Efficacy and Safety of an Injectable Combination Hormonal Contraceptive for Men.

CONTEXT: The development of a safe and effective reversible method of male contraception is still an unmet need. OBJECTIVE: Evaluation of suppression of spermatogenesis and contraceptive protection by coadministered im injections of progestogen and testosterone. DESIGN: Prospective multicentre study. SETTING: Ten study centers. PARTICIPANTS: Healthy men, aged 18-45 years, and their 18- to 38-year-old female partners, both without known fertility problems. INTERVENTION: Intramuscular injections of 200-mg norethisterone enanthate combined with 1000-mg testosterone undecanoate, administered every 8 weeks. MAIN OUTCOMES MEASURES: Suppression of spermatogenesis by ejaculate analysis, contraceptive protection by pregnancy rate. RESULTS: Of the 320 participants, 95.9 of 100 continuing users (95% confidence interval [CI], 92.8-97.9) suppressed to a sperm concentration less than or equal to 1 million/mL within 24 weeks (Kaplan-Meier method). During the efficacy phase of up to 56 weeks, 4 pregnancies occurred among the partners of the 266 male participants, with the rate of 1.57 per 100 continuing users (95% CI, 0.59-4.14). The cumulative reversibility of suppression of spermatogenesis after 52 weeks of recovery was 94.8 per 100 continuing users (95% CI, 91.5-97.1). The most common adverse events were acne, injection site pain, increased libido, and mood disorders. Following the recommendation of an external safety review committee the recruitment and hormone injections were terminated early. CONCLUSIONS: The study regimen led to near-complete and reversible suppression of spermatogenesis. The contraceptive efficacy was relatively good compared with other reversible methods available for men. The frequencies of mild to moderate mood disorders were relatively high.

Copy number variation and microdeletions of the Y chromosome linked genes and loci across different categories of Indian infertile males.

We analyzed 34 azoospermic (AZ), 43 oligospermic (OS), and 40 infertile males with normal spermiogram (INS) together with 55 normal fertile males (NFM) from the Indian population. AZ showed more microdeletions in the AZFa and AZFb regions whereas oligospermic ones showed more microdeletions in the AZFc region. Frequency of the AZF partial deletions was higher in males with spermatogenic impairments than in INS. Significantly, SRY, DAZ and BPY2 genes showed copy number variation across different categories of the patients and much reduced copies of the DYZ1 repeat arrays compared to that in normal fertile males. Likewise, INS showed microdeletions, sequence and copy number variation of several Y linked genes and loci. In the context of infertility, STS deletions and copy number variations both were statistically significant (p = 0.001). Thus, semen samples used during in vitro fertilization (IVF) and assisted reproductive technology (ART) must be assessed for the microdeletions of AZFa, b and c regions in addition to the affected genes reported herein. Present study is envisaged to be useful for DNA based diagnosis of different categories of the infertile males lending support to genetic counseling to the couples aspiring to avail assisted reproductive technologies.

Male contraceptive efficacy of Ricinus communis L. extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Ricinus communis L. (Rc), of Euphorbiaceae family is a widespread plant in tropical regions and it is used in traditional medicines as an antifertility agent in India and different parts of the world. AIM OF THE STUDY: The aim of the present study is to revalidate the ethnobotanical knowledge by evaluating the activity of only crude stem bark extracts of Rc. In this study, effects of extracts on male contraceptive efficacy were experimented in vitro with human sperm sample. The work is based on primordial and contemporary therapeutic uses of this plant. MATERIALS AND METHODS: In this study, dose of petroleum ether extract, ethyl acetate extract, acetone extract and lyophilised aqueous extract of Rc were added to fresh human semen in 1:1 volumetric ratio. As the aqueous extract showed a promising result in 1:1 ratio, therefore, the Hypo-osmotic swelling test (HOS), Nuclear chromatin decondensation test (NCD) and Acrosomal status and function test (AFT) were also carried out with the aqueous extract of Rc. RESULTS: The sperm immobilisation effects of the extract appeared immediately in a dose-dependent manner when the samples were treated with four different extracts of this plant. At a concentration of 100mg/mL, 100% (p<0.001 and p<0.05) sperms lost their progressive motility. At a concentration of 300 mg/mL, 100% (p<0.001 and p<0.05) became immotile when treated with aqueous extract. There was 88% (p<0.001 and p<0.05) morphological deformities in sperm sample due the effect of aqueous extract when they were tested for HOS and 91% (p<0.05) sperms behaved against NCD as compared to control group. Also there was a distinct decline (p<0.05) in AFT with increase in dosage concentration. CONCLUSION: The findings of the study revealed that aqueous stem bark extract of the plant showed dose dependent loss of sperm motility by influencing the morphological deformation, blockage in nuclear envelope and distinct declination in acrosomal status of spermatozoa. This research, thus, opens up scope for future exploration of bark of the plant as commercial source of new male contraceptive.

Complete sperm suppression in rats with dienogest plus testosterone undecanoate is facilitated through apoptosis in testicular cells.

Complete suppression of the production of sperm in rats with dienogest (DNG, 40 mg/kg body weight [bw]) plus testosterone undecanoate (TU, 25 mg/kg bw), every 45 days, was found to be associated with a significant increase in germ cell apoptosis. Caspase 3 activity and expression in testis were simultaneously upregulated. Rise in the activities of caspase 8 and 9 was associated with overexpression of upstream marker proteins from extrinsic (Fas [Fatty acid synthase], FasL [Fatty acid synthase ligand], and caspase 8) and intrinsic (Bax [Bcl2-associated-x protein], Bcl2 [B-cell lymphoma 2], and caspase 9) pathways of apoptosis. Apart from the germ cells, interstitial cell apoptosis was also observed along with a decline in the number of functional Leydig cells. It is therefore concluded that complete suppression of the production of sperm with DNG + TU is facilitated mainly through the removal of precursor germ cells through apoptosis. The process is largely modulated by upregulation of upstream and downstream marker proteins from intrinsic as well as extrinsic pathway of metazoan apoptosis.

Complete sperm suppression induced by dienogest plus testosterone undecanoate is associated with down-regulation in the expression of upstream steroidogenic enzyme genes in rat testis.

BACKGROUND: We had shown that dienogest (DNG) + testosterone undecanoate (TU) induced complete sperm suppression in rats when administered together every 45 days. On the other hand, individual drugs given alone in a similar fashion failed to achieve the same result. STUDY DESIGN: The present study was therefore undertaken to determine the reason for such a differential sperm suppression and to correlate it with the expression of steroidogenic enzyme genes in the rat testis. RESULTS: Administration of DNG (40 mg/kg body weight [bw]) + TU (25 mg/kg bw) every 45 days for a duration of 90 days induced spermatogenic arrest, leading to a significant reduction in testicular weight and number of precursor germ cells. Flow cytometric analysis further confirmed the same result, leading to a significant shift in the distribution of haploid cells. Measurement of testosterone (serum and intratesticular) was significantly low. Complete sperm suppression coincided with significant down-regulation in the expression of upstream steroidogenic enzyme genes represented serially by cytochrome P450 side-chain cleavage, P450 17α-hydroxylase, 3ß-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein (StAR) in the testis. On the other hand, rats administered with either DNG or TU alone demonstrated incomplete sperm suppression in which the expression of all the above genes remained characteristically nonuniform. CONCLUSION: Taken together, the above findings corroborate the fact that regulation of expression of three of the upstream steroidogenic enzymes genes and the StAR protein in rat testis is crucial in leading to complete sperm suppression as observed with DNG+TU treatment.

Simultaneous accumulation of hyaluronan binding protein 1 (HABP1/p32/gC1qR) and apoptotic induction of germ cells in cryptorchid testis.

In the experimentally cryptorchid rat, spermatogenic arrest is associated with the formation of multinuclear giant cells, leading to large-scale apoptosis and elimination of germ cells from the seminiferous epithelium. Using this model, the role of Hyaluronan Binding Protein 1 (HABP1), which expresses a stage specifically in post-meiotic cells during spermatogenesis, was examined. Cryptorchidism induced complete arrest of spermatogenesis by 2 days, and by 3-5 days many large and small multinucleated giant cells populated the affected tubules. Ultrastructure of the giant cells revealed both single and multiple chromatin aggregation, with some less compact and distorted, and others broken down into tiny fragments. These cells along with other germ cells were stained terminal deoxynucleotidyl transferase dUTP nick-end labeling positive, demonstrating strong expression of Bax and Heat Shock Protein 70. Simultaneously, there was an up-regulation of the proprotein form of HABP1 in these cells and a decrease in the mature form of protein. The above findings indicate a possible role for HABP1 proprotein in apoptosis induction of germ cells in the cryptorchid testes.

Extended intervention time and evaluation of sperm suppression by dienogest plus testosterone undecanoate in male rat.

BACKGROUND: The potential of using dienogest [DNG, 40 mg/kg body weight (bw)] plus testosterone undecanoate (TU, 25 mg/kg bw) in rats for development of a once-a-month male hormonal contraceptive has been reported earlier in our laboratories. STUDY DESIGN: In the present study, we report a separate efficacy evaluation of the same combination, DNG (40 mg/kg bw) and TU (25 mg/kg bw) in which interval of drug administration has been extended further to 45 and 60 days instead of every 30 days. RESULTS: Complete sperm suppression was observed in rats sacrificed either 60 or 90 days after DNG+TU administration, for two injections at 45-day interval. The neutral α-glucosidase activity in these treated rats remained in the normal range. Germ cell loss due to apoptosis was frequently observed both after 60 or 90 days of combination treatment. Significant decline in serum gonadotropin and testosterone, both serum and intratesticular levels, were observed in the treated rats. Following stoppage of treatment (given at 45-day interval) after two (0 and 45 days) or three injections (0, 45 and 90 days), complete restoration of spermatogenesis was observed by 120 and 165 days, respectively. The sperm suppression, however, could not be sustained when the period of combined drug administration was extended from every 45 to 60 days. CONCLUSIONS: Dienogest plus testosterone undecanoate in the above doses retained contraceptive effectiveness when administered every 45 days but not 60 days. The spermatogenic arrest was completely reversible once drug treatment is stopped. The dose and the frequency of intervention can be extrapolated in future clinical trials.

Altered levels of seminal nitric oxide, nitric oxide synthase, and enzymatic antioxidants and their association with sperm function in infertile subjects.

OBJECTIVE: To investigate the association between specific reactive nitrogen species and sperm function among infertile subjects. DESIGN: Controlled trial. SETTING: Fertility clinic at the National Institute of Health and Family Welfare, New Delhi, India. PATIENT(S): Proven fertile volunteers and subjects attending the above clinic. INTERVENTION(S): Tests for hypo-osmotic swelling (HOS), nuclear chromatin decondensation (NCD), acrosomal status (AS) were performed, and total nitric oxide (NO), nitric oxide synthase (NOS), superoxide dismutase (SOD), and catalase were assayed in infertile subjects subgrouped on the basis of routine sperm parameters. MAIN OUTCOME MEASURE(S): Correlation between sperm function and total NO, SOD, catalase, and NOS levels in semen. RESULT(S): Most of the infertile subjects were found to possess high NO and NOS activity in the semen. Leukocytospermic subjects, despite having normal sperm motility and viability, demonstrated subnormal sperm function scores. Nitric oxide in semen was inversely associated with sperm function in normospermia, asthenospermia, leukocytospermia, leukocytoasthenospermia, and teratospermia. In oligospermia with normal or subnormal motility, NO was positively associated with sperm function. Attenuated activities of either SOD or catalase or both were observed in all infertile subjects except those with normospermia. CONCLUSION(S): Subnormal sperm function in normospermia, asthenospermia, and leukocytospermia may be due to the cytotoxic effect of increased NO, whereas in oligospermia the same may be due to low or suboptimal levels.

N-acetylcysteine counteracts oxidative stress and prevents hCG-induced apoptosis in rat Leydig cells through down regulation of caspase-8 and JNK.

We have earlier reported that following persistent stimulation with hCG, oxidative stress-induced apoptosis in rat Leydig cells was mainly achieved through the extrinsic pathway. In the present study, the role of N-acetylcysteine (NAC) in counteracting the oxidative stress and the mechanisms of inhibition of apoptosis under such conditions were investigated. NAC (1 mM) intervention with repeated hCG stimulation (50 ng/ml, four times, each with 30 min challenge) prevented the decline in Leydig cell viability and the rise in lipid peroxidation and reactive oxygen species. Simultaneously, the activities of the enzymes glutathione-S-transferase, catalase, superoxide dismutase and the intracellular glutathione and antioxidant capacity of the treated cells improved significantly. Apoptotic markers Fas, FasL, and caspase-8, up-regulated following repeated hCG exposure, were significantly down-regulated following NAC co-incubation. While Bcl-2 expression was fully restored, Bax and caspase-9 remained unchanged. NAC treatment induced down-regulation of upstream JNK/pJNK and down-stream caspase-3 in the target cells. Taken together, the above findings indicate that NAC counteracted the oxidative stress in Leydig cells induced as a result of repeated hCG stimulation, and inhibited apoptosis by mainly regulating the extrinsic and JNK pathways of metazoan apoptosis.

Sperm function and seminal oxidative stress as tools to identify sperm pathologies in infertile men.

Routine semen analysis, which includes assessment of sperm motility, viability, and morphology, does not always provide complete diagnostic information as men demonstrating standard scores on these specific parameters sometimes remain infertile. In infertile men who had been subgrouped on the basis of sperm count, motility, morphology, and presence of leukocytes, an evaluation of sperm function and reactive oxygen species (ROS) was found to be effective tools to detect sperm pathologies.

Is abnormal sperm function an indicator among couples with recurrent pregnancy loss?

OBJECTIVE: To determine whether or not sperm function parameters are altered in male partners of couples with a history of idiopathic recurrent pregnancy loss (RPL). DESIGN: In comparison with proven fertile volunteers, sperm function parameters like hypo-osmotic swelling (HOS), acrosomal status (AS), and nuclear chromatin decondensation (NCD) were assessed in vitro from male partners of couples with a history of idiopathic RPL. SETTING: Infertility clinic and andrology laboratory at National Institute of Health and Family Welfare. PATIENT(S): Male partners of couples with a history of idiopathic RPL and proven fertile male volunteers (control). INTERVENTION(S): Standard semen analysis, assessment of sperm morphology, and sperm function with tests such as HOS, AS, and NCD. MAIN OUTCOME MEASURE(S): Sperm paameters, such as HOS, AS, and NCD, were assessed in semen samples from RPL in comparison with the proven fertile control group. RESULT(S): Semen samples from the idiopathic RPL group showed below normal test scores in 57.1% of the cases for all three sperm parameters. The highest aberration (83% of cases) in sperm attributes was observed in NCD, followed by AS (45.7%) and HOS (42.9%). In contrast, abnormality in sperm morphology was limited to 5.7% of the cases. Subnormal sperm function is directly proportional with subnormal sperm motility (<50%) in 23% of the cases. Even in semen samples with normal sperm motility, sperm function scores were below normal in 31.4% of the RPL group. CONCLUSION(S): Reduction in test scores of sperm function, like HOS, AS, and NCD, in male partners of couples with idiopathic RPL suggests that sperms with altered or lowered functional competencies, if they fertilize the oocytes, may lead to the development of an unsustainable embryo resulting in early pregnancy loss. Normal sperm motility does not always ensure normal sperm function scores.

Development of a rapid, sensitive, and reproducible laboratory test kit for the assessment of plasma membrane integrity of human sperm.

OBJECTIVE: To develop a rapid, sensitive, and reproducible hypoosmotic swelling test kit for the assessment of plasma membrane integrity of human sperm in vitro. DESIGN: Prospective comparison of results with the World Health Organization (WHO) method, performed simultaneously. SETTING: Infertility center in a major city in India. PATIENT(S): Couples who presented for infertility evaluation. INTERVENTION(S): The sperm tail-coiling pattern representing sperm plasma membrane integrity was analyzed by using different concentrations of NaCl and also with plain double distilled water (ddH(2)O). Hypoosmotic swelling solution with 2% NaCl in ddH(2)O equivalent to 68 Osm/L was selected for further analysis because it provided the highest and the qualitatively best type (g) of tail coiling among the various other options tried in comparison. MAIN OUTCOME MEASURE(S): A rapid laboratory test kit for the assessment of plasma membrane integrity of sperm was developed that is equally sensitive and reproducible as that described in the WHO protocol. The test was validated by using different normal and subnormal semen samples and in comparison with the standard WHO protocol. RESULT(S): After the screening with different hypoosmotic solutions by using semen samples from fertile volunteers, NaCl (2%) provided the highest and the best types of typical tail coiling, characteristic of sperm with good plasma membrane integrity. The study was then extended to 60 normozoospermic semen samples, which demonstrated a hypoosmotic swelling response of 69.5% +/- 5.23% and 71.5% +/- 4.89%, as per WHO or the present modified method, respectively (coefficient of correlation, r = 0.741). In 13 oligospermic and 18 teratozoospermic subjects, the hypoosmotic swelling response of sperm as per the new method were 36.4% +/- 5.75% and 34.2% +/- 7.78%, respectively, which were comparable to those obtained through the WHO method. There was a 15% drop in viability of sperm after the hypoosmotic challenge (5 min), irrespective of the hypoosmotic solution used. No other morphological alterations in sperm were observed after the hypoosmotic challenge. CONCLUSION(S): The new test kit can be used routinely in laboratories for assessment of plasma membrane integrity of sperm in vitro.