CONTRACEPTION
MALE MEDIATED ANTIFERTILITY
TERATWRNIC
COMPOUND
N. Sethi,
POTENTIAL
SMA IN RABBIT
EVALUATION
(ORYCTOLAGUS
M.S. (USA), Path. Board R.K. Srivastava, Ph.D. R.K. Singh, Ph.D.
OF
NEW
CUNICULUS)
(USA)
Division of Toxicology Central Drug Research Institute Lucknow - 226 001, India
Abstract The male mediated teratogenic potential of a new antifertility compound, styrene maleic anhydride (SMA), was evaluated in rabbits. No teratogenic potential was observed at the doses of 1.25 mg, 2.5 mg and 5.0 mg used in our experiment. Introduction Previously, studies were done on the antifertility activity and safety evaluation of a new polymer, SMA, in rats and monkeys (1,2). In this communication, the results of evaluation of male mediated teratogenic potential on the above compound are given. All correspondence
to the first author.
Submitted for publication April 10, 1990 Accepted for publication April 30, 1990
AUGUST 1990 VOL. 42 NO. 2
215
CONTRACEPTION
Materials and Methods Twelve adult male rabbits of proven fertility, good health and average body weight, 1.5 + 0.1 kg, were obtained from our animal breeding colony and watched for 10 days. Apparently healthy and disease-free rabbits were randomly divided into 4 groups of 3 animals each. The polymer SMA, dissolved in 0.1 ml DMSO, was injected into the lumen of the vas deferens at dose levels of 1.25 mg (Group II), 2.5 mg (Group III), and 5.0 mg (Group IV) in each vas deferens; while the control rabbits (Group I) were injected with 0.1 ml DMSO per vas deferens. The contraceptive dose (CD) of the polymer in the rabbit is 1.25 mg/vas deferens, and in our testing, the doses of 1 x CD, 2.5 x CD, and 5 x CD were the ICMR (3). taken as per guidelines of Standard procedures (4,5) were followed for the injection of SMA in rabbits. Matins
of animals
The polymer SMA remains in position without any further intervention and does not have the tendency to become dislodged or removed during the ejaculatory process. As the chemical is not absorbed, it remains in place for a long period of time (4). The rabbits were observed for 4 weeks After 4 weeks, the vas for any gross change in behaviour. deferens were flushed with 0.1 ml DMSO following the same procedure as the surgery described earlier. The rabbits were left for another 6 weeks to complete healing (which took almost 2 weeks) and to regain fertility. The fertility of the males was tested (Table I). Female rabbits weighing 2.4 & 0.1 kg obtained from our animal breeding colony were kept in air-conditioned quarters (22 + 2OC), 60% relative humidity, illuminated 12124 hrs from 6:00 A.M. to 6:00 P.M., and supplied with food and water ad libitum. Colony-bred adult nulliparous female rabbits were mated with previously treated proven fertility males in the daytime and copulation was observed, occurrence of insemination was confirmed by the presence of sperm in vaginal smears, and the day when presence of sperm was first noticed was designated day zero of pregnancy. After 3-4 attempts, the pregnant rabbits of each group were weighed and caged individually. Forty-eight females were divided into 4 groups of 12 in each group. Body weight of animals was recorded at days 0, 7, 14, 21, 28, and 30 of the postmating period, and gain in body maternal weight was calculated. The pregnant female rabbits were observed daily throughout the gestation period for a change in appetite, intake, water general observable behaviour, e-g-, irritation/depression, or any vaginal bleeding. On day 30 of gestation, caesarean delivery was performed and the
216
AUGUST 1990 VOL. 42 NO. 2
U
5
E F3
iG
p L
?!I
A
zi :+
Fertility performance of male rabbits treated with saline, solvent and flushed SMA
Open
Open
Open
Open
____________________~~~~~~~~______---_______________~~~~---~~~~~-_~_~~~~~~~__~~~~~-~
100
10
3
30 days
Flushed dissolved polymer
IV
98
10
Flushed dissolved polymer
III
3
30 days
30 days
98
99
10
10
3
3
30 days
Flushed dissolved polymer
DMSO
Solvent only,
II
I (Control)
____________________-___----________________________________________________________
___________________-~~-~~~~~~~~~~~~~~~_____--_____________~~-~~-~~_~~~~~~~~~~~~~~~~~ Patency No. of of the Females with No. of % Treatment into System Implantation Fertility Test Period Males the Vas Groups
Table I.
P
Y
H 2 b
CONTRACEPTION implantation/implantation sites, resorptions, number of live and still born fetuses, and size, corpora lutea, fetus was abnormality of each weight, sex, and gross Half of the fetuses were fixed in 70% alcohol and recorded. examined for visible abnormalities following the sectioning method of Wilson (6), and the remaining fetuses were cleared in 1% KOH solution and stained by Dawson's alizarin red technique (7) for visualization of osseous defects (6). Observations General
observable
and Results
behaviour
The virgin females which were mated earlier with treated males did not show any change in appetite, water or observable behaviour, intake, general e-g., depression/irritation, during the gestation period. There was no vaginal bleeding in either the drug-implanted or control rabbits. Mortalitv None of the pregnant rabbits, either drug-treated or of the control group, died during the gestation period. There observed or morbidity in the pregnant was no lethargy rabbits. Maternal
sain in bodv weiaht
The maternal body weight gain at weekly intervals of different treated as well as control groups was almost the same. There was steady gain in body weight in all animals of all groups (Table II). Size. weisht.
and sex of fetuses
In fetuses of Groups II, II and IV, the mean body weights were decreased as compared to controls. These decreases were not statistically significant. Average fetal body length (crown-rump) and width (mid-body cavity) in treated and control groups were similar in all groups (Table III). Gross observations Abnormalities, such as everted claw, wrist drop, etc., were seen in fetuses of the control as well as treated rabbits. These are commonly observed gross abnormalities in fetuses delivered in our colony. Visceral
observations
For slicing, transverse sections of the fetuses through different regions were taken. No abnormality was recorded
218
AUGUST 1890 VOL. 42 NO. 2
co
Y
5.0 mg
IV
2.233+ 0.213
2.0402 0.088
2.197+ 0.349
2.2312 0.188
2.170+ 0.230
1.969-t 0.101
2.143+ 0.326
2.101+ 0.196
2.319+ 0.196
2.239+ 0.314
2.1255 0.071
2.299+ 0.215
2.407+ 0.175
2.306+ 0.331
2.485+ 0.183
2.3672 0.315
2.277+ 0.110
2.iaa+
0.070
2.437+ 0.232
2.3725 0.220
____________________~---------------------_________________________~~~~~~~~~~~~~~~~~
2.5 mg
III
1.0 ml DMSO per V.D.
1.25 mg
(Control)
II
I
2.4992 0.200
2.3995 0.295
2.296+ 0.104
2.4612 0.232
Maternal Body Weight (Kg), Mean + SD _________________________I_c____________~~~~-~~~~~~~~~~~~~~~ Groups 30 days 0 day 7 days 14 days 21 days 28 days __-_____-___________~~-~-~~~~~~~~~~~-~~~~~__~_~_~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~
mg SMA/V.D./ Rabbit
__-__
Weekly maternal (mean + SD) body weight of rabbits of different groups of SMA teratology study
_____-____-______-__~--~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~
TABLE II.
P
7
Teratological observations of SMA-injected and control rabbits
1.25 mg
III
82
76
68
82
76
68
57
66
71
11
10
11
39.621+ 4.079
40.761+ 4.385
41.1932 4.317
3.312 0.28 3.302 0.25
8.932 0.72
3.292 0.24 9.09+ 0.85
9.29+ 0.73
IV
65 65 54 11 41.8042 9.23+ 3.242 5.0 mg 1.51 0.78 4.432 ____________________~~~~~~~~~~~~~~~~~_~_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
1.25 mg
0.1 ml DMSO per V.D.
II
I (Control)
____________________~~~~~~~~~~~~~-----~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Average Average Average Fetal Body Fetal Body Total No. Length (cm) Width (cm) Fetal Total No. Implan- Total No. No. of Doses (SMA (Crownfrom MidBody Corpora tation Live mg/V.D./ ResorpBody Cavity, Wt.(g), Rump), Groups Rabbit) Lutea Sites Births tions Mean + SD Mean + SD Mean + SD ____________________~~~~~~~~~~~~~~~~~~~~~~~~~~--------------------~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
TABLE III.
CONTRACEPTION in either rabbits. Skeletal
the
control
or
SMA-implanted
progeny
of
the
observations
No skeletal defects were observed in the control or All the bones of the skull, SMA-implanted rabbit tissues. ribs, and legs were normal in all the fetuses. Discussion studies (8,9) in rabbits and dogs have Previous indicated that the use of rigid devices of various types of vas deferens perforation. resulted in numerous cases Devices with flexible end tubes were found to essentially eliminate this perforation problem (lo-13), but these devices, however, were not satisfactory since some of them moved within the lumen of the vas deferens. The problem of leakage around the device is reported to be eliminated when tissue ingrowth occurs (14). More recently, chemical occlusion of vas has been considered (15) and SMA appears to be the most effective method. The polymer SMA can be used to block the lumen of Fertility vas deferens over an extended period of time. control is maintained even when the spermatozoa are present in the semen. The intravasal polymer can be flushed out by a second injection of the solvent DMSO (13). The very small dose of DMSO has not been shown to cause contraceptive or toxic effects (16). It is concluded from this experiment that the polymer does not have any effect on pregnant rabbits and their five the newborns, given in amounts up to times contraceptive dose. Acknowledgements Senior Deputy Director We thank Dr. B.N. Saxena, General, ICMR, New Delhi, for funding the study, and Prof. S.K. Guha, Head, Department of Biomedical Engineering, IIT, New Delhi, for kindly supplying the polymer. Miss Anupma Srivastava is acknowledged for continuous help during the study. References 1.
Safety evaluation of Sethi N, Srivastava RK, Singh RK. a male injectable antifertility agent, styrene maleic Contraception 1989;39:217-26. anhydride, in rats.
AUGUST l!BO VOL. 42 NO. 2
221
CONTRACEPTION 2.
Sethi N, Srivastava RK, Singh RK, Bhatia GS, Sinha N. Chronic toxicity of styrene maleic anhydride, a male monkeys (Macaca mulatta). contraceptive, in rhesus Contraception (submitted for publication).
3.
Standard protocol for preclinical toxicity ICMR 1987. of a new compound/drug/vaccine submitted by Dr. (Mrs.) Division of Toxicology, N. Sethi, Scientist-in-Charge, C.D.R.I., Lucknow, and approved by the Indian Council of Medical Research, New Delhi (1987).
4.
HP, Mahajan Guha SK, Singh S, Ray AR, Misro MM, Injectable chemical Vasudovan P. nonocclusive Contraception 1979;30:467-73. contraception in male-I.
5.
Fine structural studies Kennedy SW, Heidger PM. Anat Ret 1979;194:159-80. rat vas deferens.
6.
for administering agents and Wilson JG. Methods detecting malformations in experimental animals. In: Teratology principles and techniques. (Eds., JG Wilson, J Warkany) University of Chicago Press; Chicago, 1985; 262-277.
7.
A note on the staining of the skeleton of Dawson AB. cleared specimens with alizarine red S. stain. Technol 1926;1:123-24.
8.
Development Free MJ. occlusion device. In: Harper and Row: Maryland,
9.
Zaneveld LJD, Brueschke EE, Burns M, Rodzen R, Wingfield JR, Maness JH. Development of a reversible vas deferens occlusive device. IV. Rigid prosthetic devices. Fertil Steril 1975;26:29-30.
10.
LJD, Rodzen R, Mayerhofer K, Brueschke EE, Zaneveld Burns M, Maness JH, Wingfield JR. Development of a reversible vas deferens occlusive device: Flexible Fertil Steril 1975;26:40-52. prosthetic devices.
11.
Brueschke EE. Reversible Med 1976;17:103-15.
12.
Brueschke EE, Wingfield JR, Zaneveld LID. with intravasal devices. The soft valve. 1976;359-65.
13.
DS. Freeman C, Coffey Sterility in induced by injection of chemical agents Fertil Steril 1973;24:884-90. deferens.
222
of a reversible Control of male 1975;124-29.
occlusive
of the
intravasal fertility.
device.
J Reprod New studies Invest Urol
male into
animals the vas
AUGUST 1990 VOL. 42 NO. 2
CONTRACEPTION 14.
Kraft LA, Polidoro JP, Culver RM, Hahn DW. Intravas studies in rabbits. device on sperm II. Effects fertility and histology of the reproductive output, Contraception 1978;18:239-51. tract.
15.
Chemical Setty BS, Dasgupta PR, Kar AD. vas in rats. Contraception 1972;6:329-34.
16.
Toxicologic update Rubin LF. Ann NY Acad Sci 1983;6-IO.
AUGUST 1990 VOL. 42 NO. 2
of dimethyl
occlusion
of
sulphoxide.
223
MALE MEDIATED ANTIFERTILITY
TERATWRNIC
COMPOUND
N. Sethi,
POTENTIAL
SMA IN RABBIT
EVALUATION
(ORYCTOLAGUS
M.S. (USA), Path. Board R.K. Srivastava, Ph.D. R.K. Singh, Ph.D.
OF
NEW
CUNICULUS)
(USA)
Division of Toxicology Central Drug Research Institute Lucknow - 226 001, India
Abstract The male mediated teratogenic potential of a new antifertility compound, styrene maleic anhydride (SMA), was evaluated in rabbits. No teratogenic potential was observed at the doses of 1.25 mg, 2.5 mg and 5.0 mg used in our experiment. Introduction Previously, studies were done on the antifertility activity and safety evaluation of a new polymer, SMA, in rats and monkeys (1,2). In this communication, the results of evaluation of male mediated teratogenic potential on the above compound are given. All correspondence
to the first author.
Submitted for publication April 10, 1990 Accepted for publication April 30, 1990
AUGUST 1990 VOL. 42 NO. 2
215
CONTRACEPTION
Materials and Methods Twelve adult male rabbits of proven fertility, good health and average body weight, 1.5 + 0.1 kg, were obtained from our animal breeding colony and watched for 10 days. Apparently healthy and disease-free rabbits were randomly divided into 4 groups of 3 animals each. The polymer SMA, dissolved in 0.1 ml DMSO, was injected into the lumen of the vas deferens at dose levels of 1.25 mg (Group II), 2.5 mg (Group III), and 5.0 mg (Group IV) in each vas deferens; while the control rabbits (Group I) were injected with 0.1 ml DMSO per vas deferens. The contraceptive dose (CD) of the polymer in the rabbit is 1.25 mg/vas deferens, and in our testing, the doses of 1 x CD, 2.5 x CD, and 5 x CD were the ICMR (3). taken as per guidelines of Standard procedures (4,5) were followed for the injection of SMA in rabbits. Matins
of animals
The polymer SMA remains in position without any further intervention and does not have the tendency to become dislodged or removed during the ejaculatory process. As the chemical is not absorbed, it remains in place for a long period of time (4). The rabbits were observed for 4 weeks After 4 weeks, the vas for any gross change in behaviour. deferens were flushed with 0.1 ml DMSO following the same procedure as the surgery described earlier. The rabbits were left for another 6 weeks to complete healing (which took almost 2 weeks) and to regain fertility. The fertility of the males was tested (Table I). Female rabbits weighing 2.4 & 0.1 kg obtained from our animal breeding colony were kept in air-conditioned quarters (22 + 2OC), 60% relative humidity, illuminated 12124 hrs from 6:00 A.M. to 6:00 P.M., and supplied with food and water ad libitum. Colony-bred adult nulliparous female rabbits were mated with previously treated proven fertility males in the daytime and copulation was observed, occurrence of insemination was confirmed by the presence of sperm in vaginal smears, and the day when presence of sperm was first noticed was designated day zero of pregnancy. After 3-4 attempts, the pregnant rabbits of each group were weighed and caged individually. Forty-eight females were divided into 4 groups of 12 in each group. Body weight of animals was recorded at days 0, 7, 14, 21, 28, and 30 of the postmating period, and gain in body maternal weight was calculated. The pregnant female rabbits were observed daily throughout the gestation period for a change in appetite, intake, water general observable behaviour, e-g-, irritation/depression, or any vaginal bleeding. On day 30 of gestation, caesarean delivery was performed and the
216
AUGUST 1990 VOL. 42 NO. 2
U
5
E F3
iG
p L
?!I
A
zi :+
Fertility performance of male rabbits treated with saline, solvent and flushed SMA
Open
Open
Open
Open
____________________~~~~~~~~______---_______________~~~~---~~~~~-_~_~~~~~~~__~~~~~-~
100
10
3
30 days
Flushed dissolved polymer
IV
98
10
Flushed dissolved polymer
III
3
30 days
30 days
98
99
10
10
3
3
30 days
Flushed dissolved polymer
DMSO
Solvent only,
II
I (Control)
____________________-___----________________________________________________________
___________________-~~-~~~~~~~~~~~~~~~_____--_____________~~-~~-~~_~~~~~~~~~~~~~~~~~ Patency No. of of the Females with No. of % Treatment into System Implantation Fertility Test Period Males the Vas Groups
Table I.
P
Y
H 2 b
CONTRACEPTION implantation/implantation sites, resorptions, number of live and still born fetuses, and size, corpora lutea, fetus was abnormality of each weight, sex, and gross Half of the fetuses were fixed in 70% alcohol and recorded. examined for visible abnormalities following the sectioning method of Wilson (6), and the remaining fetuses were cleared in 1% KOH solution and stained by Dawson's alizarin red technique (7) for visualization of osseous defects (6). Observations General
observable
and Results
behaviour
The virgin females which were mated earlier with treated males did not show any change in appetite, water or observable behaviour, intake, general e-g., depression/irritation, during the gestation period. There was no vaginal bleeding in either the drug-implanted or control rabbits. Mortalitv None of the pregnant rabbits, either drug-treated or of the control group, died during the gestation period. There observed or morbidity in the pregnant was no lethargy rabbits. Maternal
sain in bodv weiaht
The maternal body weight gain at weekly intervals of different treated as well as control groups was almost the same. There was steady gain in body weight in all animals of all groups (Table II). Size. weisht.
and sex of fetuses
In fetuses of Groups II, II and IV, the mean body weights were decreased as compared to controls. These decreases were not statistically significant. Average fetal body length (crown-rump) and width (mid-body cavity) in treated and control groups were similar in all groups (Table III). Gross observations Abnormalities, such as everted claw, wrist drop, etc., were seen in fetuses of the control as well as treated rabbits. These are commonly observed gross abnormalities in fetuses delivered in our colony. Visceral
observations
For slicing, transverse sections of the fetuses through different regions were taken. No abnormality was recorded
218
AUGUST 1890 VOL. 42 NO. 2
co
Y
5.0 mg
IV
2.233+ 0.213
2.0402 0.088
2.197+ 0.349
2.2312 0.188
2.170+ 0.230
1.969-t 0.101
2.143+ 0.326
2.101+ 0.196
2.319+ 0.196
2.239+ 0.314
2.1255 0.071
2.299+ 0.215
2.407+ 0.175
2.306+ 0.331
2.485+ 0.183
2.3672 0.315
2.277+ 0.110
2.iaa+
0.070
2.437+ 0.232
2.3725 0.220
____________________~---------------------_________________________~~~~~~~~~~~~~~~~~
2.5 mg
III
1.0 ml DMSO per V.D.
1.25 mg
(Control)
II
I
2.4992 0.200
2.3995 0.295
2.296+ 0.104
2.4612 0.232
Maternal Body Weight (Kg), Mean + SD _________________________I_c____________~~~~-~~~~~~~~~~~~~~~ Groups 30 days 0 day 7 days 14 days 21 days 28 days __-_____-___________~~-~-~~~~~~~~~~~-~~~~~__~_~_~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~
mg SMA/V.D./ Rabbit
__-__
Weekly maternal (mean + SD) body weight of rabbits of different groups of SMA teratology study
_____-____-______-__~--~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~
TABLE II.
P
7
Teratological observations of SMA-injected and control rabbits
1.25 mg
III
82
76
68
82
76
68
57
66
71
11
10
11
39.621+ 4.079
40.761+ 4.385
41.1932 4.317
3.312 0.28 3.302 0.25
8.932 0.72
3.292 0.24 9.09+ 0.85
9.29+ 0.73
IV
65 65 54 11 41.8042 9.23+ 3.242 5.0 mg 1.51 0.78 4.432 ____________________~~~~~~~~~~~~~~~~~_~_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
1.25 mg
0.1 ml DMSO per V.D.
II
I (Control)
____________________~~~~~~~~~~~~~-----~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Average Average Average Fetal Body Fetal Body Total No. Length (cm) Width (cm) Fetal Total No. Implan- Total No. No. of Doses (SMA (Crownfrom MidBody Corpora tation Live mg/V.D./ ResorpBody Cavity, Wt.(g), Rump), Groups Rabbit) Lutea Sites Births tions Mean + SD Mean + SD Mean + SD ____________________~~~~~~~~~~~~~~~~~~~~~~~~~~--------------------~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
TABLE III.
CONTRACEPTION in either rabbits. Skeletal
the
control
or
SMA-implanted
progeny
of
the
observations
No skeletal defects were observed in the control or All the bones of the skull, SMA-implanted rabbit tissues. ribs, and legs were normal in all the fetuses. Discussion studies (8,9) in rabbits and dogs have Previous indicated that the use of rigid devices of various types of vas deferens perforation. resulted in numerous cases Devices with flexible end tubes were found to essentially eliminate this perforation problem (lo-13), but these devices, however, were not satisfactory since some of them moved within the lumen of the vas deferens. The problem of leakage around the device is reported to be eliminated when tissue ingrowth occurs (14). More recently, chemical occlusion of vas has been considered (15) and SMA appears to be the most effective method. The polymer SMA can be used to block the lumen of Fertility vas deferens over an extended period of time. control is maintained even when the spermatozoa are present in the semen. The intravasal polymer can be flushed out by a second injection of the solvent DMSO (13). The very small dose of DMSO has not been shown to cause contraceptive or toxic effects (16). It is concluded from this experiment that the polymer does not have any effect on pregnant rabbits and their five the newborns, given in amounts up to times contraceptive dose. Acknowledgements Senior Deputy Director We thank Dr. B.N. Saxena, General, ICMR, New Delhi, for funding the study, and Prof. S.K. Guha, Head, Department of Biomedical Engineering, IIT, New Delhi, for kindly supplying the polymer. Miss Anupma Srivastava is acknowledged for continuous help during the study. References 1.
Safety evaluation of Sethi N, Srivastava RK, Singh RK. a male injectable antifertility agent, styrene maleic Contraception 1989;39:217-26. anhydride, in rats.
AUGUST l!BO VOL. 42 NO. 2
221
CONTRACEPTION 2.
Sethi N, Srivastava RK, Singh RK, Bhatia GS, Sinha N. Chronic toxicity of styrene maleic anhydride, a male monkeys (Macaca mulatta). contraceptive, in rhesus Contraception (submitted for publication).
3.
Standard protocol for preclinical toxicity ICMR 1987. of a new compound/drug/vaccine submitted by Dr. (Mrs.) Division of Toxicology, N. Sethi, Scientist-in-Charge, C.D.R.I., Lucknow, and approved by the Indian Council of Medical Research, New Delhi (1987).
4.
HP, Mahajan Guha SK, Singh S, Ray AR, Misro MM, Injectable chemical Vasudovan P. nonocclusive Contraception 1979;30:467-73. contraception in male-I.
5.
Fine structural studies Kennedy SW, Heidger PM. Anat Ret 1979;194:159-80. rat vas deferens.
6.
for administering agents and Wilson JG. Methods detecting malformations in experimental animals. In: Teratology principles and techniques. (Eds., JG Wilson, J Warkany) University of Chicago Press; Chicago, 1985; 262-277.
7.
A note on the staining of the skeleton of Dawson AB. cleared specimens with alizarine red S. stain. Technol 1926;1:123-24.
8.
Development Free MJ. occlusion device. In: Harper and Row: Maryland,
9.
Zaneveld LJD, Brueschke EE, Burns M, Rodzen R, Wingfield JR, Maness JH. Development of a reversible vas deferens occlusive device. IV. Rigid prosthetic devices. Fertil Steril 1975;26:29-30.
10.
LJD, Rodzen R, Mayerhofer K, Brueschke EE, Zaneveld Burns M, Maness JH, Wingfield JR. Development of a reversible vas deferens occlusive device: Flexible Fertil Steril 1975;26:40-52. prosthetic devices.
11.
Brueschke EE. Reversible Med 1976;17:103-15.
12.
Brueschke EE, Wingfield JR, Zaneveld LID. with intravasal devices. The soft valve. 1976;359-65.
13.
DS. Freeman C, Coffey Sterility in induced by injection of chemical agents Fertil Steril 1973;24:884-90. deferens.
222
of a reversible Control of male 1975;124-29.
occlusive
of the
intravasal fertility.
device.
J Reprod New studies Invest Urol
male into
animals the vas
AUGUST 1990 VOL. 42 NO. 2
CONTRACEPTION 14.
Kraft LA, Polidoro JP, Culver RM, Hahn DW. Intravas studies in rabbits. device on sperm II. Effects fertility and histology of the reproductive output, Contraception 1978;18:239-51. tract.
15.
Chemical Setty BS, Dasgupta PR, Kar AD. vas in rats. Contraception 1972;6:329-34.
16.
Toxicologic update Rubin LF. Ann NY Acad Sci 1983;6-IO.
AUGUST 1990 VOL. 42 NO. 2
of dimethyl
occlusion
of
sulphoxide.
223
