Toxicity and Mutagenicity Evaluation Following RISUG Contraception Reversal in Rats.

Reestablishment of fertility, after a male contraceptive method, is of great concern. In this context, RISUG (Reversible Inhibition of Sperm Under Guidance) has been evaluated for its mutagenicity following reversibility with dimethyl sulfoxide (DMSO)/sodium bicarbonate (NaHCO3) in Wistar albino rats. Animals were divided into 7 groups, namely, sham-operated control, vas occlusion with RISUG for 90 and 360 days, reversal with DMSO and NaHCO3 after 90 and 360 days, respectively. The testis, cauda epididymis, cauda epididymal spermatozoa, and serum were evaluated for apoptosis and hormonal status through various assays. RISUG was subjected to Ames test at dose levels of 10, 50, and 100 µL. Results of terminal deoxynucleotidyl transferase nick end labeling and caspase-3 assays in testes and cauda epididymis revealed that the percentage of positive cells in the experimental groups was comparable to sham-operated control. Annexin V assay in cauda epididymal spermatozoa showed slight elevation in group II ( P < 0.05), whereas in the remaining groups, minimum numbers of positive sperms were found. Hormone profile, namely, testosterone, prolactin, cortisol, prostate-specific antigen, and sperm antibody concentration, remained unchanged. In Ames test, no significant increase was observed in the number of revertant colonies on plates containing RISUG in the presence and absence of S9 mix in all 3 strains. Therefore, the reversal of RISUG-induced contraception by solvent vehicle DMSO/NaHCO3 was successful without any toxicity at the cellular levels.

Indian folklore medicine in managing men's health and wellness.

India is a home for a large variety of plants with remarkable medicinal and pharmacological value. Traditional medicine in the form of Ayurveda, Siddha and Unani has used many of these plants since ancient days for treating and curing various ailments of the body. When it comes to issues related to reproductive health, people still hesitate to discuss and/or accept it openly and hence look for alternate and natural remedies. The various tribal populations distributed across different parts of the country still use these plant extracts in various formulations for maintenance of good health. The medical utilities of several of these plants have been documented; however, there are many more, whose potential is yet to be explored. This review discusses the role of various plants grown in the Indian subcontinent that have been widely used in maintaining various aspects of reproductive health in men such as infertility, aphrodisiac, contraception, libido, sexually transmitted infections and reproductive tract cancers as well as in treating chronic disorders.

RISUG: an intravasal injectable male contraceptive.

Over the last two decades RISUG has been drawing attention in the field of male contraception. It promises to sterile men for a period of up to 10-15 years. According to recent studies in animal models, it proves to be completely reversible. Practically, there are no better options available that can assure complete sterility and precise reversibility. Regardless of so much of information available, RISUG is still holding up for many reasons, firstly, the available information engender bewilderment such as what is this copolymer, how does it work and is reversal really possible? Secondly, advancement of this outstanding invention is drastically slow and thirdly, effects of long-term contraception with RISUG and reports on evaluation of anomalies (if any) in F 1 , F 2 progenies, are lacking. In this review the lacunae as well as advances in the development of RISUG in the light of published work and available resources are pointed out. Formulation of the RISUG, its mode of action and clinical trials have been addressed with particular emphasis.

Sperm characteristics and teratology in rats following vas deferens occlusion with RISUG and its reversal.

The functional success of the reversal of vas occlusion by styrene maleic anhydride (RISUG), using the solvent vehicle, Dimethyl Sulphoxide (DMSO), has been investigated. Reversal with DMSO was carried out in Wistar albino rats 90 days after bilateral vas occlusion. The body weight, organ weight, sperm characteristics, fertility test and teratology, including skeletal morphology were evaluated in vas occlusion and reversal animals and in F(1) progenies to assess the functional success of the occlusion and reversal. Body weight, organ weight and the cauda epididymal sperm characteristics of vas occlusion and reversal animals and of F(1) progenies were comparable to control. Ejaculated spermatozoa in the vaginal smear showed detached head/tail, acrosomal damage, bent midpiece, bent tail and morphological aberrations in sperm head after vas occlusion, which returned to normal, 90 days after reversal. Monthly fertility test, post-injection showed 0% fertility, which improved gradually and 100% fertility was achieved 90 days after reversal. The fertility/pregnancy/implantation record and skeletal morphology of the offspring were comparable to control. The results suggest functional success and safety of vas occlusion reversal by DMSO.

Metabolic properties of lactobacilli in women experiencing recurring episodes of bacterial vaginosis with vaginal pH >or= 5.

While 60% of women experiencing recurring episodes of bacterial vaginosis (BV) with vaginal pH >or= 5 are depleted of resident probiotic lactobacilli, the remainder carry one or more strains of lactobacilli. Their ability to make D-lactic acid is, however, low (3.94 +/- 0.72 mM/L) compared to the D-lactic acid produced by strains from healthy vagina with vaginal pH approximately 4 (8.04 +/- 1.07 mM/L) culture supernatant of 0.5 McFarland concentration (P < 0.001).

Safety evaluation through genotoxicity and apoptotic markers following RISUG® induced contraception and its reversal in male rabbits.

The safety evaluation following vas occlusion with RISUG® and its reversal with DMSO and NaHCO3, using genotoxicity tests and apoptotic marker assays, was carried out in rabbits. Animals were divided into groups of sham operated control, vas occlusion with RISUG® for 3 & 12 months, reversal with DMSO and NaHCO3 after 3 & 12 months, respectively. Minimum incidences of micronuclei in erythrocytes and frequency of aberrant chromosomes were observed. Caspase-3 and TUNEL positive cells in testis and cauda epididymis sections were observed within control limits. Comet assay in leukocytes and testicular cells revealed damaged cell range at the control level. DNA damage in cauda epididymal spermatozoa was observed between 2-3 % by in vitro study and annexin V assay indicated a significant enhancement (p < 0.05) of positive cells in 3 months vas occlusion group. In conclusion, RISUG® induced occlusion and its reversal has not been correlated with any toxicity.

Relative suitability of DMSO and NaHCO3 for reversal of RISUG® induced long-term contraception.

Among the vas-based methods on trial, reversible inhibition of sperm under guidance (RISUG(®) ), a co-polymer of styrene and maleic anhydride is being projected as an effective alternative to No Scalpel Vasectomy. RISUG offers long-term contraception with safety, efficacy in human trials and can be delivered by no-scalpel injection. Currently, the procedure is under phase-III clinical trial. However, reversal of this vas-based drug-induced contraception needs to be established in animal models prior to clinical trials to ensure its claim as an effective alternative for vasectomy. In the present investigation, the relative suitability of dimethyl sulphoxide (DMSO) and NaHCO3 for RISUG induced long-term vas occlusion reversal was carried out in albino rats. Animals were allocated into four groups (n = 10), viz., sham-operated control (group-I), vas occlusion with RISUG for 360 days (group-II), vas occlusion with RISUG for 360 days and reversal with DMSO (group-III) and vas occlusion with RISUG for 360 days and reversal with NaHCO3 (group-IV). A variable response in fertility was observed in different groups. Absolute sterility in group III at all mating intervals, while, zero percent fertility in groups II and IV following 90 days of occlusion was observed. Following reversal restoration of fertility with DMSO at 45 days, whereas, reversal by NaHCO3 at 30 days was noticed. Ejaculated spermatozoa of RISUG injected and initial intervals of reversed animals exhibited various degrees of abnormalities. The testes exhibited focal degeneration in vas occluded animals. The occluded lumen of the vas deferens contained an eosinated polymer with exfoliated epithelium. Following vas occlusion reversal, a complete regeneration in the vas epithelium was seen. All other parameters remained unaltered. The reversal with NaHCO3 resulted into an early resumption of fertility when compared with DMSO and the procedure found to be successful, feasible and safe up to F1 generation. Thus, RISUG provides a hope for reversible male contraceptives.

Safety evaluation of long-term vas occlusion with styrene maleic anhydride and its non-invasive reversal on accessory reproductive organs in langurs.

AIM: To evaluate the safety of the long term vas occlusion with styrene maleic anhydride (SMA) and its non-invasive reversal at the level of accessory reproductive glands ARGs in langurs. METHODS: The morphology of seminal vesicle and ventral prostate was evaluated by light as well as transmission electron microscopy. Serum clinical chemistry and urine albumin were evaluated in an autoanalyzer using reagent kits. Fructose, acid phosphatase and zinc in the seminal plasma were evaluated spectrophotometrically according to the WHO manual. Serum testosterone, prostate specific antigen and sperm antibodies were evaluated by enzyme-linked immunosorbent assays (ELISA) using reagent kits and hematology was estimated according to standard procedures. RESULTS: The morphological features and secretory activity of the seminal vesicle and prostate were normal as evidenced by the presence of well-developed mitochondria, rough endoplasmic reticulum, Golgi bodies, secretory granules and normal nuclear characteristics throughout the course of investigation. Serum testosterone and prostate specific antigen remained unaltered and serum antisperm antibodies level presented negative titres. Urine albumin was nil. Total red blood corpuscles (RBC), white blood corpuscles (WBC), hemoglobin (Hb) and red cell indices, serum protein, glucose, cholesterol, creatinine, creatine kinase (CK), serum glutamate oxalate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), lactate dehydrogenase (LDH), bilirubin, urea, triglycerides and high-density lipoprotein (HDL) did not show appreciable changes following vas occlusion and after its non-invasive reversal. Although fructose, acid phosphatase (ACP) and zinc in the seminal plasma showed a significant reduction following vas occlusion, it could not be related to the morphology of seminal vesicle and prostate. CONCLUSION: SMA vas occlusion and its non-invasive reversal do not damage the accessory reproductive organs.

Perspectives of contraceptive choices for men.

Apart from condoms and vasectomy, which have several limitations of their own, no other methods of contraception are available to men. Various chemical, hormonal, vas based and herbal contraceptives have been examined and few of them have reached the stage of clinical testing. Promising leads have been obtained from testosterone buciclate/undecanoate, alone or in combination with levonorgestrel butanoate or cyproterone acetate, RISUG, an injectable intravasal contraceptive and a few herbal products, particularly the seed products of Carica papaya. It is feasible that an ideal male contraceptive, that meets out all the essential criteria will be made available to the community in the near future.

Effects of danazol on the pituitary-gonadal axis in male gerbils and mice.

Danazol administration caused lesions in the seminiferous tubules of gerbils and mice. The seminiferous epithelium became systematically depleted of spermatogonia, spermatocytes, spermatids, and finally spermatozoa. Danazol administration did not cause damage to the epididymal cells but was followed by a significant increase (p smaller than 0.01) in the diameter of the Leydig cell nuclei. The growth of androgen-dependent organs was reversibly suppressed after treatment. Resumption of normal gonadal function in the mouse occurred 32 days after discontinuation of medication. Danazol brought about transient changes resembling those of bilateral gonadectomy in the anterior lobe of the hypophysis. Inhibition of endogenous gonadotropins occurred, reflected in the increased basophilic cell percentage.

Effects of alpha-chlorohydrin on the testes and epididymides of dog: a preliminary study.

Chronic administration of alpha-chlorohydrin (8 mg/kg for 30 days, caused lesions in the testis of dog. Seminiferous tubules presented marked degenerative changes. Leydig cell hypertrophy was conspicous. Epididymal epithelium was regressed and the lumen was devoid of spermatozoa. Obstruction of the epididymal lumen was not seen. Alpha-Chlorohydrin inhibited the synthesis of RNA and sialic acid in the testis, caput epidiymis, corpus epididymis, and cauda epididymis. The total cholesterol per gram of testis was increased significantly after alpha-chlorohydrin administration. The anti-androgenic nature of alpha-chlorohydrin is suggested.

PIP: A preliminary study of the effects of alpha-chlorohydrin on the reproductive tract of male dogs is presented. 10 animals were injected with 8 mg/kg/day of the drug for 30 days. The seminiferous tubules showed considerable degeneration and shrinkage. A gradual loss of type A spermatogonia, spermatocytes, spermatids, and spermatozoa was observed in the testes. The volume of Leydig cell cytoplasm increased, showing a granular appearance, and the nuclei were enlarged. Epididymides weights were significantly lower than those of controls (p less than .02). The epididymal lumen contained no spermatozoa and there was no evidence of obstruction. The synthesis of RNA and sialic acid was inhibited in the testis, caput epididymis, corpus epididymis, and cauda epididymis. Alpha-chlorohydrin significantly increased the total amount of cholesterol per gram/testis (p less than .02). Results indicate that alpha-chlorohydrin is antiandrogenic in nature and acts directly on the testis and epididymal biochemistry.

Reversible contraception with chloroform extract of Carica papaya Linn. seeds in male rabbits.

The contraceptive efficacy and reversibility of the chloroform extract of the seeds of Carica papaya in adult male rabbits were investigated. Eighteen adult male rabbits were divided into three groups of six animals each; Group I--control, Group II--administered chloroform extract of the seeds of Carica papaya at 20 mg/animal/d for 150 d by gavage, and Group III--administered the seed extract at 50 mg/animal/d for 150 d. Body weight and organ weight, semen analysis, sperm morphology by scanning electron microscopy, semen biochemistry, histology of the testis, haematology, serum clinical biochemistry, and the fertility status of the control and the treated animals were evaluated. Body weight and the weight of the testis, epididymis, seminal vesicles, and prostate did not show appreciable changes. Sperm concentration showed a gradual decline, reached severe oligospermia (fewer than 20 million/mL) after 75 d treatment, and attained uniform azoospermia after 120 d treatment. Sperm motility and viability were severely affected after 45 d treatment and reached less than 1% after 75 d treatment. The morphology of the spermatozoa by scanning electron microscopy revealed membrane damage in the acrosome, bent midpiece, coiled tail, and detached head and tail. The levels of fructose, glycerylphosphorylcholine, acid phosphatase, and lactate dehydrogenase in the seminal plasma were unaltered. Histology of the testis revealed arrest of spermatogenesis beyond the level of spermatocytes. No toxicity was evident from the haematology and serum biochemistry parameters. The libido of the treated animals was unaffected and the fertility rate was zero. The effects were comparable in both the dose regimens (Groups II and III) and were restored to normal 45 d after withdrawal of the treatment.

Structural and functional changes in epididymis following danazol plus testosterone enanthate administration in rabbit.

Danazol in combination with testosterone enanthate was administered to adult male rabbits to assess the structural and functional activity of epididymis. Seminal, biochemical and histological studies were made throughout the experiment. Sperm motility and count decreased markedly and at the end of treatment 75% of the animals became azoospermic. Seminal plasma GPC levels decreased significantly at 45 through 75 days of treatment. At 30 days of drug exposure, phospholipid, protein and sialic acid concentrations in caput epididymides were significantly low. Epididymal weight, phospholipid and protein contents markedly dropped in all segments of epididymides after 60 days of treatment. A reduction in epithelial and stereocilia height (caput and cauda), and lumen diameter (caput and corpus) was observed. Increase in intertubular stroma was evident. Lumen was devoid of spermatozoa at 60 days of treatment. All the changes were restored to normal 75 days after cessation of treatment. The combination therapy impaired the structural and functional integrity of the epididymis.

Effects of cyproterone acetate with combination of testosterone enanthate on seminal characteristics, androgenicity and clinical chemistry in langur monkey.

Daily oral administration of 1 mg/kg b.w. of cyproterone acetate and simultaneously administered testosterone enanthate (2 mg/kg b.w./15 days; i.m.) to adult male langur monkeys over a period of 90 days caused a gradual decrease in the count (to azoospermia) and motility of spermatozoa, concurrently with an increase in the percentage of non-motile as well as abnormal and immature sperm. Semen weight, volume, seminal fluid volume and circulating testosterone levels decreased nonsignificantly. Semen pH, libido and body weight remained unimpaired. The levels of SGOT, SGPT, serum alkaline phosphatase, LDH, bilirubin, Na+, K+ and hematological values did not alter significantly. All the changes were reversible. The results indicate that the combination regimen seems to affect the fertility in two ways, i.e. by inhibiting spermatogenesis in the testis and maturation process in the epididymis without altering the androgenicity.

Ultrastructural changes in the vas deferens of langur monkeys Presbytis entellus entellus after vas occlusion with styrene maleic anhydride and after its reversal.

Ultrastructural changes in the vas deferens of langur monkeys after 150 days of vas occlusion with styrene maleic anhydride (SMA) and after 150 days of noninvasive reversal are reported. The vas deferens of the sham-operated control animals revealed active secretory and absorptive functions. The basal cells showed prominent nucleus and sparse cytoplasmic organelles, and the principal cells characterized by oval or irregular nucleus, well developed mitochondria, Golgi bodies, rough endoplasmic reticulum, secretory granules in the Golgi area, free ribosomes, and glycogen granules in the supranuclear region suggesting secretory function. Vesicles and stereocilia in the apex region suggested absorptive functions of the vas deferens. Vas occlusion by SMA resulted in exfoliation of epithelial cells, pyknotic nuclei, and vacuolated cytoplasm virtually devoid of cytoplasmic organelles and stereocilia. After noninvasive reversal, the vas epithelium regained a state of normalcy as evidenced by prominent plasma membrane, nucleus, cytoplasmic organelles, and stereocilia. The results suggest that the exfoliation of the epithelium due to vas occlusion by SMA regains normalcy after 150 days of noninvasive reversal.

Ultrastructural changes in the spermatozoa of langur monkeys Presbytis entellus entellus after vas occlusion with styrene maleic anhydride.

Changes in the physical characteristics of semen and ultrastructure of the spermatozoa of langur monkeys after vas occlusion with styrene maleic anhydride (SMA), a polymer with pH-lowering action, are reported. Vas occlusion resulted in severe reversible hypospermia. Severe oligospermia was observed in the majority of animals (five of eight) in the first ejaculation, 30 days after vas occlusion, and in two animals in the second ejaculation, 60 days after vas occlusion. Subsequent monthly ejaculations for 5 months revealed uniform azoospermia. The voided spermatozoa were immotile and supravital staining confirmed necrospermia. Scanning electron microscopy (SEM) revealed severe coiling of tail, rupture of acrosomal envelope, and bent midpiece associated with damaged mitochondrial sheath. Observations by transmission electron microscopy (TEM) revealed vacuolization in the nucleus, membrane damage in the acrosome, loss of segmented columns, and numeric aberrations in the centriole of the neck, as well as degeneration of mitochondrial sheath and axoneme in the midpiece, and absence of outer plasma membrane in the midpiece and tail. The results indicate that the necrospermic status of the spermatozoa during initial ejaculations may offer instant sterility after vas occlusion with SMA.

Gossypol-induced hypokalemia and role of exogenous potassium salt supplementation when used as an antispermatogenic agent in male langur monkey.

In our earlier study, we have observed that hypokalemia in langur monkeys, following gossypol acetic acid (GAA) treatment (5 mg dose level) when used as an antispermatogenic agent, and potassium salt supplementation partially maintained body potassium level of the animals. The aims of the present investigation was to confirm further occurrence of hypokalemia in the monkey (comparatively at two higher dose levels) and the role of potassium salt in preventing occurrence of gossypol-induced hypokalemia. Highly purified gossypol acetic acid alone at two dose levels (7.5 and 10 mg/animal/day; oral) and in combination with potassium chloride (0.50 and 0.75 mg/animal/ day; oral) was given for 180 days. Treatment with gossypol alone as well as with the supplementation of potassium salt resulted in severe oligospermia and azoospermia. Animals receiving gossypol alone showed significant potassium deficiency with signs of fatigue at both dose levels. Enhanced potassium loss through urine was found in potassium-deficient animals, whereas animals receiving gossypol acetic acid plus potassium salt showed normal serum potassium with a less significant increase in urine potassium level during treatment phases. Other parameters of the body remained within normal range except gradual and significant elevation in serum transaminases activity. The animals gradually returned to normalcy following 150 and 180 days of termination of the treatment.

PIP: An earlier study conducted by the authors indicated that body potassium levels were partially maintained in male langur monkeys treated with gossypol acetic acid (5 mg) and potassium salt supplementation. The present study sought to confirm the persistence of hypokalemia at two higher dosage levels (7.5 and 10 mg/animal/day) and assess the role of exogenous potassium salt (0.50 and 0.75 mg/animal/day) in preventing gossypol-induced hypokalemia. The two dosages of highly purified gossypol acetic acid were administered alone and in combination with potassium chloride for 180 days. All regimens produced severe oligospermia and azoospermia. However, monkeys who received gossypol alone showed significant potassium deficiency with signs of fatigue at both doses. On the other hand, animals receiving gossypol acetic acid and potassium salt supplementation showed normal serum potassium with a less significant increase in urine potassium level during treatment. Also noted was a gradual but significant elevation in the activity of serum transaminases. All parameters returned to normal 150-180 days after treatment termination. The hypokalemic effect documented in this study with gossypol alone may be due to renal leakage and gastrointestinal disturbances.

Antispermatogenic effects of tolnidamine in langur (Presbytis entellus).

Tolnidamine (50 mg/kg body weight; twice a week; oral) was administered for 90 days to adult male langur monkeys (Presbytis entellus entellus Dufresne) to assess its contraceptive potential. Semen weight, volume, seminal fluid volume, colour, pH and libido remained unchanged. Sperm motility, vitality and morphology were impaired with the advancement of treatment. Sperm density reduced to severe oligospermia following 75-90 days of treatment. Increased number of immature germ cells were also noticed. Resumption of changes to pretreatment range was observed following 90 days of cessation of treatment. However, sperm density remained low all through the recovery period of 150 days. Seminal fructose, ACP, LDH and citric acid concentrations did not change markedly. A significant depletion in GPC and magnesium levels was recorded during treatment and early recovery periods. Alterations in germ cells and Sertoli cells were also observed. A progressive but reversible rise in serum creatinine was evident. Other clinical parameters and body weight response revealed no drug-related alterations. In conclusion, tolnidamine medication induced irreversible inhibition of spermatogenesis.

Testis functions and sexual potentia in langur monkey treated with a combination steroidal contraceptive formulation.

Administration of a combination formulation of danazol (100 mg/day; oral) plus testosterone enanthate (TE) (50 mg/15 days; i.m.) for 30 to 60 days in adult male langur monkeys resulted in the reversible suppression of testicular function without affecting the sexual potentia. Testicular weight and volume decreased significantly along with the mass atrophy of germinal epithelium and impaired morphology of Leydig and Sertoli cells. A conspicuous shrinkage of seminiferous tubules, Leydig cell nuclei and Sertoli cell nuclei was noted. Elevation of testicular cholesterol, total lipids, glycogen and phosphatases activity with the depletion of total proteins, nucleic acid, sialic acid and fructose was noteworthy. All changes were maintained during maintenance dose studies (danazol: 50 mg/day; oral plus TE: 50 mg/15 days; i.m.) for 60 days. Resumption of all measures to normal was evident following 120 days of recovery. It can be concluded that the exogenous TE substitutes the serum testosterone levels to maintain extratesticular androgen actions even after interference by danazol of Leydig cell function along with spermatogenesis inhibition.

Long-term sequelae of tolnidamine on male reproduction and general body metabolism in rabbits.

The long-term effects of tolnidamine on male reproduction and general body metabolism were studied in rabbits (Oryctolagus cuniculus). The study was divided into three groups of 10 animals each. The first group (A) received vehicle alone to serve as controls. The second and third groups (B and C) of animals were administered tolnidamine orally at 50 mg/kg body weight/week and 50 mg/kg body weight/day, respectively, for a period of 150 days. The animals of group B exhibited a sperm density of 23.60 million/mL +/- 4.87 million/mL (vs 453.00 million/mL +/- 65.30 million/mL in group A) after 150 days of treatment. In group C, all animals were azoospermic after 135 days of treatment. A reversible impairment of sperm motility, vitality and morphology was noticed. Semen weight, volume, color, pH, libido, and circulatory levels of testosterone remained unchanged. In group B animals, sperm density did not return to control levels even at 150 days after cessation of treatment (37.40 million/mL +/- 4.46 million/mL, vs 380.00 +/- 40.80 million/mL in group A). However, spermatozoa reappeared in animals treated daily (group C) after 30 days of recovery but remained < 5 million/mL during the entire recovery period. A reversible, significant depletion was recorded in seminal glycerylphosphorylcholine (GPC) levels. Fertility was unimpaired in group B animals when compared with those in group A. In group C, fertility was reduced to zero after 150 days of treatment and at 90 days and 150 days after cessation of treatment. No significant alterations were observed in other semen biochemical, hematologic, or blood/serum biochemical parameters with either dose regimen. It is concluded that tolnidamine administration induced dose dependent, irreversible inhibition of sperm production without altering general body metabolism in male rabbits.