Search

SAMMA, a mandelic acid condensation polymer, exhibits a broad antimicrobial activity against several sexually transmitted pathogens including human immunodeficiency virus (HIV). Here we demonstrated that SAMMA suppressed HIV transmission by dendritic cells (DCs), one of the first target cells for primary infection. The greatest inhibitory effect was achieved when SAMMA was present during the co-culture with target cells. The inhibitory effect of SAMMA on DC-mediated HIV transmission was not due to cytotoxicity. Analysis of the level of DC-associated HIV p24 antigen revealed that SAMMA prevented HIV internalization by DCs when the virus was pre-incubated with the compound. In contrast, pre-incubation of DCs with SAMMA followed by wash-off did not affect the amount of cell-associated HIV p24 antigen. In addition, SAMMA blocked HIV glycoprotein-mediated cell-cell fusion. This study suggests that SAMMA prevents HIV infection through multiple mechanisms.

SAMMA is licensed for development as a contraceptive microbicide. Understanding mechanisms of its biological activity is prerequisite to designing more active second generation products. This study examined Ca(2+) involvement in SAMMA-induced premature acrosomal loss (SAL) in noncapacitated human spermatozoa. SAMMA causes acrosomal loss (AL) in a dose-dependent manner (ED(50) = 0.25 microg/mL). SAL requires extracellular Ca(2+) (ED(50) = 85 microM). SAL is inhibited by verapamil (nonspecific voltage-dependent Ca(2+) channel blocker; IC(50) = 0.4 microM), diphenylhydantoin and NiCl(2) (T-type [Ca(v)3.x] channel blockers; IC(50) 210 microM and 75 microM, respectively). Verapamil blockade of L-type (Ca(v)1.x) channels is use-dependent; activated channels are more sensitive to inhibition. However, verapamil inhibition of SAL does not increase after repeated SAMMA stimulation. SAL is unaffected by 10 microM nifedipine (selective L-type channel blocker). This contrasts to 40% inhibition (P < .001) of AL induced by 1 microM thapsigargin (Ca(2+)-ATPase inhibitor; releases intracellular Ca(2+) stores, promotes capacitative Ca(2+) entry). SAL is unaffected by 1 microM BAPTA-AM (intracellular Ca(2+) chelator), and 50 microM 2-APB (blocks InsP3 receptors and store-operated channels). This contrasts with thapsigargin-induced AL, inhibited nearly 65% by BAPTA-AM (P < .005) and 91% by 2-APB (P, .001). The results suggest that SAL is mediated by Ca(2+) entry through channels pharmacologically similar to the T-type (Ca(v)3.2) class. This process appears distinct from that caused by physiological stimuli such as progesterone or zona pellucida-derived proteins. SAMMA's contraceptive activity may be caused by induction of premature AL through dysregulation of Ca(2+) signaling.

Aim: To compare the effectiveness of an acid-buffering formulation gel (ACIDFORM) with metronidazole gel in the treatment of symptomatic bacterial vaginosis (BV)., Methods: After a confirmed diagnosis of BV according to the criteria established by Nugent and Amsel, 30 nonpregnant women were enrolled in a randomized, double-blind clinical study. The women were randomly assigned to receive either 5 g ACIDFORM gel (n = 13) or 10% metronidazole gel (n = 17) intravaginally once daily for five consecutive days. Participants were evaluated in two follow-up visits (7-12 days and 28-35 days after treatment). Therapeutic success was defined as the presence of less than three of Amsel's criteria. If three or more criteria were present at first or second follow-up visit, the woman was excluded from the study and treated orally with metronidazole. Nugent scores were recorded at each visit but these were not used to define cure., Results: At the first follow-up visit, 15 (88%) of the women in the metronidazole group were cured compared with only three (23%) in the ACIDFORM group (P < 0.001). The remaining 12 women (10 of the ACIDFORM group and two of the metronidazole group) were considered as failure and were treated orally with metronidazole. At the second follow-up visit, two of the ACIDFORM-treated women and six of the metronidazole-treated women presented recurrent BV. Four women in the ACIDFORM group and one in the metronidazole group reported occasional burning and itching during product use., Conclusion: ACIDFORM gel was significantly less effective than high-dose metronidazole gel for the treatment of symptomatic BV.

Microbicides are a new category of compounds being developed as a prophylactic approach for the prevention of transmission of sexually transmitted diseases (STDs), including the human immunodeficiency virus (HIV). These are primarily being developed as women-controlled methods, with the target of designing new compounds or formulations that can be used without the knowledge of a male partner. Microbicide screening can be initially based on their hyaluronidase-inhibiting (HI) activity, as this enzyme plays a major role in the sperm and microbe penetration into the substrate. Derivatives of hesperidin, a citrus flavonoid glycoside, have been reported in the literature for their HI effects. Hesperidin was thereby sulphonated under strictly controlled conditions and the active fraction isolated and characterized, based on its HI activity. This derivative was screened for antimicrobial and enzyme-inhibitory activities, specifically for the reproductive tract. Sulphonated hesperidin (SH) was found to completely inhibit the sperm enzymes hyaluronidase, giving an indication toward its contraceptive effects. It was also been found to inhibit various sexually transmitted pathogens, including Chlamydia trachomatis, Neisseria gonorrhoea, HIV, and Herpes Simplex virus type 2 (HSV-2). Its safety assessment was based on its noninterference in sperm motility and its penetration through the cervical mucus, and no effect on the growth of lactobacilli, the normal vaginal flora. It was also found to be nontoxic to the HIV substrate cells (MT2 cells). The study concludes that sulphonated hesperidin can be developed as a potential microbicide for a dual prophylaxis of contraception and transmission of STDs and AIDS.

Purpose: Polystyrene sulfonate (PSS) is a novel noncytotoxic antimicrobial contraceptive agent. A gel formulation of PSS was found safe for vaginal administration in phase I clinical trials. The purpose of the current study was to develop and evaluate novel bioadhesive vaginal film formulations of PSS., Methods: PSS films were prepared by solvent evaporation and optimized for various physical, mechanical, and aesthetic properties. Further, films were evaluated for various biological activities and safety., Results: Vaginal films containing 300 mg PSS per unit have been developed, using generally regarded as safe (GRAS) listed excipients. The films are colorless, transparent, thin, soft, and tough, dissolve rapidly in physiologic fluid to form a smooth, viscous and bioadhesive solution that could be retained in the vagina for prolonged intervals. Sperm function inhibition (hyaluronidase and cervical mucus penetration) and antimicrobial activities against human immunodeficiency virus (HIV) and herpes simplex virus (HSV) by PSS films were found comparable to PSS. Also, films did not inhibit normal vaginal microflora (Lactobacillus) and were noncytotoxic as indicated by negligible sperm immobilization and cytotoxicity to host cell assays., Conclusions: Rapidly dissolving bioadhesive vaginal film formulation of PSS with desired physical, mechanical, aesthetic, and biological properties is a suitable candidate vaginal microbicide for prevention of sexually transmitted disease (STDs) and is ready for toxicological and clinical evaluation.

This study evaluated contraception by formulated Ushercell, a uniquely high-molecular-weight form of cellulose sulfate, in the rabbit. Variables included (1) dose effectiveness, (2) duration of effectiveness, and (3) formulation excipients. Vaginally applied carboxymethyl-cellulose-based Ushercell gel is contraceptive. A 6% gel is active for at least 18 h; partial activity is observed for at least 24 h. With an application-insemination interval of 0.5 h, Ushercell as low as 0.1% is contraceptive. Contraception is incomplete with 2% Ushercell and an application-insemination interval of 24 h. Ushercell formulations containing a relatively high concentration of Carbopol are ineffective contraceptives, whether the gel is applied before insemination or is premixed with spermatozoa before insemination. Contraceptive activity is restored in Ushercell formulations with lower Carbopol content. This study shows that formulated Ushercell is an effective, long-lasting contraceptive and, hence, is bioavailable when vaginally applied. Activity is dependent on the type and relative concentration of formulation excipients. These data support a projected successful outcome of further clinical trials.

Topical microbicides designed to prevent acquisition of sexually transmitted infections are urgently needed. Nonoxynol-9, the only commercially available spermicide, damages epithelium and may enhance human immunodeficiency virus transmission. The observation that herpes simplex virus (HSV) and human immunodeficiency virus bind heparan sulfate provided the rationale for the development of sulfated or sulfonated polymers as topical agents. Although several of the polymers have advanced to clinical trials, the spectrum and mechanism of anti-HSV activity and the effects on soluble mediators of inflammation have not been evaluated. The present studies address these gaps. The results indicate that PRO 2000, polystyrene sulfonate, cellulose sulfate, and polymethylenehydroquinone sulfonate inhibit HSV infection 10,000-fold and are active against clinical isolates, including an acyclovir-resistant variant. The compounds formed stable complexes with glycoprotein B and inhibit viral binding, entry, and cell-to-cell spread. The effects may be long lasting due to the high affinity and stability of the sulfated compound-virus complex, as evidenced by surface plasmon resonance studies. The candidate microbicides retained their antiviral activities in the presence of cervical secretions and over a broad pH range. There was little reduction in cell viability following repeated exposure of human endocervical cells to these compounds, although a reduction in secretory leukocyte protease inhibitor levels was observed. These studies support further development and rigorous evaluation of these candidate microbicides.

In recent years, AIDS and sexually transmitted diseases (STDs) have become a burgeoning problem and are spreading at an alarming rate. Microbicides are being developed as a new therapeutic category for prevention of transmission of sexually transmitted infections (STIs) and HIV. Many of the microbicide formulations (MF) may fail to elicit a protective response either because of a lack of efficacy or inadequate formulation. Manufacturing a stable, efficacious, safe, and optimal product is the main objective of formulation development programs. Preformulation parameters (PP), as discussed in Part I of this series, influence formulation development significantly and should be considered carefully before designing a formulation strategy. Initially, based on PP and market research, a target product profile (TPP) is generated, which defines product attributes that can be normally classified as "essential" and "desirable." A complex and dynamic process begins thereafter that takes into consideration myriad factors starting from selection of delivery system, selection of excipients, compatibility study, prototype composition, selection of process and optimization, stability testing, scale up, manufacturing under good manufacturing practices (GMP), and packaging development. Prototype formulations are evaluated for several performance characteristics (e.g., dispersion behavior, bioadhesion, retention, spreading, rheology). These compositions are also subjected to biologic evaluation by various in vitro and in vivo models. Such a well-planned, well-coordinated, and well-implemented formulation development program not only accelerates overall development but also minimizes failures in subsequent clinical development studies. The objective of this review is to highlight the importance of formulation science, outline the steps involved in this process, and explore how these can be exploited for achieving optimal MF.

Objective: To investigate the in vivo activity of poly(sodium 4-styrene sulfonate) (T-PSS) gel formulations as topical microbicides., Methods: The ability of the gel formulations to reduce the incidence of infection when applied prior to pathogen challenge was examined in mouse models of vaginal herpes simplex type 2 (HSV-2) and Chlamydia trachomatis infection, and rectal HSV-2 infection., Results: In the vaginal HSV-2 challenge studies, 10% T-PSS gel provided significant protection against infection, even when administered 60 min prior to virus challenge (P < 0.0001). Both 5% and 10% T-PSS gel formulations significantly reduced the incidence of upper genital tract C. trachomatis infection in animals treated up to 5 min before challenge (P < 0.001). However, no protection against C. trachomatis infection was seen in animals treated 30 min before challenge. In mice challenged rectally with HSV-2, both the 5% and 10% T-PSS gels significantly reduced infection at 20 s (P < 0.01 for both). However, only the 10% gel provided significant protection when administered 5 min before challenge (P < 0.01)., Conclusions: T-PSS gel formulations have promising in vivo activity as topical microbicides.

The rapid spread of HIV/AIDS in the female population increases the urgency of developing new formulations that offer protection from this disease as well as other sexually transmitted infections. In many cultures, women do not readily accept touching their genitals or inserting products into their vaginas. Information on this subject was collected during a study involving 635 women in Brazil to determine the preferred attributes of vaginal products. Seventy-six percent would use an idealized contraceptive method that offered dual protection even though it could only be inserted with a finger and 96% would use this method if it could only be placed with an applicator. Qualitative analyses of responses to open questions suggest that the majority of Brazilian women studied did not like to touch their vagina with their finger or to insert devices. Although the introduction of safe and effective vaginal microbicides into many cultural settings can be successful, it should be accompanied by significant efforts to educate women about their bodies.

Microbicides, the compounds and formulations that can prevent transmission of sexually transmitted diseases (STDs)/HIV are being pursued actively as a promising AIDS intervention. The drug development chain for a topical microbicide differs significantly from that of any systemic or topical compound/formulation regarding to time line, cost, activities, and milestones. This is in part because of the lack of standard in vitro models to assess efficacy, and complex ethical issues in clinical trials of microbicides. Several factors, including changes in the physiology of the cervix and vagina with age and menstrual cycle, intercourse, as well as leakage of the formulation from the vagina may affect their design, development, and performance. Selection and development of optimal microbicide delivery systems (gel/cream, pessary, film, tablet, foam, etc.), their inactive ingredients, manufacturing details, and packaging system are dependent on the properties of active drug, or their preformulation parameters (PP). The PP of the active drug substance needs to be evaluated in initial stages of drug discovery and development so that the most suitable delivery system can be selected. Some PP of microbicide agents include physical state, organoleptic properties (color, odor, appearance, taste, etc.), molecular weight, aqueous solubility, hygroscopicity, acidity/alkalinity, permeability and absorption characteristics, stability in solid/solution state, and inherent bioadhesiveness. Thus, a well-coordinated, planned, and implemented preformulation program can help in not only accelerating microbicide formulation development, but also to minimize unforeseen failures in subsequent stages of the development. The objective of this review is to highlight the significance of PP, suggesting a systematic preformulation program.

Objective: To assess the contraceptive properties, antimicrobial activity, and safety of mandelic acid condensation polymer (SAMMA)., Design: Experimental study of SAMMA's in vitro and in vivo properties., Setting: Academic research laboratories., Patient(s): Healthy volunteers for semen donation in an academic research environment., Intervention(s): Inhibition of sperm function indicators, conception, sexually transmitted infection-causing pathogens (including HIV), and lactobacilli was evaluated. Safety indicators were studied., Main Outcome Measure(s): Quantitation of SAMMA's effect on microbial infectivity or multiplication and on sperm function in vitro; evaluation of contraceptive efficacy in vivo; assessment of safety in vitro and in vivo., Result(s): Mandelic acid condensation polymer is not cytotoxic toward lactobacilli, microbial host cells, and spermatozoa. The compound inhibits hyaluronidase and acrosin, induces sperm acrosomal loss, and is contraceptive in the rabbit model. Mandelic acid condensation polymer prevents infectivity of HIV and herpesviruses 1 and 2 and, to a lesser extent, of Chlamydia trachomatis. It inhibits the multiplication of Neisseria gonorrhoeae. Mandelic acid condensation polymer is not mutagenic, has low acute oral toxicity, and is safe in the rabbit vaginal irritation assay., Conclusion(s): Mandelic acid condensation polymer inhibits sperm function, is contraceptive, has broad-spectrum antimicrobial activity, and is highly safe. Further development as a microbicide is warranted.

Presently marketed vaginal barrier methods are cytotoxic and damaging to the vaginal epithelium and natural vaginal flora when used frequently. Novel noncytotoxic agents are needed to protect men and women from sexually transmitted diseases. One novel candidate is a mandelic acid condensation polymer, designated SAMMA. The spectrum and mechanism of antiviral activity were explored using clinical isolates and laboratory-adapted strains of human immunodeficiency virus (HIV) and herpes simplex virus (HSV). SAMMA is highly effective against all CCR5 and CXCR4 isolates of HIV in primary human macrophages and peripheral blood mononuclear cells. SAMMA also inhibits infection of cervical epithelial cells by HSV. Moreover, it exhibits little or no cytotoxicity and has an excellent selectivity index. SAMMA, although not a sulfonated or sulfated polymer, blocks the binding of HIV and HSV to cells by targeting the envelope glycoproteins gp120 and gB-2, respectively, and also inhibits HSV entry postattachment. SAMMA is an excellent, structurally novel candidate microbicide that warrants further preclinical evaluation.

The vaginal route of administration offers a promising option for local and systemic delivery of drugs. Conventional vaginal formulations are associated with limitations of poor retention, leakage, and messiness, thereby causing inconvenience to users. To overcome these limitations, formulations that adhere to the vaginal mucosa for a sufficient period of time need to be developed. Bioadhesion and retention are desirable characteristics of a vaginal formulation to achieve desired efficacy. These properties can be built in during formulation development by the use of bioadhesive polymers. In the present study, assemblies for in vitro measurement of bioadhesive strength and retention characteristics of vaginal formulations have been developed. A modified simulated vaginal fluid (SVFM) was used to simulate vaginal conditions for bioadhesion studies. Cellophane hydrated with SVFM and isolated sheep vaginal mucosa were used as model membranes. The bioadhesive potential of various polymers and their combinations was evaluated. Among the polymers evaluated, xanthan gum (XG), sodium alginate (SA), Polycarbophil (PC), and their combinations (XG + SA and XG + PC) were found to possess significant bioadhesive strength. In retention experiments, XG, SA, and combinations (XG + SA and XG + PC) were retained in isolated sheep vaginal tissue, while PC exhibited poor retention under experimental conditions. Based on the results of the study conducted, XG, SA, and combinations (XG + SA and XG + PC) have been proposed as potential candidates for developing bioadhesive vaginal drug delivery systems.

This is the first report demonstrating the in vitro inhibitory activity of two novel microbicides (cellulose sulfate and polystyrene sulfonate) against bacterial vaginosis (BV)-associated bacteria. Vaginal application of these microbicides not only may reduce the risk of acquisition of human immunodeficiency virus and other sexually transmitted infection-causing organisms but may also decrease the incidence of BV.

The spread of sexually transmitted infections (STIs) and limited methods for control of pregnancies presents high risks to the reproductive health of women. Methods controlled by women and directed toward disease prevention and contraception are needed. We report on preclinical studies of the biological properties of sodium cellulose sulfate (Ushercell) currently being developed for use as a topical contraceptive antimicrobial agent. Ushercell was evaluated with tests designed to identify its contraceptive and antimicrobial properties. Ushercell inhibits hyaluronidase (reversible; IC50 = 1.7 mg/mL), impairs sperm penetration of cervical mucus (approximately 70% inhibition at 1 mg/mL), and acts as a stimulus for acrosomal loss (IC50 = 52 ng/mL). It prevents conception in rabbits when added to spermatozoa (approximately 95% inhibition at 1 mg/mL) or when vaginally applied (complete contraception by 45 mg) before insemination. However, up to 50 mg/mL, Ushercell does not irreversibly immobilize spermatozoa, suggesting that Ushercell is not cytotoxic. Ushercell has a broad spectrum of antimicrobial activity in vitro. Inhibited microbes include human immunodeficiency viruses (different laboratory strains and clinical isolates; IC50 values range from 3 to 78 microg/mL), herpes viruses, HSV-1 (IC50 = 59 ng/mL) and HSV-2 (lC50 = 24 ng/mL), Neisseria gonorrhoeae (IC50 = 2 microg/mL), and Chlamydia trachomatis (IC50 = 78 microg/mL). In contrast, Ushercell does not inhibit growth of beneficial vaginal bacteria, Lactobacillus gasseri, at 5 mg/mL. These results suggest that the antimicrobial effects of Ushercell are selective, and not likely mediated by nonspecific cytotoxic mechanisms. These data provide the basis for further clinical development of Ushercell as a vaginal agent to prevent unplanned pregnancy and STIs.

Host cell infection by sexually transmitted disease (STD)-causing microbes and fertilization by spermatozoa may have some mechanisms in common. If so, certain noncytotoxic agents could inhibit the functional activity of both organisms. High molecular mass poly(sodium 4-styrenesulfonate) (T-PSS) may be one of these compounds. T-PSS alone (1 mg/ml) or in a gel (2% or 5% T-PSS) completely prevented conception in the rabbit. Contraception was not due to sperm cytotoxicity or to an effect on sperm migration. However, T-PSS inhibited sperm hyaluronidase (IC(50) = 5.3 microg/ml) and acrosin (IC(50) = 0.3 microg/ml) and caused the loss of acrosomes from spermatozoa (85% maximal loss by 0.5 microg/ml). T-PSS (5% in gel) also reduced sperm penetration into bovine cervical mucus (73% inhibition by 1 mg gel/ml). T-PSS (5% in gel) inhibited human immunodeficiency virus (HIV; IC(50)= 16 microg gel/ml) and herpes simplex viruses (HSV-1 and HSV-2; IC(50) = 1.3 and 1.0 microg gel/ml, respectively). The drug showed high efficacy against a number of clinical isolates and laboratory strains. T-PSS (5% in gel) also inhibited Neisseria gonorrhea (IC(50) < 1.0 gel/ml) and Chlamydia trachomatis (IC(50) = 1.2 microg gel/ml) but had no effect on lactobacilli. These results imply that T-PSS is an effective functional inhibitor of both spermatozoa and certain STD-causing microbes. The noncytotoxic nature should make T-PSS safe for vaginal use. T-PSS was nonmutagenic in vitro and possessed an acute oral toxicity of >5 g/kg (rat). Gel with 10% T-PSS did not irritate the skin or penile mucosa (rabbit) and caused no dermal sensitization (guinea pig). Vaginal administration of the 5% T-PSS gel to the rabbit for 14 consecutive days caused no systemic toxicity and only mild (acceptable) vaginal irritation. T-PSS in gel form is worthy of clinical evaluation as a vaginal contraceptive HIV/STD preventative.

Hesperidin, a bioflavonoid, is an abundant and inexpensive by-product of Citrus cultivation. A deficiency of this substance in the diet has been linked with abnormal capillary leakiness as well as pain in the extremities causing aches, weakness and night leg cramps. No signs of toxicity have been observed with the normal intake of hesperidin or related compounds. Both hesperidin and its aglycone hesperetin have been reported to possess a wide range of pharmacological properties. This paper reviews various aspects of hesperidin and its related compounds, including their occurrence, physical and chemical properties, analysis, pharmacokinetics, safety and toxicity and the marketed products available. A special emphasis has been laid on the pharmacological properties and medicinal uses of these compounds., (Copyright 2001 John Wiley & Sons, Ltd.)

The high-molecular-weight sulfated or sulfonated polysaccharides or polymers cellulose sulfate, dextran sulfate, and polystyrene sulfonate were tested for microbicidal activity against bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 11 (HPV-11) and type 40 (HPV-40). In vitro assays included the BPV-1-induced focus-forming assay and transient infection of human A431 cells with HPVs. The compounds were tested for microbicidal activity directly by preincubation with virus prior to addition to cell cultures and indirectly by addition of virus to compound-treated cells and to virus-coated cells to test inactivation of the virus after virus-cell binding. The data indicated that all three compounds showed direct microbicidal activity with 50% effective concentrations between 10 to 100 microg/ml. These concentrations were nontoxic to cell cultures for both assays. When a clone of C127 cells was tested for microbicidal activity, approximately 10-fold-less compound was required to achieve a 50% reduction in BPV-1-induced foci than for the uncloned parental C127 cells. Pretreatment of cells with compound prior to addition of virus also demonstrated strong microbicidal activity with dextran sulfate and polystyrene sulfonate, but cellulose sulfate required several orders of magnitude more compound for virus inactivation. Polystyrene sulfonate prevented subsequent infection of HPV-11 after virus-cell binding, and this inactivation was observed up to 4 h after addition of virus. These data indicate that the polysulfated and polysulfonated compounds may be useful nontoxic microbicidal compounds that are active against a variety of sexually transmitted disease agents including papillomaviruses.

Vaginal prophylactic methodology may prevent heterosexual transmission of the HIV and other sexually transmitted disease-causing organisms as well as unplanned pregnancies. A new delivery system (ACIDFORM) was designed with acid-buffering, bioadhesive, and viscosity-retaining properties to (1) maintain the acidic vaginal milieu (the low pH inactivates many pathogens and spermatozoa), (2) form a protective layer over the vaginal/cervical epithelium (minimizing contact with pathogenic organisms), and (3) provide long-term vaginal retention. A Phase I clinical study with ACIDFORM provided initial information about its safety and showed the formation of a layer over the vaginal/cervical epithelium [1; Amaral et al., Contraception 1999;60:361-6]. To study the properties of the gel (without active ingredient) in more detail, ACIDFORM's acid-buffering, bioadhesive, viscosity-retaining, and spermicidal properties were compared in vitro to marketed formulations, and its long-term stability was assessed. ACIDFORM, either when titrated with NaOH or when mixed directly with semen, is highly acid buffering and much more effective than Aci-Jel, a commercial acid-buffering vaginal product. ACIDFORM adheres well to two model membranes (excised sheep vagina and cellophane) and is more bioadhesive than Conceptrol, Advantage S, Replens, Aci-Jel, and K-Y jelly. On dilution, ACIDFORM also retains its viscosity better than these marketed products. ACIDFORM is spermicidal and is stable for at least 2 years. These results suggest that ACIDFORM has advantages over presently marketed vaginal delivery systems. The gel may either be useful by itself as an antimicrobial contraceptive product or as a formulation vehicle for an active ingredient with antimicrobial and/or contraceptive properties.

In view of the need for improved vaginal formulations that are contraceptive, that may prevent transmission of sexually transmitted infections, or both, a new delivery system (base formulation; called Long Acting, Sustained Release of Spermicide, or LASRS) was developed that contains bioadhesive and other ingredients with a long history of safety, and was designed to provide long-lasting vaginal retention of the formulation and to minimize possible vaginal irritation caused by incorporated active ingredients. Nonoxynol-9 (N-9) was added as an active ingredient to study the vaginal irritating properties of the formulation and to assess its long-term effectiveness by postcoital spermicidal tests. In the first series of experiments, in vitro studies showed that the formulation spreads rapidly over a cellulose membrane, forming a bioadhesive layer that remained for at least 12 hours. The second series of experiments addressed the safety of the LASRS suppository in rabbits and primates. Even with a very high concentration of N-9 (20.5%), LASRS caused only mild/moderate but acceptable irritation in the rabbit. No vaginal irritation occurred in the primate at an even higher concentration (22.5%). During the third series of experiments, the long-lasting vaginal retention properties were evaluated by postcoital spermicidal tests in the primate. LASRS with N-9 was highly spermicidal even when mating was delayed for 12 hours after placement of the formulation. Spermicidal activity was also observed when 1) mating was delayed for 24 hours after insertion of the formulation, and 2) if the females were mated 2 or even 3 times without reinsertion of the suppository before collection of the vaginal contents. In the final series of tests, the postcoital spermicidal properties of menfegol, another cytotoxic spermicide, were evaluated as were several modifications in the base formulation. Menfegol produced essentially the same results as N-9. Altering the base formulation proved to be nonbeneficial because a decrease in the long-term spermicidal effectiveness was obtained. These results suggest that the LASRS suppository has good vehicle properties for the delivery of active ingredients to the vagina.

Sexually transmitted diseases (STDs) have been a subject of discussion both among scientists and in the mass media, especially because of their association with the human immunodeficiency virus (HIV). We studied the adoption of specific protective behaviors for the prevention of STDs among women, as well as the associations between these behaviors and socioeconomic and demographic variables. This was a descriptive study based on secondary data from a previous study carried out in Campinas, São Paulo State, Brazil. A total of 635 women were selected using the social network ("snowball") technique. Subjects were classified into four groups: adolescents and adults of upper middle and lower socioeconomic status, respectively. Condoms were the STD prevention method most frequently mentioned by interviewees. A negative association was observed between having a steady partner and condom use in all the groups. The main reason mentioned for not using condoms was "having a single partner and trusting him". Among adolescents, a positive association was observed between schooling above the 8th grade and condom use, and a negative association was observed between age and condom use. Among adults, only condom use in general was also positively associated with socioeconomic status.

A commercial preparation of a sodium polystyrene sulfonate (designated as N-PSS; its molecular weight is 500000 daltons) was tested as an inhibitor of sperm function and as a preventive agent for conception and the transmission of sexually transmitted diseases. The polymer is an irreversible inhibitor of hyaluronidase and acrosin; its IC50 values are 5.7 microg/mL and 0.5 microg/mL, for hyaluronidase and acrosin, respectively. N-PSS is also a stimulus of human sperm acrosomal loss. It produces maximal acrosomal loss at 2.5 microg/mL. Contraception in rabbits is nearly complete when rabbit spermatozoa are pretreated with 0.5 mg/mL of N-PSS before artificial insemination; however, N-PSS does not immobilize spermatozoa at concentrations as high as 50 mg/mL. N-PSS has broad spectrum antiviral and antibacterial activities. Infection by human immunodeficiency virus and herpes simplex virus are inhibited by N-PSS; 3-log reductions are produced by 7 microg/mL and 3 microg/mL, respectively. N-PSS is active against Chlamydia trachomatis and Neisseria gonorrhoeae. At 1 mg/mL, N-PSS inhibits chlamydial infectivity by more than 90%. N-PSS produces a 3-log reduction in gonococcal growth at 15 microg/mL. In contrast, N-PSS (5 mg/mL) does not affect the growth of Lactobacillus (normal component of the vaginal flora). N-PSS can be classified as a noncytotoxic contraceptive antimicrobial agent. These properties justify bringing a polystyrene sulfonate into clinical trials for its evaluation as a preventive agent for conception and several sexually transmitted diseases.

Pilot clinical trials were performed with a new vaginal suppository called "Long Acting, Sustained Release of Spermicide" ("LASRS"). No visual or colposcopic lesions or patient complaints occurred as a result of using LASRS with increasing doses of nonoxynol-9 (up to 20%) for 5 days or of applying the highest dose of nonoxynol-9 (20%; total 400 mg) for 8 h. Colposcopic or visual lesions were also not induced when LASRS with 20% nonoxynol-9 was used for 7 consecutive days by the study participants except for those who developed symptomatic monilia vaginitis. Symptoms were reported although these were mostly minor. A long-lasting, bioadhesive, translucent layer (film) of formulation formed over the vaginal and cervical surfaces. Postcoital spermicidal studies showed LASRS to be highly effective for prolonged periods of time. Although intercourse was delayed for 5 to 8.5 h after insertion of the formulation, an average of only 0. 2 motile sperm/HPF could be found in cervical mucus. These studies suggest LASRS to possess advantages over presently marketed formulations by having long-term efficacy and by forming a bioadhesive, presumably protective layer over the genital tract epithelium. The results also suggest the formulation to decrease the vaginal irritation caused by nonoxynol-9 as noted by colposcopy. These pilot data support a more extensive study with the LASRS suppository.

Presently marketed vaginal barrier agents are cytotoxic and damage the vaginal epithelium and natural vaginal flora with frequent use. Novel noncytotoxic agents are needed to protect women from sexually transmitted diseases. One candidate compound is a high-molecular-mass form of soluble poly(sodium 4-styrene sulfonate) (T-PSS). The antimicrobial activity of T-PSS was evaluated in primary culture systems and in a genital herpes murine model. Results obtained indicate that T-PSS is highly effective against herpes simplex viruses, Neisseria gonorrhoeae, and Chlamydia trachomatis in vitro. A 5% T-PSS gel protected 15 of 16 mice from vaginal herpes, compared with 2 of 16 mice treated with a placebo gel. Moreover, T-PSS exhibited little or no cytotoxicity and has an excellent selectivity index. T-PSS is an excellent candidate topical antimicrobial that blocks adherence of herpes simplex virus at low concentrations, inactivates virus at higher concentrations, and exhibits a broad spectrum of antimicrobial activity.

Vaginal formulations may have "dual" protective activity, against sexually transmitted diseases/AIDS and unplanned pregnancy. The attributes that women find acceptable or unacceptable for such dual protective methods were investigated. More than 50% of the women would not accept messiness, but it was more accepted for dual protective methods than for contraceptives. Very few women would use a dual protective method if it caused vaginal irritation, itching, swelling, or burning, problems associated with presently marketed methods. More than half of the women would use it if it appeared on the penis of their partner or required refrigeration. Use of an applicator to insert the formulation was generally preferred over a manual method. Most women preferred the formulation to be colorless or white, about 16% liked light colors, and about 10% liked darker colors. Almost half of the women were willing to pay up to $5.00 per application of a dual protective formulation, about 15% $3.00, and 30% $1.00. Dual protective methods seem highly acceptable and women would pay much more for them than for condoms. However, these methods should be free of problems usually associated with presently marketed formulations.

Sexually transmitted diseases, including AIDS, and unplanned pregnancies continue to be a serious worldwide problem. A number of organizations are developing woman-controlled vaginal formulations to prevent these problems. However, little information is available regarding the types of products women prefer even though such knowledge is essential to obtain widespread use. This is the first of several articles that describe the results of a consumer preference study for such vaginal formulations performed in Campinas, São Paulo State, Brazil. Because no published methodology was available, the instruments and interview techniques were developed first and procedures established for the identification and participation of research subjects. After preparation of a questionnaire, a pilot study was performed to evaluate it, to establish the interview technique, and to determine the optimal method for subject recruitment. Based on the results, the approach was selected and applied to 635 subjects from different age and socioeconomic groups. The developed methodology and questionnaire, the advantages and the problems encountered, are presented.

A study was carried out to identify characteristics that women would want for an idealized vaginal contraceptive, and the possible association of these characteristics with age and socioeconomic status. The study was done in Campinas, São Paulo State, Brazil. A total of 635 women were selected by age and socioeconomic status, using the "social network" technique. Almost half were adolescents (15-19 years old) and the rest were adults (20-45 years old). Half were of low socioeconomic status and the rest of medium-high status. The data were analyzed with SPSS-PC and EPI-INFO 6.0. Logistic regression and chi 2 were used for the analysis. Despite some differences found between age and socioeconomic status in regard to the characteristics desired for the idealized method, most of the participants expressed the same preferences. The results indicate that women would like the idealized method to be a cream, rather than a suppository, with no odor or flavor, to be colorless, to be placed in the vagina with an applicator well before coitus, and to offer protection against sexually transmitted diseases including AIDS.

Novel vaginal formulations are under development to combat the increasing incidence of sexually transmitted diseases, including AIDS, and also unplanned pregnancies. A study was performed to determine women's preferences for different dosage forms (gel, cream, ovule/suppository, film, foam, tablet), width, length, and color of an applicator, and various types of packages. The study was conducted in Campinas, Brazil. A total of 635 women were interviewed, including both adolescents and adults and low and middle-high socioeconomic groups. The large majority of the women preferred a gel over a cream; both were preferred over the other methods. When asked which method they would not use, the film was most frequently identified, followed by the tablet and ovule. The primary reasons for selecting a particular dosage form were ease of use, absence of odor or the presence of a pleasant one, absence of color, and insertion with an applicator. The major reasons for not using a method were discomfort, "plastic" appearance, distrust of effectiveness, difficulty with insertion, messiness, and rigidity/hardness. The majority of the women liked the applicator shown. The prefilled single dose applicator was by far the preferred packaging. This information should aid in the development of consumer-friendly, vaginal formulations.

Cysteamine (beta-mercaptoethylamine, or MEA) is a thiol-reducing agent and has anti-HIV activity. Because of these properties, cysteamine was evaluated as a vaginal contraceptive and tested for its effects on sperm function and on other sexually transmitted microbes. Cysteamine was contraceptive in the rabbit. Conception was inhibited completely when sperm were pretreated with 500 microg/ml cysteamine and was inhibited by more than 60% when 7.5 mg cysteamine was applied vaginally as a suspension in 50% K-Y Jelly. Cysteamine had multiple effects on spermatozoa. Both acrosin (EC 3.4.21.10) and hyaluronidase (EC 3.2.1.35) were reversibly inhibited by cysteamine. Calculated IC50 values were 370 microg/ml and 150 microg/ml for acrosin and hyaluronidase, respectively. Cysteamine behaved as a poor spermicide when activity was measured by the 30-second Sander-Cramer test. However, sperm motility was inhibited completely when cysteamine was preincubated for 10 minutes prior to motility evaluation, at concentrations as low as 50 microg/ml. The calcium ionophore A23187-induced human acrosome reaction was inhibited by cysteamine (IC50 = 0.5 microg/ml). Neither herpes simplex virus nor Neisseria gonorrhoeae was affected by cysteamine at concentrations as high as 500 microg/ml and 100 microg/ml, respectively. Cysteamine appears to have no effect on normal vaginal flora (i.e., lactobacillus). These results, together with published data, strongly support the further development of cysteamine as a topical contraceptive anti-HIV agent.

The acrosome reaction is an important marker for human sperm function. Since different laboratory techniques may be used for the detection of this exocytotic process, the purpose of the present study was to compare three common markers [Pisum sativum agglutinin (PSA), concanavalin A (ConA), double staining] and transmission electron microscopy for identification of acrosomal changes. Preliminary findings had demonstrated that similar results were achieved with Trypan Blue and Hoechst 33258 staining. Therefore, supravital stainings were omitted. In various experiments, human spermatozoa were treated with two concentrations (10 and 3.3 microM) of calcium ionophore A23187 for 15, 30 and 60 min after capacitation for 3 and 6 h at 37 degrees C. The percentages of spermatozoa with acrosomal loss detected by fluorescein isothiocyanate (FITC)-ConA were consistently lower than those obtained by double staining or FITC-PSA, which showed comparable results. Following 6 h of capacitation and incubation with 10 microM ionophore for 1 h at 37 degrees C, 25.9 +/- 15.7% of all spermatozoa showed almost complete loss of the acrosomal content. Binding of FITC-ConA to the acrosomal region was observed in 27.0 +/- 13.2% of spermatozoa obtained from the same sample. FITC-ConA and double staining or FITC-PSA detect different stages of the acrosome reaction and may be helpful for a differentiated evaluation of this sperm function.

For the evaluation of sperm functions it is important to assess the acrosome reaction after induction with various stimuli. Acrosome reaction tests normally include the capacitation of spermatozoa, treatment with an inducer, and detection of acrosomal loss by dyes, lectins or antibodies. Since most of these methods are time-consuming or require expensive equipment, paramagnetic beads coated with Pisum sativum agglutinin (PSA) were investigated for their usefulness in facilitating the detection of human sperm acrosome reaction. Binding of PSA beads to the acrosomal region increased significantly after incubation of capacitated spermatozoa with 10 microM A23187 (20.3 +/- 6.7% [mean +/- SD, absolute binding], n = 21), 1 mM dibutyryladenosine cyclic monophosphate (17.1 +/- 8.5%, n = 25) and 10 mM phorbol myristate acetate (21.1 +/- 12.5%, n = 10). Bead binding was significantly reduced by pre-incubation with a protein kinase inhibitor. Beads bound to Concanavalin A (ConA) were also attached to the acrosomal region after induction of the acrosome reaction by A23187 or dbcAMP, but a lower number of spermatozoa were bound to ConA-beads than to PSA beads. Pre-treatment of spermatozoa with alpha-methyl-D-mannoside before addition of the PSA beads markedly decreased bead binding, which indicates its mannose-specificity. Electron microscopic examinations demonstrated that PSA beads mainly bound to membrane structures of spermatozoa that were undergoing, but had not completed the acrosome reaction.

The human acrosome reaction (AR; sperm exocytosis) is absolutely required for fertilization. In the course of further characterizing the AR and its control, an AR-inhibiting glycoprotein (ARIG) from human seminal plasma was purified by differential centrifugation, carboxymethyl cellulose chromatography, chromatofocusing, and Sephacryl S300 gel filtration. A highly purified protein with a molecular weight of 74,000 was obtained as determined by gel filtration and SDS-PAGE. ARIG eluted in a narrow pH range (6.2-5.4) during chromatofocusing, corresponding to a pl of 5.8 +/- 0.4. It had covalent modifications, including internal disulfide bonds, and both complex N-linked and O-linked oligosaccharide chains. Lectin analysis suggested that sialic acid was absent and that the complex oligosaccharide chains had sequences containing galactose, galactosamine, and/or glucosamine in a beta 1-4 linkage. Mannose residues were also present. When ARIG was added to in vitro-capacitated human spermatozoa 30 min prior to the calcium ionophore A23187, the AR was significantly inhibited (ID50 = 8.5 micrograms/ml). In addition, ARIG reduced sperm exocytosis in response to atrial natriuretic peptide (a guanylate cyclase activator) and to the protein kinase C activators phorbol myristate acetate and dioctanoylglycerol. The ability of ARIG to block the human AR induced by a variety of agonists and the fact that biological activity of the protein was lost after removal of its sugar moieties suggests that it may function as a general inhibitor of sperm exocytosis and that its interaction with spermatozoa may be mediated by carbohydrate-binding proteins on the sperm cell.

Atrial natriuretic peptide (ANP), found in mammalian ovarian granulosa cells and oocytes (Kim et al., 1992, 1993), induces the human acrosome reaction (Anderson et al., 1994). The purpose of the present study was to determine whether ANP, as egg-derived peptides from sea urchins, can act as a chemoattractant to human spermatozoa. Small lengths of capillary tubing that contained different concentrations of ANP were suspended over a suspension of washed spermatozoa. The number of spermatozoa that entered the tubing was determined. More than twice the number of spermatozoa moved into the tubing that contained a maximally effective concentration of ANP, as compared with tubing that contained only medium. The concentration of ANP that produced a half-maximal effect was 0.7 nM. The effect was blocked by LY83583, an inhibitor of guanylate cyclase. ANP produced more than a twofold increase in the rate of cGMP formation, an effect that was blocked by LY83583. Human ANP (5-27), a fragment of the intact peptide, had no chemoattractant activity. These findings suggest that a specific sperm receptor exists for the chemoattractant activity of ANP that is associated with guanylate cyclase. The chemoattractant activity of ANP is independent of the presence of extracellular calcium ions and is independent of the action of ANP as a stimulus of the acrosome reaction. There is no association between the chemoattractant activity of follicular fluid and the follicular fluid concentration of ANP. These data suggest that factors besides ANP are responsible for the chemoattractant activity of follicular fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: To determine if the acrosome reaction of human spermatozoa can occur without prior incubation to induce capacitation or in calcium-free medium., Design: Noncapacitated (washed ejaculated) or capacitated (incubated for 3 hours in the presence of albumin) human spermatozoa were treated with either solubilized zonae pellucidae (ZP); a calcium ionophore (A23187); or activators of protein kinases A, G, and C, and the acrosomal status was monitored by the double stain technique. During agonist treatment, the capacitated spermatozoa were in medium either with or without calcium ions (Ca2+). The noncapacitated spermatozoa were always in Ca(2+)-containing medium. Sperm motility was monitored throughout the experiments., Results: Solubilized ZP and kinase activators induced acrosomal exocytosis of capacitated spermatozoa (both in Ca(2+)-containing and Ca(2+)-free medium) and of noncapacitated spermatozoa. The lack of added Ca2+ or capacitation reduced the percentage of spermatozoa that reacted in response to solubilized ZP but not to the kinase activators. Ionophore A23187 stimulated acrosomal loss from noncapacitated spermatozoa to the same extent as capacitated spermatozoa in Ca(2+)-containing medium but had no effect on capacitated spermatozoa in Ca(2+)-free medium., Conclusions: Human spermatozoa do not require incubation under capacitating conditions or the presence of extracellular Ca2+ before they can undergo the acrosome reaction in response to certain agonists. Therefore, Ca2+ influx and/or preincubation to induce capacitation are not absolute requirements for the in vitro agonist-induced acrosome reaction. However, these conditions can optimize the acrosome reaction response to zona proteins. The intracellular mechanisms leading to the acrosome reaction appear to be functional in noncapacitated spermatozoa but membrane changes probably are required before certain extracellular ligands such as zona proteins can exert their maximal effect.

Objective: To determine if the solubilized human zona pellucida (ZP)-induced acrosome reaction is mediated by protein kinases., Design: Capacitated spermatozoa were incubated with inhibitors of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent kinase (KT5720), Ca(2+)- and phospholipid-dependent kinase (Calphostin C), and cyclic guanosine 3':5'-monophosphate (cGMP)-dependent kinase (KT5823) and then treated with a corresponding kinase stimulator (dibutyryl cAMP, phorbol 12-myristate 13-acetate and dibutyryl cGMP, respectively) to determine the effect on the acrosome reaction. Appropriate controls were performed. Zonae obtained from the unfertilized oocytes of women attending an IVF program were solubilized using acidic NaH2PO4, and the effect of solubilized ZP on the acrosome reaction was tested in dose-response fashion. Comparative studies with solubilized, zona-free oocyte-treated spermatozoa were performed. The effect of the kinase inhibitors on the solubilized ZP-induced acrosome reaction was then determined., Results: No significant stimulation of the acrosome reaction by kinase stimulators occurred when spermatozoa were pretreated with inhibitors of the kinases, in contrast to the controls. Capacitated spermatozoa incubated with 2, 4, and 6 solubilized ZP showed a dose-dependent increase in the acrosome reaction. Solubilized oocytes had no effect on the acrosome reaction. Pretreatment of spermatozoa with kinase inhibitors significantly lowered the acrosome reaction induced by solubilized ZP but not completely. When a "cocktail" of the three inhibitors was used, a significant reduction in the acrosome reaction occurred in comparison with single inhibitor treatment., Conclusion: The present data indicate a role for human ZP-induced activation of multiple second messenger pathways, involving kinases A, C, and G in the human sperm acrosome reaction.

Atrial natriuretic peptides (ANPs) from several species induced the human acrosome reaction. The maximal response was highest for human ANP (18.6% above unstimulated or baseline values) and decreased progressively for peptides derived from animals lower on the phylogenetic scale. ANP concentrations required for a half-maximal effect in noncapacitated spermatozoa ranged from 0.07 to 0.38 nM. ANP induced the acrosome reaction in capacitated spermatozoa, but the concentration required was higher than in noncapacitated cells. The response in noncapacitated spermatozoa was independent of added extracellular Ca2+ and was completely inhibited by 1 microM LY83583 (inhibits particulate guanylate cyclase). However, 10 microM N omega-nitro-L-arginine (inhibits soluble guanylate cyclase) had no effect. ANP (80 pM) and 3 microM 1,2-dihexanoyl-sn-glycerol each induced a nearly half-maximal acrosome reaction. Added in combination, they produced no increased response, suggesting antagonism. Follicular fluid had variable levels of immunoreactive ANP. Average ANP content was nearly zero in samples that contained no oocyte at the time of aspiration but was higher (6.9 pM; 90% confidence limits = 1.67-28.72 pM) in follicular fluid containing oocytes that did not fertilize in vitro. Highest concentrations of ANP were present in follicular fluid containing oocytes that fertilized in vitro (72.8 pM; 90% confidence limits = 38.1-139.1 pM). These data suggest that noncapacitated spermatozoa can acrosome react without added extracellular Ca2+ in response to an extracellular ligand. Also, human spermatozoa appear to contain receptors for ANP similar to those found in other cell types. The ANP content of follicular fluid might partly explain the ability of follicular fluid to induce the acrosome reaction.

Objective: To determine the effects of peritoneal fluid (PF) from patients with endometriosis on capacitated sperm., Design: Capacitated donor sperm were allowed to migrate into straws filled with pooled PF from patients with unexplained infertility without endometriosis (n = 4) and those with mild (n = 4) and moderate-severe (n = 4) endometriosis, respectively, for 3 hours., Main Outcome Measures: Sperm motility, sperm membrane integrity, and acrosomal loss were determined., Results: Statistically, no significant differences were found among the various sperm parameters determined., Conclusion: Peritoneal fluid from patients with endometriosis does not appear to adversely affect capacitated spermatozoa.

The albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), lysozyme, lactoferrin, alpha 1-antitrypsin, alpha 1x-antichymotrypsin, and neutral proteinase levels of uterine fluid and serum of IUD-bearing women were studied in relationship to the phase of the menstrual cycle, the length of IUD implantation and the presence of reported side effects. Selection of these proteins was based on their potential importance in IUD-induced contraceptive action and/or side effects. Generally, only small differences were found in the serum levels of these proteins during the cycle, with different length of implantation or between patients with and without side effects. However, transudation of proteins (albumin, IgG, alpha 1-antitrypsin, alpha 1x-antichymotrypsin) from blood into the uterine cavity was enhanced by the IUD, especially during the postmenstrual and premenstrual periods. The IUD enhanced the local secretory response even more, i.e., the increased release of proteins from the endometrium (lysozyme, neutral proteinase, IgA) and from leukocytes (lysozyme, lactoferrin). Protein changes occurred as the period of insertion increased. These changes in the uterine milieu may account in part for the contraceptive action of the IUD. The secretory proteins rather than the transudation products differed between patients who showed IUD-associated side effects vs. those who did not, implying that IUD-related pathology is primarily associated with changes in local secretory response. The possible role of each protein in the mechanism of IUD action is presented in view of the present findings.

Objective: To provide a simple, universally applicable method of quality assurance for sperm counting, thereby reducing intercounting chamber variation., Design: By using a known concentration of latex beads, the sperm:bead ratio can be used to calculate the actual sperm count., Main Outcome Measures: The mean sperm and bead counts were determined in both a Spot-lite hemocytometer (Baxter Diagnostics, McGaw Park, IL) and a Makler chamber (Polymedco Inc., Yorktown, NY) from 21 different ejaculates mixed with a known concentration of beads. The hemocytometer chamber was used as the standard counting chamber because it consistently yielded a low variation in sperm count. The adjusted sperm concentration of the Makler chamber was calculated using the following formula [hemocytometer beads]/[Makler beads] x [Makler sperm]., Results: Observed mean +/- SD sperm counts were significantly different between the hemocytometer chamber (110.6 +/- 66.2 x 10(6)/mL) and Makler chamber (173.3 +/- 103.5 x 10(6)/mL). However, calculated Makler chamber sperm counts (118.1 +/- 76.1 x 10(6)/mL) was not statistically different from observed hemocytometer sperm counts., Conclusion: This novel approach to sperm counting using a known concentration of latex beads as a reference material can be used to reduce variation in sperm counting between observers, counting chambers, and possibly computerized sperm analyzers.

The osmo-sensitivity of the human sperm acrosome reaction was investigated by determining the effect of hyper- and hypo-osmolal conditions on the ionophore A23187- and dbcAMP-induced reaction of both capacitated and non-capacitated spermatozoa. Hyper-osmolal conditions inhibited the agonist-induced reactions of both types of spermatozoa. Hypo-osmolal conditions caused a spontaneous loss of acrosomes from capacitated but not from non-capacitated spermatozoa. The loss of acrosomes under hypo-osmolal conditions was further enhanced by dbcAMP but not by ionophore A23187. Although significant decreases in sperm viability were occasionally observed at the high and low osmolalities, these decreases were not consistent and could not account for the observed loss of acrosomes. It is concluded that the human sperm acrosome reaction is osmo-sensitive. The acrosome reaction stimulated by ionophore A23187 (raises intracellular Ca2+) and dbcAMP (activates protein kinase A which causes protein phosphorylation) appears to involve water entry downstream from the action of these agonists. Preincubation in albumin (capacitation) causes human spermatozoa to lose their acrosomes under hypo-osmolal conditions. Finally, capacitation is not an essential prerequisite to the acrosome reaction as long as agonists are used that by-pass certain membrane-related events.

Objective: To determine the effect of sodium alginate encapsulation of rodent embryos on in vitro embryonic cleavage rates, implantation rates, and livebirth rates, and to find the in vivo degradation time for the capsules., Design: Studies were conducted using both CB6F1 mice and Golden Syrian hamsters., Results: Capsules made with 3.0% sodium alginate degraded in vivo within 24 to 48 hours after transfer. In vitro embryonic cleavage of encapsulated embryos was not impaired, nor were implantation rates in CB6F1 mice. Finally, 8.6% of transferred encapsulated embryos resulted in livebirths., Conclusions: Encapsulation of rodent embryos in 3.0% sodium alginate is not detrimental to embryonic development, implantation rates, or fetal development. Because the capsule degrades within 48 hours after transfer, encapsulating embryos may be beneficial for human in vitro fertilization and embryo transfer.

The goals of this study were to determine the effect of the nonionic detergent, Triton X-100, on the recovery of acrosin in the clinical assay (Kennedy et al, 1989), since previous investigations have used higher concentrations for acrosin extraction from spermatozoa; and to establish the minimal acrosin activity in fertile men. The recovered acrosin activity was dependent on the concentration of Triton. A peak in acrosin activity was obtained at 0.01% to 0.02% Triton, the approximate critical micelle concentration (CMC; 0.015%). The bimodal effect of Triton was not due to substrate/buffer alterations, to the degree of acrosomal disruption as assessed by light and transmission electron microscopic examination, or to its effect on the kinetic properties of acrosin, as determined by spectrophotometric analysis of acid-extracted enzyme. However, Triton affected the conversion of proacrosin to acrosin, with peak activation occurring at 0.01% to 0.02% detergent. The acrosin activity of a group of presumably fertile men (as established by the production of offspring under natural conditions) varied from 18 to 42 microIU/10(6) spermatozoa, as assessed by the clinical assay containing 0.01% Triton. Furthermore, men who had initial acrosin values in the low normal range (18 to 25 microIU/10(6) sperm) were observed for 11 months. The acrosin activity of their ejaculates never fell below 17 microIU/10(6) sperm. Thus, it can be tentatively assumed that the minimal levels of acrosin for naturally fertile men are 17 to 18 microIU acrosin/10(6) sperm in this assay.

The objective of this study was to determine whether the metabolism of arachidonic acid via the cyclooxygenase pathway, the lipoxygenase pathway, or both has a pivotal role in the human sperm acrosome reaction. To do so, the stimulatory effect of arachidonic acid and a number of its metabolites, as well as the inhibitory effect of cyclooxygenase and lipoxygenase inhibitors on the acrosome reaction, was evaluated. Arachidonic acid, prostaglandin E2, and prostacyclin (PGI2) induced the acrosome reaction when added to 3-hour preincubated (capacitated) spermatozoa. The arachidonic acid-induced acrosome reaction was dependent upon extracellular calcium. Leukotriene B4 and 15-HPETE only induced the acrosome reaction when present throughout the preincubation period, indicating that they may enhance the capacitation process rather than the acrosome reaction. Thromboxane did not affect the acrosome reaction under any of the conditions tested. Inhibitors of cyclooxygenase (indomethacin, phenylbutazone) and lipoxygenase (phenidone, nordihydroguiaretic acid) or FPL 55712 (a leukotriene antagonist) did not prevent the arachidonic acid-stimulated acrosome reaction. Furthermore, 5, 8, 11, 14-eicosatetraynoic acid (ETYA), the acetylenic analog of arachidonic acid that inhibits arachidonic acid metabolism, induced an acrosome reaction equivalent to that of arachidonic acid. These results strongly suggest that the acrosome reaction induced by exogenous arachidonic acid is not mediated via either the cyclooxygenase pathway or the lipoxygenase pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective: To compare the variability of sperm acrosin and hypoosmotic swelling to the more standard semen variables in relationship to controlled periods of sexual abstinence using a defined group of men., Design: Ten men abstained sequentially for 1, 2, 3, 4, 5, and 10 days and produced an ejaculate after each time period. The ejaculate variables measured at each time point were sperm acrosin, hypoosmotic swelling, volume, sperm number, sperm concentration, sperm motility, sperm morphology, pH, and white blood cells (WBCs). Comparisons were performed between the values obtained at each abstinence period., Results: The percentage of hypoosmotically reactive spermatozoa did not vary significantly with the abstinence period. Sperm acrosin remained similar up to 5 days of abstinence but decreased almost twofold after 10 days of abstinence. The sperm volume and concentration increased gradually with the length of abstinence, being approximately twofold higher after 10 days of abstinence than after 1 day of abstinence. The total sperm number increased about fourfold from 1 day of abstinence to 10 days of abstinence. The percent normal sperm forms tended to increase until 5 days of abstinence but decreased after 10 days of abstinence. The WBC count showed only a small increase with longer abstinence periods. The pH remained essentially the same., Conclusions: The length of abstinence affects the various semen variables differently. An abstinence period of up to 10 days does not alter the hypoosmotic swelling test results. However, the sperm acrosin values decrease after prolonged abstinence so that the abstinence period needs to be taken into consideration when performing this assay.

The in vitro acrosome reaction of noncapacitated human spermatozoa was induced by both calcium ionophore (A23187) and dibutyryl adenosine cyclic monophosphate (Bu2cAMP), a membrane permeant cyclic nucleotide analog, in a dose-dependent manner. Maximal frequencies of acrosome-reacted spermatozoa above baseline values (12%; 90% confidence limits = 10.6 to 14.2%) were similar for Bu2cAMP and A23187 (24.5% and 25.1%, respectively). The concentration of Bu2cAMP required for a half-maximal response was 14.3 mumol/L, while that for A23187 was 24.5 pmol/L. The ability of A23187 to induce the acrosome reaction depended on the presence of calcium ion in the incubation medium. The A23187-induced reaction was prevented by the inclusion of human serum albumin in the medium; the inhibitory effect of albumin was partially reversed after preincubation of spermatozoa for 3 hours under capacitating conditions. In contrast, the Bu2cAMP-induced acrosome reaction was unaffected by either Ca2+ or albumin. Pulsed addition of Bu2cAMP enhanced the frequency of acrosome-reacted spermatozoa. This effect appeared to be influenced by pulse frequency: additions made every 5 minutes produced a greater maximal response than additions made every 2 minutes or every 15 minutes. The maximum theoretical acrosome reaction above baseline values (12%) was 88% of the total number of cells, accounting for almost the entire sperm population. Pulsed addition of A23187 did not increase the frequency of acrosome-reacted spermatozoa above values obtained from single equimolar additions of this agent. These data indicate that: (1) intracellular mechanisms for the human acrosome reaction are functional in noncapacitated spermatozoa; (2) the acrosome reaction can be separated from the process of capacitation; and (3) the acrosome reaction is affected by the pattern, as well as the type, of activation.