| | A short-term evaluation of semen and accessory sex gland function in phase III trial subjects receiving intravasal contraceptive RISUGReceived 29 August 2001; received in revised form 13 September 2002; accepted 18 September 2002. Abstract Following the intravasal injection of a new male contraceptive RISUG (reversible inhibition of sperm under guidance) in volunteers, routine semen analysis, semen biochemistry and germ cell morphology were evaluated in comparison with the corresponding preinjection samples for a maximum period of 6 months. Sperm counts in all 25 subjects before injection varied from 45 to 120 × 106/ml. Out of 25 subjects, 6 became azoospermic after 1 month, 15 after 2 months, 3 after 3 months and 1 after 4 months of contraceptive injection. The mean volume of the ejaculates was found to be less as compared to preinjection samples. Occasional sperm or sperm heads and immature germ cells were identified in only a few postinjected subjects. However, no pregnancy was reported in these subjects during the study period. Abnormal morphology found in most of the sperm, but not in the accompanying immature germ cells, may be due to a charge-related effect on the former but not on the latter cells. Neutral α-glucosidase, the biochemical marker for epididymis, was estimated to be significantly lower in the seminal plasma of all the postinjected subjects. On the other hand, acid phosphatase activity and fructose levels in the seminal plasma were found to be in the normal range. Based on the above findings, it is concluded that at least for the present study period, RISUG, a new male contraceptive, is effective as a partially occluding agent in the vas deferens.
1. Introduction  The reproductive and child health (RCH) program in India emphasizes quality development and delivery of contraceptive services. In order to strengthen the cafeteria approach for contraceptives, it recommends the availability of a number of good quality contraceptives for both males and females. Although development of a male contraceptive has long been a priority amongst the scientific community, the contraceptive choice for men is very much restricted, limited to only a few barrier methods or to a terminal method like vasectomy. Immunological or hormonal approaches in the male are less preferred since periodic intervention would be necessary for maintaining contraceptive efficacy. An ideal alternative would be allowing a one-time contraceptive intervention lasting for years with minimum side effects and with the possibility of a risk-free option for easy reversal. RISUG (reversible inhibition of sperm under guidance) [1], a new male contraceptive, has been developed and is currently undergoing phase III clinical trial. The contraceptive consists of a specific preparation of a copolymer styrene maleic anhydride dissolved in dimethyl sulphoxide (DMSO). It is delivered into the vas deferens following exposure or directly through a no-scalpel approach. The polymer does not have any tissue sclerosing or adherent properties but is retained inside the lumen of the vas deferens without being dislodged. The polymer affects the plasma membrane of the sperm head leading to leaching out of the enzymes acrosin and hyaluronidase [1]. The damaged sperm are thus rendered incapable of fertilization. The effective dose for the contraceptive injection was earlier established [2], [3]. The present study was aimed to evaluate the semen and accessory sex gland function of the subjects in the phase III clinical trial, limited to a period of 6 months. 2. Materials and methods 2.1. Subject selection, pretreatment evaluation and RISUG injection Twenty-five subjects were included in the study according to the criteria as reported in the phase II trial of the drug [3]. In brief, healthy adult male volunteers with normal reproductive profiles were chosen for the study. The criteria for induction were as follows: (a) age varied from 25 to 40 years; (b) two or more healthy children; (c) good health; (d) residing in an area close to trial center [Lok Nayak Jay Prakash (LNJP) Hospital, New Delhi, India] and (e) good semen profile. Subjects with sperm count less than 30 million/ml, more than 30% abnormal sperm morphology, and less than 50% sperm motility were not included in the study. Pretreatment semen data on sperm count, sperm density, sperm and germ cell morphology and sperm motility were obtained from each subject. A written informed consent was received from each subject prior to the contraceptive injection. The protocol of injection was followed as described earlier [3]. Briefly, following skin disinfection, a small incision (∼7 mm in length) was made 15–20 mm above the upper pole of the testis to expose the spermatic cord. A single skin incision was used to deliver the drug into the vas deferens of either side. The cord was incised and the vas deferens was isolated without damaging the adjacent blood vessels. With the help of a 23-gauze needle, 60-mg styrene maleic anhydride (SMA) dissolved in 120 μL of DMSO was injected into the vas deferens of each subject. After the injection, the spermatic sheath was closed with a single catgut suture knot. The subjects were advised abstinence for a period of 1 week following administration of the contraceptive in the vas deferens. Then freedom of unrestricted interactions with the female partner was allowed. However, the subjects were advised to take advantage of a barrier method during coitus until azoospermia was confirmed through semen analysis. 2.2. Semen collection and analysis Semen samples were collected from all subjects prior to the injection and at the intervals of 1, 1 1/2, 2 weeks and once every month until 6 months after the injection. Standard semen analysis was carried out according to World Health Organization guidelines [4]. 2.3. Sperm and germ cell morphology Since most of the subjects were azoospermic after the injection, semen samples were processed by centrifugation and only the deposit was smeared on glass slides for Papanicolaou staining. Sperm and various types of immature germ cell morphology were evaluated using a phase contrast microscope [5]. 2.4. Neutral α-glucosidase activity Neutral α-glucosidase activity in seminal plasma was estimated as described [6]. In brief, 15 μL of seminal plasma were added to 100 μL of p-Nityrophenyl a-D Glucopyranoside (Sigma Chemical Co., St. Louis, MO, USA) (5 g/L in phosphate buffer, 0.01 M, pH 6.8). Distilled water replaced the seminal plasma as water blank. Five microliters of castenospermine (1 mM), the inhibitor for α-glucosidase, along with semen, served as the semen blank. All the tubes were vortexed and incubated for 2 h at 37°C. Incubation was terminated by adding color reagent I (Na2CO3, 0.1 M) to each tube and vortexing. Absorbance was read at 405 nm along with different concentrations of p-Nitrophenyl (PNP; Sigma Chemical Co.) standard (stock: 5 mM/L) diluted in color reagent II (color reagent I containing 1% sodium dodecyl sulfate (Sigma Chemical Co.). 2.5. Acid phosphatase activity Acid phosphatase activity in seminal plasma was estimated biochemically [7]. Briefly, seminal plasma was diluted 10,000 times in citrate buffer (0.09 M, pH 4.8) and an aliquot of 10 μL of the sample was added in the assay tube containing 0.1 ml of the freshly prepared substrate (P-nitrophenol phosphate, 4 mg/ml) solution. Semen was replaced with distilled water to serve as blanks. All the tubes were incubated at 37°C for 30 min. The reaction was terminated by adding 1 ml of NaOH (0.1 M). Absorbance was read at 405 nm against assay blanks for all the tubes containing either unknown or different concentrations of PNP (stock: 5 mM/L) standards. 2.6. Fructose concentration For fructose estimation, colorimetric determination with indol was adopted [4]. Briefly, 20 μL of seminal plasma was diluted to 16 times with dH2O and deproteinized by addition of a de-proteinizing agent (1.8% ZnSO4 in 0.1 M NaOH). Following centrifugation, 200 μL of the clear supernatant was placed in separate tubes along with various concentrations of fructose standards (stock: 2.24 mM). An equal volume of the indole reagent (25 mg of indol in 100 ml of dH2O containing 200 mg of benzoic acid) was added to each tube. All the tubes were incubated at 60°C for 20 min. The reaction was terminated by addition of 2 ml of HCl (32%) and absorbance was read at 470 nm. 2.7. Statistical analysis Data were analyzed by Student’s t-test. A probability of <0.05 was considered as statistically significant. 3. Results Table 1 shows the postinjected period of achieving azoospermia in different subjects. Out of 25 subjects, 6 reach azoospermia after 1 month, 15 after 2 months, 3 after 3 months and 1 after 4 months. Prior to contraceptive injection, sperm counts ranged from 45 to 120 × 106/ml and motility 60 to 70%. Decline in semen volume was significant (p < 0.001) by 2 months of contraceptive injection. However, there was no further decline in volume for the rest of the study period (Fig. 1). No pregnancy was reported during the period of the study. Presence of occasional immotile sperm was characterized by abnormal morphology of various types, such as bent and coiled tail, amorphous head with large elongated tail, double and tapering head etc. (Fig. 2). Immature germ cells with normal morphology were observed in most of the samples (Fig. 3). These cells were uniformly observed in almost all samples, azoospermic or otherwise. Acid phosphatase activity and fructose levels in the seminal plasma did not change significantly (Fig. 5, Fig. 6) in the pre- and postinjection samples. However, neutral α-glucosidase activity in the seminal plasma of the treated subjects declined significantly (p < 0.001) compared to preinjection control levels (Fig. 4). | | |  | Subject no. | Preinjection sperm count (million/mL) | Azoospermia achieved (months) | |
|---|
| S1 | 90 | 1 | |
| S2 | 110 | 3 | |
| S3 | 83 | 2 | |
| S4 | 45 | 2 | |
| S5 | 45 | 1 | |
| S6 | 68 | 2 | |
| S7 | 50 | 1 | |
| S8 | 62 | 1 | |
| S9 | 80 | 2 | |
| S10 | 50 | 2 | |
| S11 | 80 | 2 | |
| S12 | 65 | 3 | |
| S13 | 55 | 2 | |
| S14 | 80 | 2 | |
| S15 | 95 | 2 | |
| S16 | 88 | 2 | |
| S17 | 120 | 2 | |
| S18 | 82 | 2 | |
| S19 | 110 | 4 | |
| S20 | 90 | 2 | |
| S21 | 84 | 2 | |
| S22 | 70 | 2 | |
| S23 | 70 | 3 | |
| S24 | 76 | 1 | |
| S25 | 55 | 1 | | | | |
4. Discussion The findings here represent the first comprehensive short-term analysis of semen, sperm and germ cell morphology and semen biochemistry in subjects from the phase III clinical trial of RISUG undertaken at the Lok Nayak Jai Prakash Hospital, New Delhi, under the active monitoring of the Indian Council of Medical Research, New Delhi. The semen data evaluated for the 25 subjects are part of the data from a total of 50 subjects targeted to be included in the phase III trial of the contraceptive. Development of a male contraceptive with high efficacy, safety and with the scope of easy reversibility has been a challenge for those working in the field. Conventional hormonal protocols demonstrated comparatively large window periods prior to achieving azoospermia and with adverse side effects. Immunological methods, too, failed to achieve the desirable degree of success due to lack of uniform maintenance of antibody titers over a period of time in a given individual or lack of effective immunologic responses observed in individuals resulting in a wide range of amplitudes in antibody titers unable to sustain contraceptive efficacy. The development of RISUG was aimed to achieve contraceptive efficacy at the shortest possible time and with minimal side effects. The existence of a lengthy vas deferens was exploited for delivering the contraceptive through noninvasive intervention. The details of the procedures adopted for administration of the contraceptive has earlier been reported in the phase II clinical studies [3]. While for 12 subjects, only 2 were reported to be azoospermic after 1 month of contraceptive administration in the phase II study, the present phase III study resulted in 6 out of 25 subjects attaining azoospermia by 1 month and another 15 by 2 months postadministration of the contraceptive (Table 1). The results appear encouraging when compared to the data in the phase II study. The shortening of the window period for complete azoospermia in phase III may be attributed to a better delivery system and expertise gained as compared to earlier occasions. Evaluation of sperm and germ cell morphology was not carried out in the phase I and II trials but were included for the first time in the protocols of the phase III trial. Poor sperm quality as a factor has been implicated in cases of recurrent abortion and predominance of genetic abnormalities in the offspring [8], [9]. It was seen that in the semen containing occasional sperm from the treated subjects, only a few complete sperm were present. These spermatozoa were mostly immotile and abnormal in shape. In rare occasions and with extensive screening, one or two immotile but normal sperm were confirmed. Abnormal morphology was limited to sperm only. Thus, the chances of fertilization by RISUG-affected immotile and abnormal spermatozoa would be minimal. The contraceptive, in close contact with biological fluids, was expected to create a charged environment surrounding it. The altered morphology in sperm might be due to a charge-related effect on the negatively charged sperm surface versus the positively charged immature germ cells. There was a distinct drop in the volume of semen in the post-contraceptive-injected subjects as compared to the preinjection period. Semen biochemistry studies in these subjects indicated normal functioning of both prostrate and seminal vesicle (Fig. 5, Fig. 6). This finding was in agreement with the recently reported nuclear magnetic resonance study on semen from subjects administered with RISUG for more than 8 years [10]. Since accessory gland secretions account for 95% of the total volume of the ejaculate, it appears that the decline in semen volume might be due to a lack of contribution from testicular or epididymal secretions. This is supported by the fact that neutral α-glucosidase activity in the semen was found to be significantly (p < 0.001) declined (Fig. 4). Not a single subject showed α-glucosidase activity in the normal range, indicating that either partial or near total blockage occurred at the site of administration, at least in the initial periods. The presence of immature germ cells, occasional sperm or both, confirm only partial patency of the system. The hydrophilic nature of the polymer (styrene maleic anhydride) used in RISUG would result in an initial swelling of the polymer at the site of administration. The microporosity of the polymer ordinarily would have allowed free flow of contents from the proximal epididymis. Under actual conditions, however, the opposite was observed in the present study. Thus, the mechanism of action of inducing azoospermia, at least for the study period as mentioned above, may be attributed in part to a partial blockage and in part to the charge-related effects of the polymer, which causes the majority of sperm to disintegrate. A few of those that escape such an effect were found to be morphologically abnormal without motility and thus incapable of fertilization. In any case, no pregnancy was reported in the female partners of the treated subjects throughout the study period. RISUG, a new male contraceptive, has been shown to be efficacious in inducing azoospermia in 84% of the subjects within 2 months as evaluated in the present study. The presence of occasional abnormal sperm along with low α-glucosidase activity indicates a partial blockage of the system, which does not seem to affect normal accessory sex gland function. Acknowledgements The phase III clinical trial at LNJP Hospital was supported by the Indian Council of Medical Research. The technical assistance from Mr. M. Ibrahim is greatly acknowledged. References [1].
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Reproduction. 2001;122:431–436. MEDLINE a Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Mehrauli Road, Munirka, New Delhi 110067, India b Family Welfare Centre (Male), Lok Nayak Jay Prakash Hospital, Daryagang, New Delhi 110002, India  Corresponding author. Tel.: +91-11-616-5959; fax: +91-11-610-1623.
PII: S0010-7824(02)00442-0 © 2003 Elsevier Science Inc. All rights reserved. | 
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