Spermicidal Activity Research Articles

Spermicidal Activity of Bivittoside D from Bohadschia vitiensis

A recent revelation about increased susceptibility to HIV by use of nonoxynol-9 (N-9) has called for identification of novel molecules with potent sperm-attenuating activity and lower side-effect profile, as suitable alternatives. The present study was designed to investigate spermicidal activity in Bohadschia vitiensis whole-body extracts followed by isolation and characterization of bioactive molecule. Bohadschia vitiensis (Semper) was collected from the Southern Andaman coast of India. Freshly collected marine animals were extracted with methanol. A portion of the crude extract was fractionated into four fractions by macerating with hexane, chloroform, and n-butanol successively. All fractions were evaluated for spermicidal activity. Because maximum activity was localized in the n-butanol soluble fraction, it was chromatographed over a silica gel column, and elution with chloroform-methanol-water (35:10:2, v/v) yielded the major compound bivittoside D (400 mg). Bivittoside D [molecular weight (MW) 1426] is a lanostane triterpenoid with six monosaccharide units. The structure of the compound was established on the basis of physicochemical data, acid hydrolysis of saponin, identification of sugar units and aglycon, melting point, and by comparison with data reported in the literature. The aqueous methanol extract of the Bohadschia vitiensis caused 100% mortality of human sperm at 0.01% concentration in vitro, whereas N-9 (reference control) exhibited an equivalent activity at 0.05%. On further fractionation, activity was localized in n-butanol soluble fraction from which the major compound purified was a lanostane triterpenoid called bivittoside D. Bivittoside D was found to be a more potent spermicide (approximately 2.3 times) than N-9 and killed 100% human sperm at the concentration of 350 muM in approximately 20 sec in vitro. Supravital staining and hypoosmotic swelling test revealed sperm membrane permeabilization by bivittoside D as the major mode of spermicidal action. However, bivittoside D was much safer than N-9 towards normal vaginal flora (Lactobacillus) in vitro, although it affected the viability of HeLa cells like other surfactants. Bivittoside D from B. vitiensis can adequately replace N-9 in vaginal contraceptives to make them more vaginally safe and ecofriendly.

Surfactants as Microbicides and Contraceptive Agents: A Systematic In Vitro Study

BackgroundThe urgent need for cheap and easy-to-use protection against both unwanted pregnancies and sexually transmitted diseases has stimulated considerable interest in the use of surfactants as microbicides, anti-viral, and contraceptive agents in recent years. In the present study we report a systematic in vitro evaluation of the microbicidal, anti-viral and contraceptive potential of cationic, anionic, zwitterionic, and non-ionic surfactants.Methodology/Principal FindingsToxicity was evaluated in mammalian columnar epithelial (MDCK) cells, human sperm cells, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Streptococcus agalactiae and Enterococcus faecalis. The inhibition of adenovirus and lentivirus infection of MDCK cells was also tested. A homologous series of cationic surfactants, alkyl-N,N,N-trimethylammonium bromides (CnTAB), with varying alkyl chains were shown to be bactericidal and fungicidal at doses that were related to the surfactant critical micelle concentrations (CMC), all of them at concentrations significantly below the CMC. In general, bacteria were more susceptible to this surfactant group than C. albicans and this organism, in turn, was more susceptible than MDCK cells. This suggests that the CnTAB may be useful as vaginal disinfectants only in so far as bacterial and fungal infections are concerned. None of the surfactants examined, including those that have been used in pre-clinical studies, showed inhibition of adenovirus or lentivirus infection of MDCK cells or spermicidal activity at doses that were sub-toxic to MDCK cells.Conclusions/SignificanceThe results of this study lead us to propose that systematic analysis of surfactant toxicity, such as we report in the present work, be made a mandatory pre-condition for the use of these substances in pre-clinical animal and/or human studies.

Chenopodium album seed extract-induced sperm cell death: exploration of a plausible pathway

BackgroundThis study was conducted for to explore the plausible pathway of Chenopodium album seed extract (CAE)-mediated sperm cell death. Study DesignThe role of CAE for its spermicidal action was assessed by (a) measuring lipid peroxidation, protein carbonyl content and intracellular glutathione content in CAE exposed sperm cells; (b) assaying antioxidant enzymes like catalase and superoxide dismutase (SOD); (c) analyzing protein expressions by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis; (d) fluorimetric measurement of intracellular H2O2 level and generation of reactive oxygen species (ROS) in CAE-treated sperm cells; and (e) DNA ladder formation study. ResultsCAE-induced sperm death is due to (a) lipid peroxidation of the sperm cell membrane, oxidation of some critical cellular proteins and depletion of intracellular reduced gluthathione, indicating production of ROS; (b) activation of Mn-SOD and inactivation of catalase favoring endogenous accumulation of H2O2; (c) generation of O2− at an enhanced rate during oxidative stress as evidenced by increased Mn-SOD activity and protein expression; (d) accumulation of ROS in spermatozoa reflected in the fluorimetric experiments; and (e) increased production of O2− and H2O2 induced apoptosis-like death in sperm cells as observed by DNA ladder formation. ConclusionThe sperm death mediated by CAE is due to oxidative damage of cellular macromolecules by in situ generation of ROS.

Spermicidal activity of sulfonylureas and meglitinide analogues: role of intrasperm Ca2+ elevation

Intrasperm calcium concentration ([Ca2+]is) is known to play a vital role in regulating motility and viability of ejaculated spermatozoa. K ATP channel blockers are reported to block K ATP channels, leading to depolarization of the cell membrane. This activates the voltage-gated calcium channels, resulting in enhanced Ca2+ influx, which eventually elevates the intracellular [Ca2+] level. Hence, it can be hypothesized that drugs acting on K ATP channels could possess the ability to elevate [Ca2+]is. Sulfonylureas such as glibenclamide or gliclazide, as well as meglitinide analogues such as repaglinide, produced a dose- and time-dependent decrease in viability, each requiring 7.5 mM, 10 mM and 6.5 mM, respectively, to produce death of all sperm cells immediately upon addition to ejaculated human semen samples. The reduction in sperm viability was accompanied by an elevation of [Ca2+]is and was affected by removal of extracellular Ca2+. Significantly (P < 0.05) less time was required to elevate [Ca2+](is) and produce complete loss of sperm viability when any of these drugs were added to sperm cells simultaneously with selected agents affecting Ca2+ homeostasis. Thus, the spermicidal activity of these drugs attributed to elevation of [Ca2+]is and their synergism can be potentially exploited for developing contact spermicidal formulations.

Update on nonoxynol-9 as vaginal spermicide

Nonoxynol-9 (N-9) is a typical surfactant. For more than 30 years that very property of N-9 has been successfully exploited for its spermicidal action. It is available as an over-the-counter, locally acting vaginal spermicide. The suitability of N-9 as a spermicide is elaborated in this article. The reasons why N-9 may fail as a contraceptive are discussed. In spite of many drawbacks, which are mentioned in the article, N-9 is still often resorted to as a locally acting contraceptive. The review ends with suggestions to alter the molecular structure of N-9 and to adjust the dosages.

Solvent free synthesis of new-1-acetyl-3-( 4-fluoronaphthyl)-5-substituted aryl pyrazolines as spermicides

In this paper we wish to report the solvent free synthesis of 1-acetyl-3-(4-fluoronaphthyl)-5-substituted aryl pyrazolines by using simple and efficient Green Chemistry techniques (Grindstone method and microwave irradiation). These methods have been developed for the rapid solvent free synthesis of title compounds from corresponding chalcones and hydrazine hydrate or phenylhydrazine in acetic acid. Considerable increase in the reaction rates have been observed with better yields compared with sample prepared from conventional method. Structure of synthesized new compounds have been confirmed on the basis of spectral (IR, 1 H NMR) and analytical data. Representative compounds have been screened for their spermicidal activity in HF cattles and Murrah buffalo.

Spermicidal bacteriocins: Lacticin 3147 and subtilosin A

Spermicidal compounds that also exhibit antimicrobial properties would be extremely attractive agents as they could be used to not only prevent unwanted pregnancy but also to combat the growing prevalence of sexually transmitted infections (STI). One class of compounds that are potential candidates for development of dual-acting contraceptive products are antimicrobial peptides (AMPs). Herein, we report preliminary studies carried out to investigate the spermicidal activity of two bacteriocins, lacticin 3147 and subtilosin A, on bovine, horse/pony, boar and rat sperm.

In vitro spermicidal activity of peptides from amphibian skin: Dermaseptin S4 and derivatives

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 potency of dermaseptin DS4, an antimicrobial peptide derived from frog skin. To assess the structure–activity relationship between the native DS4 and its derivatives, a set of chemically modified peptides was synthesized and evaluated. Normal human semen samples were used to detect the spermicidal activity of the new compounds. HeLa cultures were used to determine the safety of compounds toward their toxicity. Fluorescent-binding assays were performed to evaluate the rapidity and the irreversibility of the sperm-immobilizing activity of peptides. All DS4 derivatives elicited concentration-dependent spermicidal activity at microgram concentrations (EC 100 values: 25 μg/ml–l mg/ml). The order was K4S4 = S4a > S4 > K4S4(1–16)a > S4(6–28). In cytotoxicity assay, some compounds were found to be significantly safer than nonoxynol-9, the most widely used spermicide, and their activity was not accompanied by total loss of plasma membrane integrity as detected by fluorescent microscopy. Our data also show that increasing the number of positive charges of the peptide resulted in a reduced cytotoxicity without affecting the spermicidal effect. This study indicates that dermaseptins are spermicidal molecules that deserve to be tested as topical contraceptive with useful activities that can add to their prophylaxis, safety, and effectiveness.

Synthesis of disulfide esters of dialkylaminocarbothioic acid as potent, non-detergent spermicidal agents

S, S′-[disulfanediylbis(dialkylaminopropane-2,1-diyl)]bis- (dialkylaminothiocarbamate) ( 14– 31) were prepared and evaluated for the spermicidal activity and antifungal activity. Dialkyldithiocarbamates ( 1– 5) were reacted with epichlorohydrin to give 1-dialkylaminocarbothioic acid S-[(2,3-epithio)propyl]ester ( 7– 11), these on further reaction with a secondary amine gave S, S′-[disulfanediylbis(dialkylaminopropane-2,1-diyl)]bis- (dialkylaminothiocarbamate) ( 14– 31). Some of these compounds ( 16, 19– 21, 23, 30, 31) were found to be very potent spermicidal agents with marginal antifungal activity. Two compounds ( 20, 21) were 25 times more active than nonoxynol-9 (N-9), the spermicide currently in the market.

Optimized Benzalkonium Chloride Gel: A Potential Vaginal Microbicides

Objective To develop an optimized BZK gel with good pharmaceutical effect and less toxicity to vaginal mucosa. Methods Four methods as below were used to research the spermicidal activity of BZK gel: 1) in vitro spermicidal test; 2) in vivo spermicidal test in rabbits; 3) anti-fertility test in rabbits; 4) contraceptive test in rabbits. Mucosal irritation test was used in rats to evaluate the safety of optimized BZK gel. Microbiological assessments were used to research anti-STI pathogens (including treponema pallidum, neisseria gonorrhoeae, trichomona vaginalis, candida albicans, ureaplama urealyticum, herpes simplex virus type-2, chlamydiae trachomatis) effect of optimized BZK gel. Results In vitro spermicidal test, EC50 of BZK gel was 0.029mg/ml, a little higher than that of N-9 (0.019mg/ml). The MIC of BZK gel was 0.25mg/ml, similar to that of N-9 (0.20mg/ml). The vaginal mucosal irritation test in rats showed that 0.429% BZK gel showed only minimal vaginal irritation, and did not damage the vaginal epithelium or cause local inflammation in rats. Microbiological assessments showed that optimized BZK gel could inhibit or inactivate common STI pathogens even after 3-fold or 5-fold dilution. Conclusion Optimized BZK gel was an effective microbicides.

Potato aspartic proteases ( StAPs) exert cytotoxic activity on bovine and human spermatozoa

To evaluate the in vitro spermicidal activity of Solanum tuberosum aspartic proteinases (StAPs) on bovine and human sperm. Controlled laboratory study. Three research laboratories at a university of biologic science. ANIMAL(S) AND DONOR(S): Frozen semen from five Aberdeen Angus bulls and six proven fertile men volunteers. The effect of StAPs on sperm motility was studied in vitro by incubation of different concentrations of StAPs with sperm suspensions, and motility was assessed by direct microscopic observation. Membrane integrity was analyzed by SYTOX Green uptake after incubation with different StAP concentrations. The effect of StAPs was evaluated by human erythrocyte lysis, as a control in somatic cells. The StAPs binding was monitored by fluorescence. Total and progressive sperm motility; hypoosmotic swelling test and SYTOX Green uptake as a measure of membrane damage; fluorescein isothiocyanate-labeled StAP binding by an optical microscopy. The StAPs reduced sperm motility in a dose-dependent manner, and 25 microM of StAP1 and 35 microM of StAP3 completely abolished the progressive motility. The StAPs were able to bind in the postacrosomal and midpiece region only in bovine sperm. Also, StAPs caused spermatozoa agglutination. In vitro cell toxicity was observed by a dose-dependent increase in hypoosmotic swelling negative sperm and SYTOX Green uptake in both human and bovine spermatozoa; however, no toxic effect was observed on erythrocytes. The spermicidal effect of StAPs involves plasma membrane permeabilization.

IDENTIFICATION OF ANTIMICROBIAL ACTIVITY FROM GOAT CERVICAL EPITHELIUM

1. In mammals, antimicrobial peptides are present in epithelial cells of the respiratory, gastrointestinal and urinogenital tracts. In the present study, we report the identification and partial characterization of an antimicrobial peptide in extracts prepared from goat cervical epithelial tissue. 2. The bioactivity, as determined by radial and disc diffusion assays, was identified in a peptide of molecular mass 4.5 kDa purified by cation exchange and gel filtration techniques. The purified peptide exhibited antibacterial activity against Escherichia coli (Gram-negative) as well as Bacillus subtilis (Gram-positive) bacteria. 3. The minimum inhibitory concentration of the peptide estimated by liquid broth assay for E. coli and B. subtilis suspensions was 31 and 50 microg/mL, respectively, a result implying that the peptide is more efficient in inhibiting Gram-negative than Gram-positive bacteria. 4. Because the peptide has no spermicidal activity, we propose that it is responsible for protecting the female genital tract against bacteria. 5. Whether the peptide has potentials for use in the cryopreservation, storage and transport of semen remains to be determined.

Nuevas opciones en anticoncepción: posible uso espermicida de plantas colombianas

AbstractCurrently there exists increasing preoccupation concerning sexual and reproductive health among teenagers; in spite of the availability of different contraceptive methods, the number of undesired pregnancies is steadily increasing. Among the products presently available for birth control, spermicides are a means that can be totally controlled by the woman and are very reliable compared to other contraceptives in common use. However, they cause irritation in the vaginal epithelium due to their tensoactive effect on cellular membranes which might enhance the risk of acquiring sexually transmissible diseases. In searching for new alternatives, it was observed that a wide variety of plants have spermicidal activity. Hence it is interesting to consider potential contraceptives of vegetable origin, as they may constitute a key tool to prevent undesired pregnancies in general, and in particular in vulnerable groups such as teenagers and young women.

Spermicidal activity of extract from Cestrum parqui

Objective The leaf extract of Cestrum parqui was examined for its effects on sperm motility in vitro. Methods Washed sperm were prepared by discontinuous buoyant density gradient centrifugation and incubated with varying concentrations of extract from C. parqui (40–250 μg/mL) at 37°C and 5% CO 2. Sperm motility and viability were assessed at different intervals ranging from 5 to 240 min. Morphological changes in human ejaculated spermatozoa after exposure to the extract were evaluated under transmission electron microscope. Results A dose- and time-dependent effect of this extract on sperm motility and viability was observed. The maximal spermicidal effect was observed with a 250-μg/mL dose of the extract. Transmission electron microscope revealed a significant damage to sperm membrane in both head and acrosomal membranes, notable swelling and disruption. Conclusion The present study indicates that this natural extract has potential spermicidal effect in vitro.

Novel disulphide esters of carbothioic acid as potent, non-detergent spermicides with low toxicity to Lactobacillus and HeLa cells in vitro

The design, synthesis, characterization and evaluation of a novel series of non-detergent spermicides has led to the discovery of two unique molecules (DSE-36 and DSE-37) that were approximately 25 times more potent spermicides than nonoxynol-9 (N-9). Normal human spermatozoa were used to assess the spermicidal activity (Sander-Cramer Assay), the effect on sperm-membrane integrity [hypo-osmotic swelling test (HOST)], supravital staining and scanning electron microscopy (SEM) and the induction of apoptosis [fluorescein isothiocyanate (FITC) Annexin-V and JC-1 labelling using flow cytometry] by the new class of compounds. HeLa and Lactobacillus cultures were used to assess the cytotoxicity of compounds and their compatibility to normal vaginal flora, respectively. Compounds DSE-36 and DSE-37 exhibited a strong spermicidal activity [minimum effective concentration (MEC) = 0.002%], which was approximately 25 times more potent than that of N-9 and Sapindus saponins (MEC = 0.05%). As compared with surfactants, DSE-36 and DSE-37 were found to be safer at MEC towards the growth and survival of Lactobacilli and HeLa cells in vitro and to have a milder effect on sperm plasma membrane. At EC(50) both induced apoptosis in sperm cells as characterized by increased labelling with Annexin-V and decreased polarization of sperm mitochondria. Preliminary studies have revealed that in sharp contrast to the non-specific surfactant action of N-9, DSE-36 and DSE-37 have a highly potent, mechanism-based, detrimental action on human sperm. The unique ability of these non-detergent molecules to selectively kill sperm and spare Lactobacilli and HeLa cells at MEC values much lower than that required for N-9 indicates their potential as superior ingredients for formulation into microbicidal contraceptives.

Spermicidal Activity of Bacterial Lipopolysaccharide Is Only Partly Due to Lipid A

We have previously shown that co-incubation of Chlamydia trachomatis lipopolysaccharide (LPS) leads to premature sperm death by an apoptosis-like mechanism. It was always assumed that lipid A is the toxic component of LPS. Here we investigate the possible involvement of 3-deoxy-D-manno-octulosonic acid (Kdo), which is an additional component of the LPS in C. trachomatis. Highly motile preparations of sperm from normozoospermic patients were incubated for 6 hours with commercial sources of lipid A and Kdo. Conventional lipid A inhibitors, polymyxin B (PMB) and anti-CD14 monoclonal antibody (mAb) were used to test the ability of both lipid A and Kdo to induce an apoptotic-like response in mature sperm. Flow cytometry was used to determine apoptosis by the expression of annexin V. Caspase activity was also measured by fluorometry and by the use of a pan-caspase inhibitor and caspase-3 inhibitor. Both lipid A and Kdo at 50 micro g/mL caused significant mortality of sperm. However, although PMB and anti-CD14 mAb were inhibitory to the activity of lipid A on sperm, no such effect was seen against Kdo. In the presence of either lipid A or Kdo, sperm death was caused by an apoptotic-like effect that was caspase mediated. We conclude that Kdo shares its spermicidal properties with lipid A and seems to kill sperm in a similar manner. These results provide an explanation for higher than expected levels of spermicidal activity of LPS that are not caused by lipid A.

Amino Acid Conjugated Sophorolipids: A New Family of Biologically Active Functionalized Glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).

Chenopodium album seed extract: a potent sperm-immobilizing agent both in vitro and in vivo

Purpose Aqueous decoction of Chenopodium album seeds (CAD) was assessed for its sperm-immobilizing and contraceptive efficacy in laboratory mammals. Method Spermicidal efficacy was evaluated in vitro by a modified Sander–Cramer test. The mode of spermicidal action was assessed by (a) supravital and double fluoroprobe staining of sperm, (b) hypoosmotic swelling tests and (c) transmission electron microscopy. Contraceptive efficacy was evaluated by intrauterine and vaginal application of CAD in rats and rabbits, respectively, followed by their mating and evaluation of pregnancy outcomes. Results The minimum effective concentration of CAD that induced instantaneous immobilization of rat spermatozoa in vitro was 2 mg/mL. The mechanism of CAD action involved disintegration of sperm plasma membrane and dissolution of acrosomal cap causing sperm death. Fertilization of oocytes and establishment of implantation were prevented in the uterine horn that was administered with CAD, while these events occurred unhindered in the untreated contralateral side. In rabbit, intravaginal application of CAD significantly blocked the establishment of pregnancy. Conclusion CAD possesses appreciable spermicidal potential, which may be explored as an effector constituent of vaginal contraceptive.

Intrasperm Ca2+ modulation and human ejaculated sperm viability: influence of miconazole, clotrimazole and loperamide

Elevation of intrasperm Ca(2+) is reported to influence viability of ejaculated spermatozoa. Human spermatozoa possess inositol triphosphate (IP(3))-sensitive Ca(2+) stores, which can be targeted for increasing intrasperm Ca(2+) level. The influence of agents affecting Ca(2+) homeostasis has been investigated. Miconazole nitrate, clotrimazole and loperamide hydrochloride produced a dose- and time-dependent decrease in viability, each requiring respectively 0.5, 1.0 and 1.0 mM for producing death of all sperm cells immediately upon addition to ejaculated human semen samples. The reduction in sperm viability was accompanied by elevation of intrasperm Ca(2+) and was not affected by presence or absence of extracellular Ca(2+). Significantly (P<0.05) less time was required for producing complete loss of sperm viability and increasing intrasperm Ca(2+) when any of these drugs was added to sperm cells previously treated with selected agents affecting Ca(2+) homeostasis. This enhanced spermicidal activity of miconazole, clotrimazole and loperamide appeared to be due to further mobilization of Ca(2+) from partially depleted intrasperm Ca(2+) stores. Synergism of spermicidal activity and intrasperm Ca(2+) elevation by miconazole or clotrimazole was observed when Ca(2+) efflux from sperm cells was simultaneously inhibited by 2',4'-dichlorobenzamil hydrochloride, a Na(+)-Ca(2+) exchange inhibitor. The spermicidal activity of miconazole, clotrimazole or loperamide due to elevation of intrasperm Ca(2+) and its synergism, therefore, has great potential for exploitation of these drugs as contact spermicides.

A polyherbal vaginal pessary with spermicidal and antimicrobial action: evaluation of its safety

A polyherbal vaginal pessary (Praneem) has been formulated that has antimicrobial properties against genital pathogens in addition to spermicidal action. Thus, it has dual potential as a barrier method for contraception and for providing protection against some sexually transmitted infections. The present study reports the findings of a multicentre trial that was conducted to evaluate the safety of this product. Trials were carried out in 23 women in three centres in India: the Postgraduate Institute of Medical Education and Research, Chandigarh; Safdarjang Hospital, New Delhi; and Kamla Nehru Memorial Hospital, Allahabad. Thorough clinical and pelvic examinations were carried out as well as cervical cytology, blood biochemistry and haematology before and after use of the polyherbal pessary intravaginally once daily for 7 consecutive days. No toxicity was observed on clinical examination or by laboratory investigations. Daily intravaginal use of this pessary for 7 days had no adverse effects on cervical cytology or on metabolic and organ functions.