RISUG: An intravasal injectable male contraceptive

Abstract

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.

Full text

RISUG: An intravasal injectable male contraceptive
N.K. Lohiya, I. Alam, M. Hussain, S.R. Khan & A.S. Ansari
Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur, India
Received January 17, 2013
Over the last two decades RISUG has been drawing attention in the eld 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, rstly, 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 F1, F2 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.
Key words Clinical trials - male contraception - RISUG - reversal - sperm - styrene maleic anhydride
Indian J Med Res 140, November (Suppl.) 2014, pp 63-72
63
Review Article
Introduction
The burden of population control has been generally
borne by women while men fall behind due to lack
of efcient and acceptable contraceptive. Available
methods are mostly dependent on permanent vasectomy
and widely used condoms that are reluctantly accepted
in men’s world. In early 80s Misro et al1 came with
a revolutionary occlusive polymer which was claimed
to sterile subjects by single injection of styrene maleic
anhydride (SMA) dissolved in dimethyl sulphoxide
(DMSO), in both vas deferens; it was named as RISUG
(Reversible Inhibition of Sperm Under Guidance)
under impression that the contraception with SMA
can be reversed at any time following vas occlusion.
However, mode of action of SMA and the mechanism
of bio-adhesion and bio-sustainability in the vas
deferens was poorly explained. Early experiments
carried out in rats and monkeys showed loss of fertility
by so called pH-lowering effect and convinced to be
an effective contraceptive, however, morphological
alterations in vas deferens were also documented2-4.
A study on vas occlusion with RISUG demonstrated
100 per cent sterility within 15 days post injection in
rats providing evidence of morphological aberrations
in sperms, which included nuclear membrane damage
in acrosome, loss of segmented columns and numeric
aberration in centriole of the neck, degeneration of
mitochondrial sheath and axoneme in the mid-piece and
absence of plasma membrane in the mid-piece and tail5.
This was perhaps the rst study to document functional
reversal of contraception with RISUG through dimethyl
sulphoxide (DMSO)5; further, sodium bicarbonate

(NaHCO3) was used as another mode of reversal
which also showed complete resumption of fertility
(unpublished observation). Issues such as mutagenicity,
genotoxicity and carcinogenicity are being evaluated at
many centres in India. The clinical trials conducted in
the last two decades have been drastically slow. Phase
I clinical trial reported only the dose regimen and
efcacy in terms of sperm count along with behavioural
and general observation such as discomfort and scrotal
enlargement6. The phase II clinical trial also provided
limited information such as sperm count, behavioural
and general observation, sperm motility, however,
was added for the rst time, numbers of subjects were
also reduced by more than half compared to phase I7.
Ongoing extended phase III trial results provided some
critical information in 25 subjects which included dose
of regimen, abstinence from sex, semen volume, sperm
count, sperm morphology, germ cell morphology along
with biochemistry of fructose, acid phosphatase and
α-glucosidase8. Phase III clinical trial revealed variation
in duration before subjects became azoospermic. Of the
25 subjects, six became azoospermic after 1 month, 15
after 2 months and other 4 in 3-4 months. Aberration
in sperm morphology, for the rst time was termed
as the charge-related effect following the hypothesis
given in US patent8,9. The term ‘partial occlusion’ by
RISUG was also rstly coined in phase III clinical trial
by the same group indicating lower epididymal neutral
α-glucosidase and normal fructose level in seminal
plasma8.
Components of the vas-based injectable
contraceptive ‘RISUG’
RISUG has had a history of names; such as, an
injectable non-occlusive chemical1,2, an injectable
non-occlusive contraceptive device10, pH lowering
polymer-styrene maleic anhydride (SMA)11, an
anti-fertility agent-SMA4, RISUG12, styrene maleic
anhydride13, RISUG hydrogel14 and nally smart
RISUG15. Its constituents are as dened 60 mg of
SMA dissolved in 120 µl of DMSO (1:2)4-9. To
understand this contraceptive and its effectiveness, it
is important to know the polymer by its components
and manufacturing.
Styrene: Styrene (C6H5CH=CH2) is a derivative of
benzene of molar mass 104.15 g/mol, it evaporates
easily and has a sweet smell. The presence of vinyl
group allows styrene to be precursor to polystyrene
and several other copolymers. It was rst isolated
from a Turkish tree named oriental sweetgum
(Liquidamber orientalis). Commercially, it is produced
by dehydrogenation of ethylbenzene and oxidation
of ethylbenzene hydroperoxide. There is inadequate
information available to prove its carcinogenicity in
human, however, the US National Toxicology Program
(NTP) has described it ‘reasonably anticipated to
be a human carcinogen’16. According to hazardous
substances database (HSDB)17 the oxide form of styrene
(Styrene 7,8 oxide) binds to DNA and shows genetic
effect. The workers exposed to styrene were detected
with presence of styrene 7,8 oxide in their blood
(HSDB)18. Material safety data sheet (MSDS) shows
that exposure to styrene vapour causes irritation in eyes
and throat19. According to the monograph (monographs.
iarc.fr/index.php) on the evaluation of the carcinogenic
risk of chemical to man, styrene exposed people have
been found with increased chromosomal aberration in
their white blood cells. Further studies have shown that
the liver, kidney and the haematological abnormalities
have neither been linked to styrene toxicity nor does it
have any teratogenicity or spontaneous abortion.
Maleic anhydride: Maleic anhydride [C2H2(CO)2O]
is the acid anhydride of the maleic acid with an acrid
odour. It is manufactured by oxidation of benzene
or N-butane and has a molar mass of 98.06 g/mol. It
generates half easter (cis-HOOC–CH=CH–COOCH3)
in presence of alcohol and produces maleic acid
(cis-HOOC–CH=CH–COOH) when hydrolyzed.
Upon exposure it causes irritation in skin and eyes.
In HSDB17 maleic anhydride has been classied
as non carcinogenic, however, workers exposed in
manual processing of polyester lacquers containing
maleic anhydride without safety precautions suffered
acute poisoning which was manifested as nervous,
respiratory, and cardiovascular systems disorders20.
In another study21, workers sensitized by one acid
anhydride, trimellitic anhydride, could possibly
react immunologically to two other acid anhydrides,
phthalic anhydride (P) or maleic anhydride (M). In
ELISA based cross-inhibition studies, trimellitic
anhydride conjugated to human serum albumin (TM-
HSA) inhibited IgE binding to TM-HSA, but when 100
times more P-HSA or M-HSA was used, no signicant
inhibition occurred.
Styrene maleic anhydride (SMA): Styrene maleic
anhydride [(C8H8)n-(C4H2O3)m] also called Xiran is
a crystal clear polymer of variable molar mass and
soluble in alkaline solution and polar organic solvents.
It is a synthetic polymer built up of perfectly alternating,
styrene and maleic anhydride monomers, making it an
alternating copolymer. The copolymer is transparent,
heat resistant, with high dimensional stability and
specic reactivity of the anhydride groups. These
64 INDIAN J MED RES, NOVEMBER (SUPPL.) 2014

LOHIYA et al: RISUG AS A POTENTIAL MALE CONTRACEPTIVE 65
characters allow it to be used in plastic application,
manufacturing of poly-methyl methacrylate (PMMA),
acrylonitrile butadiene styrene (ABD) and polyvinyl
chloride (PVC). When dissolved in dimethyl suphoxide
(DMSO) it can penetrate the cells and adhere to it.
Based on this property of SMA, RISUG was created.
There is no information available about the copolymer
in HSDB.
Dimethyl sulphoxide (DMSO): DMSO is organosulphur
compound with chemical structure (CH3)2SO. It
dissolves both polar and non-polar compounds and
gives garlic like taste. It also has ability to penetrate
skin without damaging the cells and is used for
administration of many medicinal drugs by clinician.
Apart from its ability to restrict DNA to form secondary
structure, it is also used as a cryopreservative for
stem cells22. With its valuable use for its ability to
penetrate cells it has also been considered as a potential
hazardous chemical, DMSO by itself is considered
low toxic but with compounds dissolved in it can
cause severe toxicity23. DMSO is listed in hazardous
substances database, however, no serious toxicity has
been reported in human, there are animal data which
show varied responses to the DMSO. DMSO produces
widespread apoptosis in the developing central nervous
system24, and DMSO exposure to developing mouse
brains can produce brain degeneration24. Substances
dissolved in DMSO may be quickly absorbed; larger
amount of DMSO can have adverse effect on relative
tissues or skin.
Reversible inhibition of sperm under guidance
(RISUG)
There is no published chemical structure of
the RISUG available. There have been constant
advancements in the development of RISUG during
the last two decades after the technology was patented.
According to the patent9, contraceptive was referred
to as an injectable uid of a copolymer of SMA in a
solvent DMSO. The copolymer was prepared through
cobalt 60 gamma irradiation of the monomers styrene
and maleic anhydride in presence of nitrogen in ethyl
acetate at a dose of 0.2 to 0.24 megarad for every
40 g of polymer at a dose rate of 30 - 40 rad/sec. The
radiation provides a range of polymers of varying
molecular weight along with combination of biological
effect. For injectable viscosity of 1.5 - 1.9 pa relative to
DMSO correspond to a molecular weight ranging from
60,000 - 100,000 g/mol9. Inside the vas the anhydride
copolymer hydrolyzes in presence of water molecules
in the spermatic uid, this hydrolyzed SMA has a pH
of 4.0 - 4.5. It is also hypothesized that DMSO helps in
the penetration of polymer into the folds of inner wall
of vas deferens and provides retention. According to
patent information, the injection comprises 40 - 60 per
cent (w/v) of SMA providing reason that if less then
40 per cent (w/v), the SMA will freely ow inside the
vas, however, if more than 60 per cent (w/v), it will
be highly viscous, difcult to inject. Hence, the ratio
suggested for styrene and maleic anhydride was 1.2:1
and 1:1. Other ratios were also suggested with different
mode of action such as 2:1 which would function
mainly as an occlusion device, whereas, 1.5:1 would
provide excessive charge but reduced stability9. Further
details were produced in subsequent research papers
such as gamma irradiation of monomers at 0.3 Gy/s at
37oC with a total dose of 2.4 Gy and precipitation of
copolymer by petroleum ether and soxhelt distillation
using 1,2-dichloroethane25. In another study, 50 mg of
SMA was dissolved in 100 µl of DMSO (1:2) to obtain
RISUG hydrogel and kept for 48 h to dissolve properly
and then precipitated with distilled water14. Similarly,
a study detailed about the time of purging in N2 after
the SMA dissolved in ethyl acetate, which was 5 min15.
Smart RISUG contained two new components Fe3O4
and Cu, by adding 10 per cent (w/v) iron oxide and 5
per cent copper powder in mixture of SMA and DMSO
in 1:30 ratio (Fe3O4-Cu-SMA-DMSO), in addition,
the solution was continuously stirred for 48 h at 35oC,
however, molecular weight for this compound was not
provided15. Another paper in the same year by the same
authors indicated molecule bindings as SMA-Fe3O4-
Cu-DMSO which differed from the binding shown
earlier15,26. In one study styrene and maleic anhydride
mixture in ethyl acetate was shown in 1:1:7 ratio
indicating an optimum total dose of gamma irradiation
as 2.4 kGy, which was used for RISUG synthesis; this
statement differed from the previously reported 2.4
Gy irradiation27. Jha et al27 also reported that dose rate
and total dose interrelation plays an important role in
irradiation based polymeric drug and its acceptability in
biological use. Whether RISUG results in any toxicity
is not reported extensively, however, genotoxicity,
mutagenicity and carcinogenicity studies have been
carried out at many centers in India28. A recent study
on Wistar albino rats for a long-term evaluation of
genotoxicity of RISUG has revealed that it is unlikely
to produce any DNA damage following injection of
RISUG and its reversal29. Further studies are required
in this area for effective quality control of RISUG.
Mode of action
The US patent reports that the manner by which
the contraceptive works is not fully understood, and

it still continues to be ambiguous. However, a few not
completely established methods have been reported
regarding its mode of action in the following years,
such as, partial occlusion, complete occlusion, pH
lowering, charge effect, sulphur moiety and protein-
SMA agglomerate9.
Partial occlusion: As explained earlier, the ratio of
styrene and maleic anhydride and the concentration
of SMA in DMSO perform differently in a bio-
environment. Higher concentration of styrene and/
or higher w/v concentration of SMA will completely
occlude the vas deferens whereas lower concentration
of styrene alone and SMA as a whole would freely ow
inside the vas and could be easily ushed out9. This
implies that the dose regimen of 1:1 of styrene and
maleic anhydride and 40 - 60 per cent (w/v) of SMA in
DMSO would not completely block the vas deferens.
In phase III clinical trial, neutral α-glucosidase, a
biochemical marker for epididymis, was estimated to
be gradually decreasing by nearly 8-folds in a period
of 6 months post-injection, but not completely absent
in the ejaculated seminal plasma, and on the other
hand normal range of acid phosphatase and fructose
levels were recorded8. The authors concluded that the
mode of occlusion by 60 mg styrene maleic anhydride
dissolved in 120 µl of DMSO (1:2) was ‘partial’ and not
‘complete’8, however, no further study was published
to support this statement.
Complete occlusion: Studies5,30-34 have been
consistently showing evidence of azoospermia in
animal models, with doses in exactly same 1:2 ratio.
In one study, following vas occlusion by RISUG in
rat for short-term complete sterility was noted on 7th
day post-injection and azoospermia after 90 days post-
injection5. Azoospermia can only be achieved if there
are no sperms in ejaculates, in case of vas occlusion
with RISUG, where all control were proven fertile
subjects, can only show azoospermia if the vas is
completely occluded. However, before azoospermia
the animal models have shown aberration in the
ejaculated sperm morphology, which make no sense
if it is not passing through the occlusion or came in
contact of RISUG5. In case of vasectomy, the sperms
are still observed in the ejaculates for up to 6 months
post-vasectomy and there have been numbers of
pregnancies reported even after vasectomy, which
entail that there must be a pool of sperm load in distal
vas deferens. Hence it is logically admissible that the
vas may be completely occluded following injection
of RISUG in 1:2 ratio, however, further studies are
required. Flickinger35 provided evidence which was
contradicting to the hypothesis of complete occlusion,
the study showed that in case of vasectomy in rabbit
the sperms accumulated in the male duct system for
six months and later large numbers of lysosomes in
the epithelium of the caput and cauda epididymis and
the proximal vas deferens were perhaps degenerated
and engulfed by the corresponding epithelial cells. The
study also reported that due to the stress of sperm load
epithelial cells of cauda epididymis and proximal vas
deferens showed folding of the epithelial and formation
of long apical projections which was not observed in
case of vas occlusion by RISUG.
pH lowering effect: In 1985, Carr et al36, published a
study on effects of pH on sperm motility in different
species, and provided interesting information with
respect to the RISUG. The study showed varied effects
on motility at physiological pH and non-physiological
pH. The inhibition of cauda epididymal sperm motility
of dog (pH 5) was at higher pH comparing to that of
the bull (pH 4), addition of 15mM lactate shifted the
pH to nearly 2-fold higher, whereas rat, hamster and
guinea pig showed inhibition at pH 4 with or without
lactate. They also reported about human ejaculated
sperm which showed gradual increase in motility with
increasing pH, at pH 7.5 it showed optimal motility,
addition of lactate inhibited sperm motility completely
at pH 4, however, without lactate the ejaculated sperm
showed motility. The epididymal quiescence factors are
important for sperm motility and are greatly inuenced
by pH of semen, weak acids such as lactate mimics the
inhibitory effect of cauda epididymis37. The hydrolyzed
RISUG in the vas deferens is claimed to have a pH of
4.0 - 4.5 which is likely to lower the motility but would
it completely immotile the sperms is an important
question9. The damages reported in RISUG treated
sperms do not show a pH lowering effect, however,
whether or not it affected motility is not clear.
Charge effect: ‘Stability of the suspension is a
function of the charge’, this theory has highlighted
that the velocity of small particles in a suspension in
electric eld has a correlation with the phenomenon of
occulation where the velocity is minimum38. In 1903,
Lillie39 showed that in a medium, near the neutral point
sperms tend to migrate towards the positive pole and has
concluded that sperm has a negative charge. Walton’s
study showed that with increasing pH the migration
of sperm towards cathode decreases, it also indicate
that greater velocity of sperm in alkaline medium will
enable a mutual repulsion due to the charge38. Sperm
surface charge has always given importance assuming
66 INDIAN J MED RES, NOVEMBER (SUPPL.) 2014

its signicance in capacitation and fertilization of egg.
Using positively charged ferric oxide it is possible to
locate species specic negative charge distribution
on different region of sperm surface40. It is to be
noted that in seminal plasma during capacitation, the
process of removal of surface protein occurs which
eventually reduces the net negative charge on sperm
surface41. It is known that RISUG has an acidic pH
but it should still contain a positive charge to disturb
the claimed negative charge of sperm. According to
Guha9, the anhydrons copolymer, when injected in to
the vas deferens, hydrolyzes in the presence of water
molecules in the spermatic uid. The formed hydride
generate positive change which attract negatively
charged sperms and thus results in to membrane charge
imbalance. This theory has not been studied very well
and requires attention. Another hypothesis given by
Guha9 is that upon conversion to hydride, chloride ions
are no longer kept out which allows water to ow in
and cause swelling of plasma membrane and rupturing.
Guha also reported that the presence of water in the
spermatic uid generated loss of charge; however,
more studies are required in this area. Guha9 also claims
that the free owing charge would not have similar
effect on sperm; hence, the RISUG has the property of
membrane bound positive charge9.
Oxidative stress: Damaged acrosome, loss of
segmented columns and numeric aberration in centriole
of the neck, degeneration of mitochondrial sheath and
axoneme in the mid-piece and absence of plasma
membrane in the mid-piece and tail are the normal
damages found in RISUG treated sperms following vas
occlusion30,42. These damages are very much similar to
that of the damages caused by oxidative stress. The
generation of excess of intracellular or extracellular
reactive oxygen species (ROS) such as, O2¯, H2O2,
ROO•, OH• is associated with many cell damages,
including morphological defects, DNA fragmentation,
lipid peroxidation, decrease in acrosome reaction and
fusiogenic ability and impaired fertilization43-46. Human
spermatozoa are specically vulnerable to oxidative
stress due to its unsaturated fatty acid containing plasma
membrane, particularly docosahexaenoic acid. Damage
in plasma membrane of RISUG treated spermatozoa
could represent an oxidative damage47 (Figure).
When ROS attacks the double bonds associated with
these unsaturated fatty acid, lipid peroxidation chain
reaction becomes operational, which leads to loss of
membrane uidity and loss of sperm function. Lipid
peroxidation (LPO) is most common expression of
oxygen activation which is catalyzed by Fe3+, Cu+
and O2¯ 47-49, in general, the most signicant effect of
LPO in cells is perturbation of membrane structure
and function. Besides membrane effects, LPO can also
damage DNA and protein through oxidation of DNA
bases, however, there is no published report on DNA
damage in sperm treated with RISUG50. The oxidative
damage also occurs in mitochondria and mitochondrial
Fig. Diagrammatic representation of reactive oxygen species (ROS) and total antioxidant capacity (TAC) localization in human sperm. SOD,
superoxide dismutase.
LOHIYA et al: RISUG AS A POTENTIAL MALE CONTRACEPTIVE 67

DNA, in addition, the redox status of spermatozoa
is also likely to affect phosphorylation and ATP
generation51. The stimulation of NADPH-dependent
ROS generation in human appears to regulate the
acrosome reaction through tyrosin phosphorylation.
Two ROS-generating systems have been introduced,
an NADPH oxidase-like system at the sperm plasma
membrane level, and a sperm diaphorase which is an
NADH-dependent oxido reductase located in the mid
piece and integrated in to the mitochondrial respiratory
system of the sperm52. Glutathione peroxidase a
selenium-containing antioxidant, has been shown
to have a vital role in sperm mitochondria54. It is
reported by Calvin et al54, that selenium in rat sperm
is associated with cysteine-rich structural protein of
the mitochondrial capsule. Glutathione plays a likely
role in sperm nucleus decondensation and microtubule
formation in the ovum, which eventually affects
pregnancy55. The seminal plasma is a rich source of
antioxidants which restrict the oxidative insults, such
as, vitamin C, α-tocopherol, tyrosine, hypotaurine,
uric acid, albumin, superoxide dismutase (SOD) and
glutathione peroxidase 5 (GPx5), research shows that
long-term exposure to seminal plasma is detrimental to
motility and sperm survival56,57. SOD increases DNA
fragmentation of sperm, rather than decreasing the
oxidative stress58. Excess free radicals generated by
the spermatozoa of infertile patients reect underlying
defect in Sertoli cells. It is known that a complete
healthy morphology of sperm depends on oxidative
stress status (OSS) which is between ROS and total
antioxidant capacity (TAC). Observed damages in
RISUG are similar to that of the damages caused by
imbalance of oxidative stress status (OSS), it indicates
that there could be a possible crosslink between pH
lowering, charge distribution and oxidative stress
status42.
Clinical trials
After approval from the drug regulatory agency of
India (DCGI), the phase I clinical trial was initiated
at a few centers. Male volunteers were enrolled who
were injected with RISUG and became sterile for many
years. The longest duration of the RISUG bearer was
more than 10 years. The phase I and phase II clinical
trials were published in 1993 and 19976,7. An extended
phase III clinical trial has been launched and a report
has been published in 20038.
Phase I: Healthy adult male volunteers with normal
reproductive system were subjected to RISUG
injection. Following medical examination, about 7 mm
incision was made in the scrotal skin and the vas was
exposed and a 23-gauge needle was inserted pointing
distally and different doses of polymeric drug were
injected. After one week interval following injection
clinical assessment and semenology was periodically
performed6. The available information about the
subjects was for more than two years. Of the 38
subjects, none was undergone semen volume, pH,
sperm motility, viability, morphology and biochemical
analysis. The only information available from phase
I clinical trial was sperm count and behavioural
study. Injection of 5 - 40 mg SMA did not induce
contraception in any subjects, however, from 60 - 140
mg SMA showed varied responses which eventually
led to azoospermia. Best results were seen for 70 mg
SMA dose which showed azoospermia in nearly three
weeks and the subjects stayed azoospermic for 292
days6 (Table).
Phase II: A total of 12 healthy male volunteers were
similarly operated as in phase I and injected with xed
dose of 60 mg of SMA. Pre- and post-treatment semen
examinations were carried out which included sperm
count, motility and morphology. Individual data for
semen analysis were examined for a period of nearly
one and half years. The administration of 60 mg of
SMA resulted in azoospermia beyond 12 months and
an immediate contraceptive effect was seen7 (Table). A
two year clinical efcacy trial with variable doses (40,
50, 60, 65 and 70 mg of SMA) of RISUG was carried out
in 20 subjects who were other than those inducted into
the formal phase II study12. Subjects were monitored for
the maximum of 1407 days, among 20 subjects one had
a normal child after 145 days post-injection, reason for
which was given as slippage during injection. Report
suggested that all subjects maintained good health
during the course of vas occlusion with RISUG, except
one case which showed pelvic inammatory disease,
later treated successfully. It was estimated that dosages
ranging from 40 to 70 mg of SMA were effective in
giving >2 years of fertility control regardless of their
azoospermic or non-azoospermic stage.
Phase III: An extended phase III clinical trial is
ongoing. Chaki et al8, showed a short-term evaluation
of semen and accessory gland function. The number of
subjects was 25 out of 141 enrolled volunteers and the
report was limited to a period of 6 months8,28. Healthy
adult male volunteers were subjected to RISUG
administration, a dose of 60 mg SMA dissolved in
120 µl of DMSO (1:2) was administered. Semen and
biochemical analysis were done for a period of six
months post-injection. The results were predominantly
68 INDIAN J MED RES, NOVEMBER (SUPPL.) 2014

Table. Clinical trials conducted on RISUG: Summarized information
No. of
Subjects
Dose
regimen
Semen volume (ml) Sperm count
(million/ml)
Motility (%) Sperm mor-
phology
Semen biochemical parameters
Fructose µ/Mol/eja.
(estimated range)
Acid phosphate U/ml
(estimated range)
α-glucosidase mU/ml
(estimated range)
Phase I638 5 to 140
mg
not reported For 60-140
mg dose
azoospermia
was reported
during 20-
389 days post
injection
Not reported Not reported Not reported Not reported Not reported
Phase II712 60 mg not reported
All subjects
were azoo-
spermic within
5-243 days
Duration motility
Head defect,
cytoplasmic
droplet and tail
defect have
been observed
Not reported Not reported Not reported
10-193
days
sluggish-
zero- azoo-
spermic
Extended
Phase
III8
25 60 mg Period volume
(ml)
All subjects
were
azoospermic
within 30-120
days
Not reported Immotile
sperms with dif-
ferent abnormal
morphology
were observed
after RISUG
injection
Period Concentra-
tion
Period Concentra-
tion
Period Concentra-
tion
Pre-treat-
ment
3.0 ± 0.5 Pre-treat-
ment
15 ± 3 Pre treat-
ment
130 ± 50 Pre-treat-
ment
9-80
Post treat-
ment 60
days
2.0 ± 0.5
Post treat-
ment 60
days
17 ± 2
Post treat-
ment 60
days
180 ± 10
Post treat-
ment 60
days
0-8
Post treat-
ment 180
days
1.75 ± 0.5
Post treat-
ment 180
days
16.8 ± 2.2 Post treat-
ment 180
days
170 ± 10 Post treat-
ment 180
days
0-2.5
Values are mean ± SD
LOHIYA et al: RISUG AS A POTENTIAL MALE CONTRACEPTIVE 69

showing immotile and abnormal spermatozoa in all
subjects after injection of RISUG. Abnormalities
such as bent and coiled tail, amorphous head with
large elongated tail, double and tepering head, etc.
were found. Immature germ cells were reported in all
samples including azoospermic samples. Further, the
volume of semen was also distinctly declined in all
subjects. In addition, the semen biochemistry indicated
normal functioning in both prostate and seminal
vesicle, however, none of the subjects showed neutral
α-glucosidase activity within normal range, it was
reported to be signicantly lowered. The presence of
immature germ cells, occasional sperm or both, was
considered as only partial patency. The sperms escaped
from the occlusion were found morphologically
damaged and without any motility8 (Table).
Reversibility
At higher pH solution RISUG tend to dissolve
easily, pH ranging from 8-9 in a solution mainly
unstable the components of RISUG and allows it to
unbound from the wall of vas deferens. DMSO and
sodium-bi-carbonate (NaHCO3) are used to dissolve
SMA2,59. Reversibility of RISUG continued to be an
issue, studies carried out in Langur monkeys indicated
that complete reversal was achievable by non-
invasive reversal technique, the procedure involved
percutaneous squeezing of the vas deferens, along with
synchronized application of electrical stimulation to
the vas segment, supra-pubic percussion and pre-rectal
digital massage of the ampullary region of the vas, to
loosen the intravasal SMA deposits and push them
towards urethera30-33,60. However, the same technique
is difcult for use in human, as the human vas is
difcult to palpate beyond scrotum. Hence, DMSO and
NaHCO3 have been used to ush the RISUG out from
vas deferens through urethra. The reversal was carried
out by ushing the RISUG from urethra by injecting
200 - 500 µl of DMSO or 5 per cent NaHCO3 in to
the vas deferens5,59. In our earlier study a short-time
vas occlusion and reversal was conducted to evaluate
the teratological aberration and reversibility of RISUG
by DMSO in Wister albino rats. It was reported that
following vas occlusion the vaginal smear showed
detached heads and tails, acrosomal damage, bent in
mid-piece, bent tail and morphological aberrations,
which returned to normal within 90 days of reversal
with DMSO and 100 per cent fertility was recorded5.
Larger amount of DMSO can have adverse effect on
relative tissues or skin, hence, NaHCO3 could be a
better alternative for lessening the RISUG binding to
the epithelial cells of vas deferens and to ush out.
Other concerns
RISUG’s cell damaging property is not only
limited to sperm but it also damages bacteria and
possibly viruses. Another component of styrene
maleic anhydride has bactericidal property such as
Poly(styrene-alt-maleic anhydride)-4-aminophenol.
This is being considered as a modied RISUG for
female which acts as a contraceptive and bactericidal.
RISUG has been reported for exfoliation of the
epithelial cells of the vas deferens which recovers in
a few months after reversal; however, more attention
is required to conrm the time of recovery and extent
of exfoliation13. Long-term treatment with RISUG
and its impact on individual’s reproductive health and
offspring following reversal along with carcinogenicity
and mutagenicity, are major concerns. The teratological
evaluation of F1 and F2 progenies after reversal must be
carefully studied, there are only animal data available
showing no physical abnormalities in F1 progeny5.
Conclusion
At present, there are limited approaches available
for men. The search of an ideal contraceptive for male
is still an elusive goal. Among all available approaches
for men, the vas based methods are mostly appreciated,
vasectomy accounts for more than 20 per cent of the
current methods of contraception in male. The RISUG
has surely created a new concept of contraception with
great feasibility and long lasting sterility. Unfortunately,
the advancement of this injectable polymer is slow,
the clinical trials are not providing enough robust
conclusions. It is, however, understandable that
treatment with RISUG and its follow up in human
subjects is a difcult task for reasons such as, subjects
normally do not want to talk about it after injection
unless there are any complications, the follow up must
be done for a long time to report polymer’s efcacy and
side-effects, after reversal the F1 generation is difcult
to track, and overall the clinical trial for RISUG is
long run to ascertain its contraceptive and reversal
effectiveness. Although many leads have been taken
towards making of an effective male contraceptive,
many of these failed, many of these succeeded at rst
and then failed, many are still struggling for recognition,
RISUG on the other hand, provides a hope which has a
slow pace and drawbacks but it is in a right direction.
Acknowledgment
The nancial support from the Indian Council of Medical
Research (ICMR) and Ministry of Health and Family Welfare,
Government of India, New Delhi, is acknowledged.
70 INDIAN J MED RES, NOVEMBER (SUPPL.) 2014

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Reprint requests: Prof. N.K. Lohiya, Centre for Advanced Studies, Department of Zoology, University of Rajasthan
Jaipur 302 004, Rajasthan, India
e-mail: lohiyank@hotmail.com; lohiyank@gmail.com
72 INDIAN J MED RES, NOVEMBER (SUPPL.) 2014