groups of partially vaccinated individuals compared to completely vaccinated and unvaccinated populations. Finally, our results delineated a significantly higher incidence of SARS-CoV-2 acquisition in males, asymptomatic individuals, individuals with comorbidities, and those who were unvaccinated.

Lactobacilli metabolites restore E-cadherin and suppress MMP9 in cervical cancer cells.

Cervical cancer is leading cause of cancer death in females worldwide. Vaginal lactobacilli colonizing cervical area are known to play an important role in maintaining cervical physiological conditions to ward away vaginal infections including bacterial vaginosis (BV) and cancer prevention. There are limited studies to study effect of Lactobacilli isolated from different sources on cervical cancer. The objective of the study was to investigate the potential of cell-free culture supernatants (CFCs) or metabolites of twelve well-characterized Lactobacillus species from different microenvironments for their anti-proliferative properties on HPV16 and HPV18 cervical cancer cells and to investigate the mechanisms of anti-proliferative and anti-metastatic activities. Lactobacillus metabolites exerted a dose, strain and cell line-dependent effect on cervical cells as demonstrated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. The metabolites from vaginalis and L. salivarius exhibited the lowest half-maximal inhibitory concentration (IC50) on HeLa (131 and 167 ng/ml) respectively and SiHa (149 and 205 ng/ml) respectively. Lactobacilli demonstrating greater inhibitory effect produced majorly l-lactic acid and hydrogen peroxide (H2O2). Treatment with lactobacilli CFCs significantly upregulated E- cadherin levels in HeLa (p = 0.0451) and SiHa (p = 0.0051) cells and downregulated matrix metalloproteinase 9 (MMP9) levels in Hela cells (p = 0.0465) as measured by ELISA. Lactobacillus-derived metabolites could be explored as biotherapeutics for the control of HPV infections and cervical cancer.

Evaluation of cytokine profile in cervicovaginal lavage specimens of women having asymptomatic reproductive tract infections.

Reproductive tract infections (RTIs) such as vaginal candidiasis and bacterial vaginosis (BV) are common among sexually active women and can be both symptomatic or asymptomatic. The microbiota of the reproductive tract triggers immune response at the cervicovaginal interface resulting in secretion of cytokines during the course of these RTIs. The objective of this study was to evaluate the cytokine profile in cervicovaginal lavage of women having asymptomatic vaginal infections. Measurement of vaginal cytokines was done for various interleukins including IL-1ß, IL-6, IL-8, IL-10, IL-12/IL23p40, IL-17A, tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) by ProcartaPlex™ Multiplex Immunoassay. Women having vaginal Candida infection had increased concentration of IL-1ß (p=.01), IL-6 (p=.007), IL-8 (p=.327), IL-12/IL23p40 (p=.049) and IFN-γ (p=.125). The results of our study suggest that evaluation of these cytokines could be explored as an additional measure to determine host inflammatory response in women having asymptomatic vaginal candidiasis.Impact StatementWhat is already known on this subject? Studies assessing the vaginal cytokine profile to assess the vaginal milieu in various cohorts such as post-menopausal women, pregnant women, women with history of preterm birth, CIN and scheduled IVF cycle are being undertaken. Variable cytokine response has been reported in literature in women with symptomatic bacterial vaginosis and Candida infection. However, much less is known about vaginal cytokine profile in asymptomatic infection.What do the results of this study add? The results of the study show increased concentration of the pro-inflammatory cytokines IL-1ß, IL-6 IL-8, IL-12/IL23p40 and interferon gamma (IFN-γ) in women having asymptomatic Candida, vaginal leucocytosis and raised vaginal pH.What are the implications of these findings for clinical practice and/or further research? Evaluation of vaginal cytokine profile (IL-1ß, IL-6 IL-8, IL-1ß, IL-12/IL23p40 and IFN-γ) could be explored as an additional measure to determine inflammation in asymptomatic women. Vaginal cytokines (IL-1ß, IL-6 IL-8, IL-1ß, IL-12/IL23p40 and IFN-γ) could be used further for development of a point of care test.

Cycle threshold values in RT-PCR to determine dynamics of SARS-CoV-2 viral load: An approach to reduce the isolation period for COVID-19 patients.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease-2019 (COVID-19). Here, we examined the duration of clearance of SARS-CoV-2 in respiratory samples from 207 infected cases by real-time reverse-transcription polymerase chain reaction (RT-PCR). A substantial proportion of COVID-19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS-CoV-2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID-19, irrespective of their Ct values, cleared SARS-CoV-2 RNA within 14 days of the initial detection. Longitudinal assessment of RT-PCR test results in individuals requiring 15-30 days to clear SARS-CoV-2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow-up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID-19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS-CoV-2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure.

Vaginal Dysbiotic Microbiome in Women With No Symptoms of Genital Infections.

The vaginal microbiome plays a critical role in determining the progression of female genital tract infections; however, little is known about the vaginal microbiota of Indian women. We aimed to investigate the vaginal microbial architecture of women with asymptomatic bacterial vaginosis (BV) (n=20) and normal microbiota (n=19). Microbial diversity was analyzed in vaginal swabs from regularly menstruating women (18-45yrs) by 16S rRNA V3-V4 amplicon (MiSeq Illumina) sequencing. Rarefaction analysis showed a higher number of species in normal flora compared to BV. Alpha diversity as measured by Pielou's evenness revealed microbial diversity was significantly greater in BV samples than normal microbiota (p= 0.0165). Beta diversity comparison using UniFrac metrics indicated distinct microbial communities clustering between normal and BV flora. Firmicutes were the major phyla observed in vaginal specimens of normal microbiota whereas Actinobacteria, Fusobacteria, Bacteroidetes were significantly abundant in BV samples. Notably, the relative abundance of Lactobacillus was significantly high in normal microbiota. Conversely Gardnerella, Sneathia, Prevotella, Atopobium, Ureaplasma, Dialister significantly dominated dysbiotic microbiota. Relative frequency of Lactobacillus decreased significantly in BV (6%) as compared to normal microbiota (35.2%). L. fermentum, L. gasseri, L. iners, L. jensenii, L. mucosae, L. ruminis, L. salivarius, L. coleohominis was more exclusively present in normal microbiota. L. iners was detected from both the groups with a relative frequency of 50.4% and 17.2% in normal and BV microbiota respectively. Lefse analysis indicated Atopobium vaginae, Sneathia amnii, Mycoplasma hominis Prevotella disiens in the vaginal microbiota as a biomarker for dysbiosis and L. jensenii as a biomarker of a healthy microbiota. Firmicutes were negatively correlated to Tenericutes, Actinobacteria, Bacteroidetes, and Fusobacteria. Proteobacteria positively correlated to Tenericutes, and Bacteroidetes were shown to be positively correlated to Fusobacteria. Predicted functional analysis indicated differences in the functional profiles between BV and normal microbiota. Normal microbiota utilized pathways essential for phosphatidylglycerol biosynthesis I & II, peptidoglycan biosynthesis, geranylgeranyl diphosphate biosynthesis I, mevalonate pathway, CoA biosynthesis pathway I and pyrimidine nucleotide salvage; whereas BV bacteria had characteristic aromatic amino acid biosynthesis, pentose phosphate pathway, carbohydrate degradation. In conclusion, women with asymptomatic BV have vaginal microbiota significantly different than women with normal microbiota. Furthermore, the study provides insights into the vaginal microbial structure of Indian women that will enable us to explore the prospective candidates for restoring the vaginal microbiota.

Distinct Functional Traits of Lactobacilli from Women with Asymptomatic Bacterial Vaginosis and Normal Microbiota.

Asymptomatic bacterial vaginosis (BV) in reproductive-age women has serious obstetric and gynecological consequences. Despite its high incidence, the behavior of vaginal lactobacilli in asymptomatic BV is unknown. We analyzed the functional properties of previously isolated vaginal lactobacilli from asymptomatic women with normal, intermediate, and BV microbiota. Lactic acid and antimicrobial activity against seven urogenital pathogens were evaluated from lactobacilli cell-free culture supernatants (CFCs) (n = 207) after 48 h incubation in MRS. Lactobacilli isolates were used to evaluate H2O2, autoaggregation and coaggregation with C. albicans. Lactobacilli from normal microbiota produced more d-lactate than lactobacilli from intermediate and asymptomatic BV (p = 0.007). L. plantarum, L. fermentum and L. reuteri produced greater d-lactate whereas L. rhamnosus, L. crispatus, L. johnsonii were greater producers of l-lactate. Interspecies positive correlation was observed in the lactic acid contents of CFCs. Distribution of H2O2-producing lactobacilli did not vary significantly among the groups. When lactic acid isomers were considered, species from intermediate and BV microbiota clustered together with each other and distinctly from species of normal microbiota. Broad-spectrum antagonism (≥90% inhibition) against E. coli, C. albicans, S. aureus, P. aeruginosa, G. vaginalis, N. gonorrhoeae, S. agalactiae were displayed by 46.86% (97) of isolates. Our study highlights the differential functional properties of vaginal lactobacilli from women with normal microbiota and asymptomatic BV.

Vaginal microbiota of asymptomatic bacterial vaginosis and vulvovaginal candidiasis: Are they different from normal microbiota?

Vaginal microbiota contributes in maintaining and protecting the urogenital niche from infections and their sequelae. Despite extensive research, microbiome studies have often ignored asymptomatic bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). The present study aimed to explore the cultivable vaginal bacterial and mycological communities in women asymptomatic for BV and VVC using multiplex PCR and species-specific PCR. Vaginal swabs collected from 199 participants asymptomatic for urogenital infections, scored by Nugent criteria indicated 73.9% had normal microbiota, 11.6% intermediate and 14.5% BV. The most frequent Lactobacillus species in normal women were L. iners (69.4%), L. crispatus (24.5%), L. reuteri (20.4%). Women with BV colonized L. iners (62.1%); L. rhamnosus (41.4%); L. salivarius (13.7%) and L. reuteri (7.2%). Furthermore, L. crispatus was associated with normal microbiota, whereas L. iners was a frequent member of normal and dysbiotic microbiota. Lactobacillus abundance and species richness reduced in asymptomatic BV. Also L. crispatus, L. fermentum, L. acidophilus and L. delbruckii were absent in these women. L. iners significantly co-existed with other Lactobacillus species, indicating its failure in independently maintaining the healthy vaginal niche. Of 30.4% women detected with Candida, 72.1% constituted non-albicans Candida. Predominance of C. albicans increased from 18.4% in healthy to 60% in women with asymptomatic BV; whereas distribution of BV related bacteria did not vary across the groups. Heterogeneous population of lactobacilli in 80.8% of normal women calls attention towards cumulative effects of these species in safeguarding the vaginal microenvironment. Since the microbiota of asymptomatic BV was different from healthy, screening and management could be encouraged to avoid further complications of infections.

Cross sectional analysis of vaginal Lactobacillus in asymptomatic women of reproductive age in Mumbai, India.

INTRODUCTION: Lactobacillus dominated vaginal microenvironment is associated with lower risk of genital infections. Numerous studies have reported geographic and ethnic variations in vaginal microbiome structure between healthy individuals from different race and ethnicity. India has a great diversity, so it is intriguing to find out if such divergences exist in vaginal lactobacilli. The present study aimed to investigate predominant Lactobacillus species in vaginas of healthy Indian women and screen isolates for lactic acid and H2O2 production. METHODOLOGY: 203 premenopausal women asymptomatic for any vaginal complaints were recruited. The lactobacilli isolates on MRS agar were identified by Multiplex-PCR and 16sRNA gene sequencing. RAPD was used to differentiate strains of same species. H2O2 and lactic acid was evaluated on TMB-HRP MRS agar and BCP-MRS agar respectively. RESULTS: Lactobacilli were recovered from 107/109 (98.2%) women with normal microflora. L. iners 64.7% (68), L. crispatus 26.7% (28), L. reuteri 21.9% (23), L. jensenii 16.2% (17) and L. gasseri 15.2% (16) were the most frequently occurring vaginal lactobacilli in normal women. The vaginal microflora was dominated by either by a single (80%, n = 84) or a combination (20%, n = 21) of Lactobacillus species. Though most frequently identified, L. iners, coexisted only with other Lactobacillus species. All isolates were acid producers but H2O2 was produced by 94.2% isolates. CONCLUSIONS: Our study reports prevalent vaginal lactobacilli which could be explored as probiotics. Presence of heterogeneous Lactobacillus population highlights the cumulative effects of different lactobacilli maintaining vaginal health. Contrasting observations about L. iners reiterates its puzzling role in vaginal immunity, advocating further research.

Identification and characterization of anti-microbial peptides from rabbit vaginal fluid.

Antimicrobial peptides (AMPs) serve as a first line of host defense and represent an important, though poorly understood components of the innate immune system. The present study was an attempt to identify and characterize the major molecules having anti-bacterial activities from the vaginal fluid of rabbit, Oryctologus cuniculus. AMPs from the rabbit vaginal fluid (RVF) were identified in the acid extracts of pooled RVF samples after RP-HPLC purification. The protein, RVFAMP was effective against gram negative (Escherichia coli and Pseudomonas aeruginosa) and gram positive (Staphylococcus aureus and Streptococcus pyogenes) bacteria. The results of acid urea-PAGE-gel overlay assay (AU-PAGE-GOA) demonstrated clear zone of growth inhibition of E. coli corresponding to 6 and 15 kDa protein bands. LC-MS data of these proteins indicated that 15 kDa protein consists of lysozyme, lipopolysaccharide binding protein (LBP), hemoglobin-α and ß subunits (Hb-α/ß), whereas 9 kDa protein band consists of transthyretin and calcyclin while uteroglobulin and neutrophil antibacterial peptide-5 (NAMP-5) are present in the 6 kDa protein band. Of the eight proteins, Hb-α derived protein was further characterized, as it showed the highest Probability Based Mowse Score (PBMS) of 288. A 25mer peptide, RVFHbαp was active against several clinical pathogens as demonstrated by minimum inhibitory concentration (MIC) and radial diffusion assays (RDA). The interaction of RVFHbαP with bacterial liposome membrane was assessed by calcein dye leakage assay. RVFHbαP did not show cytotoxicity against human endocervical cells (End1/E6E7) or erythrocytes. RT-PCR and immunofluorescence results revealed the expression of RVFHbαP mRNA and protein in rabbit vaginal tissue. To the best our knowledge, this is the first report describing the detection of AMPs in RVFs. In conclusion, these studies indicated that vaginal epithelial cells (VEC) derived RVFHbαP may have therapeutic potential in the management of reproductive well being of rabbits. The major reason for undertaking this study in rabbits is that, it forms an excellent in vivo model system for human's studies.

Interaction of contraceptive antimicrobial peptide nisin with target cell membranes: implications for use as vaginal microbicide.

BACKGROUND: Nisin, a naturally occurring antimicrobial peptide (AMP), is currently the focus of clinical trials as an intravaginal microbicide. Therefore its mechanism of interaction with various cell membranes was studied. STUDY DESIGN: Flow cytometry was used for quantitative estimation of membrane damage by nisin which was further determined by scanning electron microscopy (SEM). Affinity of nisin for different unilamellar liposome vesicles was determined spectroflurometrically and confirmed using laser scanning confocal microscopy (LSCM). RESULTS: Propidium iodide (PI) staining by flow cytometry exhibited selective membrane permeabilizing effect of nisin on sperm and bacterial membranes which correlated with ultrastructural changes. In vitro interaction of nisin with liposome model vesicles revealed significant leakage of calcein from liposomes composed of phosphatidylcholine/phosphatidylglycerol (POPC/POPG) (e.g., bacteria) and phosphatidylcholine/phosphatidylserine (POPC/POPS) (e.g., spermatozoa) as compared to phosphatidylcholine/phosphatidylethanolamine (POPC/POPE) vesicles (e.g., red blood corpuscles). LSCM results were in complete agreement with cell membrane affinity studies. CONCLUSION: This unique property of nisin can be exploited in the development of a safe and effective vaginal microbicide for the prevention of sexually transmitted infections/acquired immunodeficiency syndrome (STIs/AIDS) and unplanned pregnancies.

Toll-like receptors and cytokines as surrogate biomarkers for evaluating vaginal immune response following microbicide administration.

Topical microbicides are intended for frequent use by women in reproductive age. Hence, it is essential to evaluate their impact on mucosal immune function in the vagina. In the present study, we evaluated nisin, a naturally occurring antimicrobial peptide (AMP), for its efficacy as an intravaginal microbicide. Its effect on the vaginal immune function was determined by localizing Toll-like receptors (TLRs-3, 9) and cytokines (IL-4, 6 , 10 and TNF-alpha) in the rabbit cervicovaginal epithelium following intravaginal administration of high dose of nisin gel for 14 consecutive days. The results revealed no alteration in the expression of TLRs and cytokines at both protein and mRNA levels. However, in SDS gel-treated group, the levels were significantly upregulated with the induction of NF-kappaB signalling cascade. Thus, TLRs and cytokines appear as sensitive indicators for screening immunotoxic potential of candidate microbicides.

Evaluation of developmental toxicity of microbicide Nisin in rats.

In the present study, we have investigated the developmental toxicity of a naturally occurring peptide, Nisin in rats in order to determine its suitability as a safe vaginal microbicide. Our earlier studies indicated that, Nisin is a dual function microbicide having contraceptive and antibacterial activities. However, as part of the safety evaluation of any vaginal microbicide, it is essential to determine its teratogenic potential in a suitable animal model before it is found suitable to enter clinical trials. Sixty pregnant rats allocated into four groups were orally administered with 10, 25 and 50 mg Nisin/kg/day from day 6 to day 15 of gestation. Individual food/water consumption and body weight changes were measured daily. Nisin did not cause maternal mortality nor did the treated animals show any clinical signs of toxicity when compared to the control animals. There were no biologically significant differences in maternal liver, kidney, thymus, ovary, gravid and empty uterine weights. Mean number of corpora lutea and implantation sites also did not differ in the treated groups when compared to their respective controls. All the fetuses were weighed, sexed and examined carefully for externally visible malformations. No gross external fetal alterations were observed at any dose tested. When stained by the double staining method, no skeletal malformations and visceral defects were observed in the fetuses. The growth and reproductive performance of the F1 progeny was also unaffected. In conclusion, Nisin shows unique clinical potential as a safe prophylactic microbicide to curb the transmission of STIs/HIV and unintended pregnancies.

Contraceptive efficacy of antimicrobial peptide Nisin: in vitro and in vivo studies.

Sexually transmitted infections and unplanned pregnancies present a great risk to the reproductive health of women. Therefore, female-controlled vaginal products directed toward disease prevention and contraception are needed urgently. In the present study, efforts were made to evaluate the contraceptive potential of Nisin. The effect of Nisin on sperm motility was assessed under in vitro and in vivo conditions. The results showed that sperm motility was completely inhibited with Nisin. The minimum effective concentration of Nisin required to immobilize sperm (80-100 x 10(6)) in vitro within 20 s was found to be 50 microg in rat, 200 microg in rabbit and 300-400 microg in monkey and human. The effect on sperm motility was observed to be dose- and time-dependent. Intravaginal administration of Nisin (200 microg) before mating during proestrus-estrous transition phase caused complete arrest of sperm motility and blockage of conception. Subacute toxicity studies in rats indicated that, repetitive intravaginal application of Nisin at the dose of 200 microg for 14 consecutive days induced no abnormalities either in the length of estrous cycle or in the morphology of vaginal epithelial cells. No histopathological abnormalities in vaginal tissue or any change in blood and serum biochemical profiles were observed. Furthermore, no adverse effects were observed on subsequent reproductive performance, neonate survival and development of pups. It is suggested that Nisin, with its antibacterial and spermicidal activities, could be developed as a potent vaginal contraceptive for future use in humans.