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Effect of a copper wire as an IUD on some rat uterine enzymes
EFFECT OF A COPPER WIRE AS AN IUD ON SOME RAT UTERINE ENZYMES
S. Chatterji and K.R. Laumas Department of Reproductive Biology All-India Institute of Medical Sciences New Delhi-110016, India
ABSTRACT To elucidate the effect of copper as an IUD, its effect on uterine weight, protein content and some enzyme activities has been studied. The weight, protein content and synthesis of proteins in the IUD horn compared with the control horn were increased in ovariectomized, ovariectomized and estrogen, progesterone and estrogen f progesterone supplemented animals, and also in the cycling and pregnant animals. Both the acid and alkaline phosphatases and 8-glucuronidase were higher in the copper IUD horn than in the control. However, under the same experimental conditions, the lactic dehydrogenase activity was decreased in the copper IUD horn. The present findings on the effect of copper IUD in increasing the weight, protein content, proteins synthesising capacity and in causing alterations in estrogen dependent enzymes suggest that the Copper IUD may be producing an estrogen-like effect in the rat uterus.
Accepted
JANUARY
for publication
1974 VOL.
November
9 NO.
1
15,
1973
CONTRACEPTION
INTRODUCTION The introduction of copper into the polyethylene IUD has led to the development of a copper IUD which reduced the pregnancy rate, bleeding and expulsion rate as compared to the commonly used Lippes loop made out of polyethylene (2, 23). A number of clinical trials are underway to test the clinical efficacy and the increased benefits of this copper IUD. Zipper -et al. (3) suggested that more effective antifertility action of copper may be due to its action on several sites such as endometrial milieu, tubal mucosa, endometrium, cervical mucus and also possibly its direct effect on the spermatozoa itself. Chang and Tatum (4) have reported that in the presence of copper, morula failed to develop into blastocyst and the effect has been found to be a total change in the intrauterine environment (5). In spite of these studies,the antifertility action of copper IUD is not yet fully understood. Copper is an important component of many enzymes and co-factors and thus it would be of importance to see whether copper affects any of the enzymes present in the uterus. This paper describes some of our results on the weight of the uterus, its protein content, protein synthesis, lactic dehydrogenase,&glucuronidase, acid and alkaline phosphatase activities of the rat uterus in the presence of a copper IUD in the hormone treated cyclic and pregnant animals.
MATERIALS ANDMETHODS Animals : Three-month-old adult female rats of the Wistar strain were used this study. The animals were kept on pellet diet obtained from Hindustan Levers Ltd., Bombay. They were ovariectomized under ether anaesthesia and a copper wire (-004 inch diameter) was inserted through the antimesometrial wall of right uterine horn according to the procedure of Chang et al. (5) . In another group, copper wire was inserted keeping the ov%_z intact. The animals were kept for 3 weeks postoperatively. Ovariectomized rats with copper ILJDin one horn Hormone treatment: Group I animals received subcutaneously were divided into four groups: 0.2 ml of olive oil; Group II received 2.0 ug estradiol-17B; Group III, 2.0 mg progesterone; Group IV, 2.0 ug estradiol-178 and 2.0 mg progesterone for seven days. Twenty-four hours after the last injection, the animals were killed by cervical dislocation. Both the control and IUD horns were excised and were weighed separately after removing the adhering tissues.
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Intact adult female rats Experiment on cycling rats: with copper IUD in one horn were followed for their estrous cycle by examining vaginal smears daily. Groups of animals on the estrus and diestrus day of their cycle were killed and uteri removed as described earlier (1). In this group Experiment on day 4 and 5 of pregnancy: intact adult female rats normally cycling and containing copper IUD in one horn were mated with male rats of proven fertility. Vaginal smears were taken and examThe day on which ined for the presence of sperms. spermatozoa were found in the smear was designated as day 1 of pregnancy. The animals were sacrificed on the morning of day 4 and 5 of pregnancy. The control and IUD horns were then removed. Homogenisation and centrifugation: The weighed control and IUD horns were separately homogenised in an all glass homogeniser in 3 ml of double distilled water and the homogenate was centrifuged at O°C in an International Centrifuge PR-2 at 1500 r.p.m. for 10 minutes in order to remove the cell debris. The supernatant was used for the estimation of different enzymes. Estimation of enzymes: assays were carried All enzyme out in duplicate on the above supernatant. Lactic dehydrogenase (LDH) was estimated according to Cabaud and Wroblewski (6). The unit of enzyme activity (LDH Unit) was expressed as the amount which caused an initial oxidation of one micro-mole of pyruvic acid/min. The results are expressed as total unit activity/horn/100 g body weight. B-Glucuronidase was assayed according to Fishman and Bernfeld (7). The unit of enzyme activity was expressed as equivalent to lug of phenolphthalein liberated in 1 hr at pH 5.0 and 37OC from .OOl M phenolphthalein mono-B-glucuronide solution in the presence of 0.028% DNA in a total volume of 1 ml. The results are expressed as total unit activity/horn/100 g body weight. Acid and alkaline phosphatases were assayed according to King and Wootton (8). The King Armstrong Unit of phosphatase was determined as the amount of the enzyme which liberated 1 mg of phenol in a given time under the conditions of the test. The results are expressed as total activity/horn/100 g body weight. Incorporation of dl-leucine-l-14C into proteins: The control and IUD uteri were separately incubated with dl-leucine-1-14C (2 PC) for 2 hr in Tris buffer medium (pH 7.4) at 370C. The uteri after incubation were homogenised in 0.6 N perchloric acid and fractionated
JANITARY 1974
VOL.
9 KO. 1
0;
CONTRACEPTION into lipid and proteins according to the modified method of Laumas and Yadava (9) . The protein fraction was divided into two parts. One part was taken for counting and the other for protein estimation by the method of Lowry et al. (10). The results have been expressed as DPM/horn/lOO g body Ki’ight. Statistical analysis: The control and copper IUD horns in the same group were compared by applying paired ‘t’ test to the difference of the control and IUD horn. The student’s ‘t’ test was applied to compare the means of two different groups of animals.
RESULTS
EFFECTOF COPPERIUD ONWETWEIGHT ANDPROTEINCONTENT: Table 1 gives the weight and the protein content of the IUD horn which was significantly increased compared with the control horn of the ovariectomized, estrogen, progesterone and estrogen f progesterone treated groups. The increase in protein content paralleled the increase in the uterine weight. Copper IUD also increased the weight and protein content of uterine horns in animals in estrus, diestrus, day 4 and day 5 of pregnancy (Table 2). EFFECTOF COPPERIUD ONTHEUTERINEENZYMES: Hormone treated
animals:
The activity of B-glucuronidase copper IUD horn compared with the horn in the ovariectomised and hormone treated animals (Table The increase in the activity of B-glucuronidase was higher in estrus than in the diestrus stage (Table 4A).
was control 3A). the
The acid and alkaline phosphatases were also increased in the IUD horn compared with the control. The activity of alkaline phosphatase was higher in both horns of the estrogen treated group compared with the corresponding horns of the control group. The activity of acid phosphatase of both horns of the progesterone treated group was higher than the corresponding control and estrogen treated group (Table 3B). The activity of LDHwas lower in the IUD horn of the experimental groups compared with control horn, except the ovariectomised animals where it was slightly higher.
68
.IANITARY 197k
VOT,. 9 NO. 1
c/c = T/T = N.S. =
P co.02 N.S. P qo.02
P so.05
T/T
P <0.02 N.S.
Control horn vs Control horn IUD horn vs IUD horn Not significant
Control/Estrogen Control/Progesterone Control/Estrogen + Progesterone
C/C
P eo.02
P eo.05 P x0.05
P <0.05 P dO.05 P <0.02
T/T
C/C
Number of animals shown in parentheses P *0,05 P ~0.02 Control horn compared with the IUD horn.
STATISTICAL ANALYSIS:
* **
+
50.3 i 6.1
Estrogen + Progesterone (S)+
3.68 f 0.3 *
3.20 k 0.3**
1.86 f 0.37
71.0 f 3.5*
34.2 k 1.2
Progesterone (S)+ 2.03 + 0.43
3.82 ?r 0.5**
2.79 + 1.20
110.4 + 2.7*
67.4 f 3.1
Estrngen (9)+
101.8 f 9.8*
2.06 ? 0.5**
1.00 + 0.40
58.8 i 5.9*
Uterine protein (mg)/Horn/lBO g Body weight IUD horn Control horn
30.3 r 2.9
Uterine weight/Horn/100 g Body weight (mg) IUD horn Control horn
Ovariectomised Control (9)+
GROUP
EFFECT OF COPPER AS AN IUD ON UTERINE WEIGHT AND PROTEIN CONTENT OF CONTROL AND IUD HORNS OF OVARIECTOMISED AND HORMONE TREATED RATS
TABLE 1
3.981 0.50* 2.95+_ 0.32* 3.40* 0.55*
3.05 k 0.20 2.09 i. 0.38 2.98 + 0.19
105.0 f 6.0*
65.3 f 4.1 50.0 k 2.4 71.7 i 1.3
Estrus (9)+
D-4 Pregnancy (S)+
D-5 Pregnancy (8)+
T/T = N.S. =
=
P=O.OS
D-4 Pregnancy/ D-5 Pregnancy
c/c
P co.05
P=O.OS
Diestrus/Estrus
Control horn vs control horn IUD horn vs IUD horn Not significant
N.S.
T/T
C/C
STATISTICAL ANALYSIS:
N.S.
P qo.02
C/C
* P x0.05 Control horn compared with the IUD horn. + Number of animals shown in parentheses.
96.0 + 4.5*
N.S.
P 10.05
T/T
2.80? 0.35*
1.54 k 0.16
62.9 i. 4.3*
47.5 ? 4.1
Diestrus (S)+
80.0 + 6.4*
Uterine Protein/Horn/100 g Body weight(mg) Control horn IUD horn
GROUP
Uterine weight/Horn/100 g Body weight(mg) Control horn IUD horn
EFFECT OF COPPER AS AN IUD ON UTERINE WEIGHT AND PROTEIN CONTENT OF CONTROL & IUD HORN OF CYCLING & PREGNANT RATS
TABLE 2
= = =
c/c
T'/T N.S.
P < 0.05 N.S. N.S.
c/c
N.S. N.S. N.S.
T/T
Controlhorn vs Controlhorn IUD horn vs IUD horn Not significant
Control/Estrogen Control/Progesterone Control/Estrogen + Progesterone
STATISTICAL ANALYSIS: P co.02 N.S. P co.05
C/C
P < 0.02 - Controlhorn ccmrpared with the ILJDhorn
*
80.7 + 3.2
Numberof animalsshown in parenthesis
92.8 t 9.0
Estrogen+ Progesterone(9)+
al.9 f 3.9
100.0 f 1.9
88.4 f.3.8
+
85.1 + 5.3
109.1 + 4.9
84.5 + 4.3
Lacticdehydrogenase activity/ Horn/100g Body weight Controlhorn IuDhom
Progesterone (a)+
Estrogen(9)+
Ovariectomised Control(9)+
GROUP
P co.01 N.S. N.S.
T/T
171.7f 13.5
101.0+ 16.3
284.9+ 30.0
116.9+ 12.4
245.0+ 17.0*
218.8f 19.4*
654.1+ 22.4*
242.8+ 13.7*
Beta-Glucuronidase activity/ Horn/100g Body weight Controlhorn IUDhom
EFFECIOF COPPERAS AN IUD ON UTERINELACTICDEHYDRCXXNASE AND BETA-GLLJCURONIDASE IN THE PRESENCEOF EXOGENOUSESTROGENAND PROGESTERONE IN RAT
TABLE 3A
50.2
12.2 ?r 3.1 22.7 + 7.0
Progesterone (8)+
Estrogen + Progesterone (9)+
P x0.02
c/c
=
T/T = N.S. =
P = 0.05 N.S. P = 0.05
P co.02 N.S. N.S.
Control horn vs Control horn IUD horn vs IUD horn Not significant
T/T
C/C
Control horn vs IUD horn
Control/Estrogen Control/Progesterone Control/Estrogen+Progesterone
-
k 8.7*
f 6.6* * i16.0
C/C
+_ 1.8 * 2.1 f 5.9
9.3 22.0 15.4
N.S. P
T/T
+ 0.6
6.1
9.4*
1.2*
o.s*
45.2 + 10.4*
44.2 +
19.1 +
25.9 f
Acid phosphatase activity/ Horn/100 g Body weight Control horn IUD horn
P = 0.05 P x0.02 P co.02
&16.1*
Number of animals shown in parenthesis
STATISTICAL ANALYSIS:
*
+
100.3
38.5 + 5.6
Estrogen (9)+
84.9
64.7
19.5 f 3.2
Alkaline phosphatase activity/ Horn/100 g Body weight Control horn IUD horn
Ovariectomised Control (9)+
GROUP
EFFECT OF COPPER AS AN IUD ON RAT UTERINE ALKALINE AND ACIP PHOSPHATASES IN THE PRESENCE OF EXOGENOUS ESTROGEN AND PROGESTERONE
TABLE 3B
3
8
= = =
244.4 -t 4.6* 958.2 + 41.0** 519.9 ? 23.0** 265.4 k 15.9**
178.0 + 7.6 441.3 + 7.3 215.4 k 5.4 168.4 ? 5.7
Beta-Glucuronidase activity/ Horn/100g Body weight IUDhom Controlhorn
N.S.
P <0.05
T/T
P co.01 P co.02 P <0.05
T/T P co.01
c/c
P co.05 - Controlhorn comparedwith the IUD horn
Controlhorn vs Controlhorn IUD horn vs IUD horn Not significant
N.S.
Day-4 Pregnancy/ Day-5 Pregnancy
C/C T/T N.S.
P co.02
c/c
Diestrus/Estrus
STATISTICAlANALYSIS:
P co.02
*
523.3 ? 35.0
**
541.4 + 13.0
Day-5 Pregnancy(8)+
456.8 i-26.5
Number of animals shown in parenthesis
537.7 + 12.1
Day-4 Pregnancy(S)+
7.4
269.2 2 42.0*
175.6 t
+
308.7 + 35.6
Estrus (9)+
7.6
160.5 +
Lactic dehydrogenaseactivity/ Horn/100g Body weight Controlhorn IUDhom
Diestms (8)+
GROUP
EFFECT OF COPPER AS AN IUDONUTERINE LACTICDEHYDRCKXNASE AND BETA-GLUCURONIDASE OF INTACT CYCLINGAND PREGNANTRAT
TABLE4A
393.2+ 15.8**
245.5f 16.5**
c/c
=
Controlhorn vs Controlhorn IUD horn vs IUD horn Not significant
P co.02
P 10.05
Day-4 Pregnancy/ Day-5Pregnancy
T/T = N.S. =
75.1 k 1.3
70.0 f 4.5
81.9 k 4.6
32.9 + 2.7
110.5 f 5.4*
123.1f 5.2**
218.9f 6.6**
102.8 k 6.9**
Acid phosphataseactivity/ Horn/l00g Body weight Controlhorn IuDhom
N.S.
P co.01
N.S.
P co.01
Controlhorn comparedwith IUD horn
P co.01
T/T
P co.05
P
*
Diestrus/Estrus
STATISTICAL ANALYSIS:
P co.02
7.6**
329.5 2 8.4**
135.9+
of animalsshown in parenthesis
**
132.3f 2.8
Day-5Pregnancy(S)+
mber
98.8 of 5.3
Day-4Pregnancy(S)+
+
99.1 f
Estrus (9)+ 3.9
39.3 f 4.9
Diestrus(8)+
Alkalinephosphataseactivity/ Horn/100g Body weight Controlhorn IUDhom
EFFECTOF COPPERAS AN IUD ON WERINE ALKALINEAND ACID PHOSPHATASES OF INTAm CYCLINGAND PREGNANTRAT
TABLE 4B
CONTRACEPTION
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1974
VOL. 9 NO. I
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CONTRACEPTION
Intact cycling and pregnant animals: The activity of B-glucuronidase was significantly higher in the IUD horn compared with the control horn of animals in diestrus, esirus, day 4 and day 5 of pregnancy. The enzyme activity was higher in both control and IUD horns of the animals in estrus compared with the corresponding horns of the animals in diestrus and pregnancy (Table 4A). The alkaline and acid phosphatasesshowed significantly higher activitiesin the IUD horn compared with the control horn of animals in diestrus, estrus, day 4 and 5 of pregnancy (Table 4B). Both control and IUD horns showed higher activity of alkaline phosphatase in the estrus than in the diestrus. Alkaline phosphatase activity was higher in both the horns on day 5 of pregnancy compared with the corresponding horns on day 4 of pregnancy. The activity of acid phosphatase on the day of estrus was higher than that in the animals in diestrus and pregnancy. The LDH activity was lower in the IUD horn compared with the control horn in estrus, day 4 and day 5 of pregnancy (Table 4A). In diestrus the activity of LDH was higher in the IUD horn than in the control horn; however,it was not significant. The activity of LDH in the IUD horn of animals on day 4 and day 5 was higher compared with the corresponding horns of the animals in estrus and diestrus. Incorporation incornoration significantly control horn (Table 5 ) .
of dl-leucine-l-14C into proteins: The of dl-leucine-l-14, into nroteins was higher in IUD hornLas compared with the of animals both in estrus and diestrus
DISCUSSION This investigation shows that the presence of a copper IUD in the rat uterus increased the uterine weight, protein content,acid and alkaline phosphatases The effect of the and 6-glucuronidase activities. copper IUD in increasing the weight and protein content of the horn is similar to what has been observed previously with a silk thread as an IUD (1,8,11,12,13). Chang -et al.(5) have suggested that copper acts in the rat uterus by inhibiting growth and development of morulae into blastocysts and,furthermore,copper reduces the response of the endometrium to a decidual reaction. Metallic copper is known to dissolve from a copper IUD
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in vivo andin the presence of uterine secretions in vitro --in weight and protein content of the (14) * The increase copper IUD uterus which was comparable to that of a silk thread IUD suggested that the dissolved couner from the copper IUD may-not be producing a generalised effect in the rat and its increased effectiveness in preventing pregnancy may be due to an increased local effect of the copper IUD on the uterus. Oster (14) has found that copper causes a scission of disulphide bonds resulting in a modification of S - S containing proteins, however, the S - S scission will only occur in molecules which are in intimate and direct contact with the copper surface suggestive of an increased local effect. Manning et al.(lS) have shown that 6-glucuronidase and alkaline phGpKtase are known to be under the influence of estrogens. The enzyme,alkaline phosphatase, seems to be involved in the process of growth and differentiation of new tissue and membrane transport (16). The effect of silk thread as an IUD has been reoorted to increase alkaline phosphatase and B-glucuronidase in the IUD horn compared with the control (1). The present finding of increased activity of B-glucuronidase, alkaline phosphatase and acid phosphatase in the copper IUD horn of the ovariectomised rats and also after treatment with estrogen indicated that it is under some sort of estrogenThis contention is further like effect 11.17.18.19.20). substantiated by the-results of higher 8-glucuronidase and alkaline and acid phosphatase activities in the IUD horn compared with the control horn of animals in diestrus, estrus, day 4 and day 5 of pregnancy. Compared with the rat, the human endometrium in the presence of Cu-T was shown to have decreased activity of alkaline phosphatase and B-glucuronidase during the secretory phase,while in the proliferative phase an increase in protein concentration and an increased acid phosphatase activity was observed (21,22). The results in the secretory phase of the cycle in the human thus appeared at variance with the rat which may be due to species variation. Considerable evidence has accumulated which points to the effect of an IUD at the endometrial level that discourages implantation. Addition of copper to the simple inert IUD enhances this antifertility effect in animals and in humans (2,23). The dissolution of copper from the copper IUD has been suggested to cause nrecinitation of albumin which sticks to-the uterine walls preventing implantation,loss of stickiness of the uterine secretions which may also prevent implantation, the formation of cupric ions which may suppress myometrial contractions and inactivation of enzymes believed to be crucial in reproductive physiology (14). The alterations in the uterine enzyme activities of acid phosphatase alkaline phosphatase and fi-glucuronidase may further contribute to the antiimplantation effect of the copper IUD.
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ACKNOWLEDCXNT Thiswork was supported by grantsfrom IndianCouncilof MedicalResearch, New Delhi,the Ford Foundation, New York,and the WorldHealthOrganization, Geneva.
REFERENCES 1.
Chatterjee, S. and Laumas, K.R.: Studies on some uterine enzymes in the presence of an intrauterine contraceptive device (IUD) in the rat. Biol. Reprod., 7 : 307-313 (1972).
2.
Zipper, J., Medel, M. and Prager, R.: Toxic action of copper and zinc on implantation rates in rabbits. 1n:Abstract of the 6th World Congress on Sterility and Fertility, Tel Aviv, May 20-27, 1968, p.154 (1968).
3.
Zipper, J., Medel, M. and Prager, R.; Suppression of fertility by intrauterine copper and zinc in rabbits. Amer. J. Obstet. 6 Gynec., 105 : 529-534 (1969).
4.
Chang, C.C. and Tatum, H.J.: A study of the antifertility effect of intrauterine copper. Contraception, 1 : 265-270 (1970).
5.
Chang, C.C., Tatum, H.J. and Kincl, F.A.: The effect of intrauterine copper and other metals on implantation in rats and hamsters. Fertil. Steril., 21 : 274 -278 (1970).
6.
Cabaud, P.G. and Wroblewski, F.W.: Calorimetric measurement of lactic dehydrogenase activity. Amer. J. Clin. Path., 30 : 234-236 (1958).
7.
Fishman, W.H. and Bernfeld, P.: Methods in Enzymology. Edited by Colowick,S.P. and Kaplan, M.O., Academic Press Inc., publishers, New York, Vol.1 (Sec. 31): p.262-269 (1962).
8.
King, E.J. and Wootton, I.D.P.: Alkaline and acid phosphatase estimation by determination of phenol with 4-amino-antipyrine. In: Micro analysis in Medical Biochemistry, 3rd Ed., p. 85-88 (1959).
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9.
Laumas, glucose uterine 389-400
K.R. and Yadava, H.S.: Metabolism of(U-14C) in the rat uterus in the presence of an intracontraceptive device, J. Reprod. Fertil., 20 : (1969).
10. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J.: Protein measurement with Folin-phenol reagent. J. Biol. Chem., 193 : 265-275 (1951). 11. Parr, E.L. and Segal, S.J.: The effect of an intrauterine contraceptive device on the weight of the uterus. Fertil. Steril., 17 : 648-653 (1966). 12. Joshi, S.G. and Sujan-Tejuja, S.: Biochemistry of the human endometrium in users of the intrauterine contraceptive device. Fertil. Steril., 20 : 98-110 (1969). 13.
El Sahwi, S. and Moyer, D.L.: Antifertility effects of the intrauterine foreign body. Contraception, 2 : l-28 (1970).
14.
Oster, G.K.: Chemical reactions of the copper intrauterine devices, Fertil. Steril., 23 : 16-23 (1972).
15.
Manning,J.P., Meli, A. and Steinetz, B.G.: Alkaline phosphatase and B-glucuronidase activity in the rat uterus during early pregnancy. J. Endocrinol., 35 : 385-391 (1966).
16
Hester, L.L., Kellett, W.W., Spicer, S.S., Williamson, H.O. and Pratt-Thomas, H.R.: Effect of the intrauterine contraceptive device on endometrial enzyme and carbohydrate histochemistry. Amer. J. Obstet. 8 Gynec., 106 : 1144-1154 (1970).
17
Pincus, G.: In discussion : Chapter on mechanism of contraceptive action of IUCD. Proceedings of the second international conference on intrauterine contraception. Oct. 2-3, 1964, New York, Excerpta Medica Foundation, International Congress, Series No.86, p.211 (1965).
18.
Kar, A.B., Goswami, A., Kamboj, V.P. and Chowdhury,S.R.: Effect of foreign body on the response of the uterus of ovariectomised rats to estrogen. Steroids, 4 : 159-174 (1964).
19.
Kar, A.B.: Mechanism of action of intrauterine contraceptive device. J. Sci. Industr. Res., 27 : 70-77 (1968).
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20.
Laumas, K.R.; Biochemical changes in the uterus in the presence of an intrauterine contraceptive device. Proceedings of the seminar on Review of Research work in India on Intrauterine Contraceptive Devices (K.R. Laumas, Editor). Published by the Indian Council of Medical Research, New Delhi, p. 3-11 (1969).
21
Hagenfeldt, K.: Intrauterine contraception with the Copper-T device 2. Influence on endometrial acid and alkaline phosphatase, 8-glucuronidase and lactic dehydrogenase activities, Contraception, 6, 191 (1972).
22
Hagenfeldt, K: Intrauterine contraception with Copper-T device.4. Influence on protein and copper concentration and enzyme activities in uterine washings. Contraception 6, 219 (1972).
23
Zipper, J., Tatum, H.J., Pastene, L., Medel, M. and Rivera, M.: Metalic copper as an intrauterine contraceptive adjunct to the 'T' device. Amer. J. Obst. Gynec. 105 : 1274-1278, 1969
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VOL. 9 NO. 1
S. Chatterji and K.R. Laumas Department of Reproductive Biology All-India Institute of Medical Sciences New Delhi-110016, India
ABSTRACT To elucidate the effect of copper as an IUD, its effect on uterine weight, protein content and some enzyme activities has been studied. The weight, protein content and synthesis of proteins in the IUD horn compared with the control horn were increased in ovariectomized, ovariectomized and estrogen, progesterone and estrogen f progesterone supplemented animals, and also in the cycling and pregnant animals. Both the acid and alkaline phosphatases and 8-glucuronidase were higher in the copper IUD horn than in the control. However, under the same experimental conditions, the lactic dehydrogenase activity was decreased in the copper IUD horn. The present findings on the effect of copper IUD in increasing the weight, protein content, proteins synthesising capacity and in causing alterations in estrogen dependent enzymes suggest that the Copper IUD may be producing an estrogen-like effect in the rat uterus.
Accepted
JANUARY
for publication
1974 VOL.
November
9 NO.
1
15,
1973
CONTRACEPTION
INTRODUCTION The introduction of copper into the polyethylene IUD has led to the development of a copper IUD which reduced the pregnancy rate, bleeding and expulsion rate as compared to the commonly used Lippes loop made out of polyethylene (2, 23). A number of clinical trials are underway to test the clinical efficacy and the increased benefits of this copper IUD. Zipper -et al. (3) suggested that more effective antifertility action of copper may be due to its action on several sites such as endometrial milieu, tubal mucosa, endometrium, cervical mucus and also possibly its direct effect on the spermatozoa itself. Chang and Tatum (4) have reported that in the presence of copper, morula failed to develop into blastocyst and the effect has been found to be a total change in the intrauterine environment (5). In spite of these studies,the antifertility action of copper IUD is not yet fully understood. Copper is an important component of many enzymes and co-factors and thus it would be of importance to see whether copper affects any of the enzymes present in the uterus. This paper describes some of our results on the weight of the uterus, its protein content, protein synthesis, lactic dehydrogenase,&glucuronidase, acid and alkaline phosphatase activities of the rat uterus in the presence of a copper IUD in the hormone treated cyclic and pregnant animals.
MATERIALS ANDMETHODS Animals : Three-month-old adult female rats of the Wistar strain were used this study. The animals were kept on pellet diet obtained from Hindustan Levers Ltd., Bombay. They were ovariectomized under ether anaesthesia and a copper wire (-004 inch diameter) was inserted through the antimesometrial wall of right uterine horn according to the procedure of Chang et al. (5) . In another group, copper wire was inserted keeping the ov%_z intact. The animals were kept for 3 weeks postoperatively. Ovariectomized rats with copper ILJDin one horn Hormone treatment: Group I animals received subcutaneously were divided into four groups: 0.2 ml of olive oil; Group II received 2.0 ug estradiol-17B; Group III, 2.0 mg progesterone; Group IV, 2.0 ug estradiol-178 and 2.0 mg progesterone for seven days. Twenty-four hours after the last injection, the animals were killed by cervical dislocation. Both the control and IUD horns were excised and were weighed separately after removing the adhering tissues.
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1971, VOL. 9 NO. 1
CONTRACEPTION
Intact adult female rats Experiment on cycling rats: with copper IUD in one horn were followed for their estrous cycle by examining vaginal smears daily. Groups of animals on the estrus and diestrus day of their cycle were killed and uteri removed as described earlier (1). In this group Experiment on day 4 and 5 of pregnancy: intact adult female rats normally cycling and containing copper IUD in one horn were mated with male rats of proven fertility. Vaginal smears were taken and examThe day on which ined for the presence of sperms. spermatozoa were found in the smear was designated as day 1 of pregnancy. The animals were sacrificed on the morning of day 4 and 5 of pregnancy. The control and IUD horns were then removed. Homogenisation and centrifugation: The weighed control and IUD horns were separately homogenised in an all glass homogeniser in 3 ml of double distilled water and the homogenate was centrifuged at O°C in an International Centrifuge PR-2 at 1500 r.p.m. for 10 minutes in order to remove the cell debris. The supernatant was used for the estimation of different enzymes. Estimation of enzymes: assays were carried All enzyme out in duplicate on the above supernatant. Lactic dehydrogenase (LDH) was estimated according to Cabaud and Wroblewski (6). The unit of enzyme activity (LDH Unit) was expressed as the amount which caused an initial oxidation of one micro-mole of pyruvic acid/min. The results are expressed as total unit activity/horn/100 g body weight. B-Glucuronidase was assayed according to Fishman and Bernfeld (7). The unit of enzyme activity was expressed as equivalent to lug of phenolphthalein liberated in 1 hr at pH 5.0 and 37OC from .OOl M phenolphthalein mono-B-glucuronide solution in the presence of 0.028% DNA in a total volume of 1 ml. The results are expressed as total unit activity/horn/100 g body weight. Acid and alkaline phosphatases were assayed according to King and Wootton (8). The King Armstrong Unit of phosphatase was determined as the amount of the enzyme which liberated 1 mg of phenol in a given time under the conditions of the test. The results are expressed as total activity/horn/100 g body weight. Incorporation of dl-leucine-l-14C into proteins: The control and IUD uteri were separately incubated with dl-leucine-1-14C (2 PC) for 2 hr in Tris buffer medium (pH 7.4) at 370C. The uteri after incubation were homogenised in 0.6 N perchloric acid and fractionated
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CONTRACEPTION into lipid and proteins according to the modified method of Laumas and Yadava (9) . The protein fraction was divided into two parts. One part was taken for counting and the other for protein estimation by the method of Lowry et al. (10). The results have been expressed as DPM/horn/lOO g body Ki’ight. Statistical analysis: The control and copper IUD horns in the same group were compared by applying paired ‘t’ test to the difference of the control and IUD horn. The student’s ‘t’ test was applied to compare the means of two different groups of animals.
RESULTS
EFFECTOF COPPERIUD ONWETWEIGHT ANDPROTEINCONTENT: Table 1 gives the weight and the protein content of the IUD horn which was significantly increased compared with the control horn of the ovariectomized, estrogen, progesterone and estrogen f progesterone treated groups. The increase in protein content paralleled the increase in the uterine weight. Copper IUD also increased the weight and protein content of uterine horns in animals in estrus, diestrus, day 4 and day 5 of pregnancy (Table 2). EFFECTOF COPPERIUD ONTHEUTERINEENZYMES: Hormone treated
animals:
The activity of B-glucuronidase copper IUD horn compared with the horn in the ovariectomised and hormone treated animals (Table The increase in the activity of B-glucuronidase was higher in estrus than in the diestrus stage (Table 4A).
was control 3A). the
The acid and alkaline phosphatases were also increased in the IUD horn compared with the control. The activity of alkaline phosphatase was higher in both horns of the estrogen treated group compared with the corresponding horns of the control group. The activity of acid phosphatase of both horns of the progesterone treated group was higher than the corresponding control and estrogen treated group (Table 3B). The activity of LDHwas lower in the IUD horn of the experimental groups compared with control horn, except the ovariectomised animals where it was slightly higher.
68
.IANITARY 197k
VOT,. 9 NO. 1
c/c = T/T = N.S. =
P co.02 N.S. P qo.02
P so.05
T/T
P <0.02 N.S.
Control horn vs Control horn IUD horn vs IUD horn Not significant
Control/Estrogen Control/Progesterone Control/Estrogen + Progesterone
C/C
P eo.02
P eo.05 P x0.05
P <0.05 P dO.05 P <0.02
T/T
C/C
Number of animals shown in parentheses P *0,05 P ~0.02 Control horn compared with the IUD horn.
STATISTICAL ANALYSIS:
* **
+
50.3 i 6.1
Estrogen + Progesterone (S)+
3.68 f 0.3 *
3.20 k 0.3**
1.86 f 0.37
71.0 f 3.5*
34.2 k 1.2
Progesterone (S)+ 2.03 + 0.43
3.82 ?r 0.5**
2.79 + 1.20
110.4 + 2.7*
67.4 f 3.1
Estrngen (9)+
101.8 f 9.8*
2.06 ? 0.5**
1.00 + 0.40
58.8 i 5.9*
Uterine protein (mg)/Horn/lBO g Body weight IUD horn Control horn
30.3 r 2.9
Uterine weight/Horn/100 g Body weight (mg) IUD horn Control horn
Ovariectomised Control (9)+
GROUP
EFFECT OF COPPER AS AN IUD ON UTERINE WEIGHT AND PROTEIN CONTENT OF CONTROL AND IUD HORNS OF OVARIECTOMISED AND HORMONE TREATED RATS
TABLE 1
3.981 0.50* 2.95+_ 0.32* 3.40* 0.55*
3.05 k 0.20 2.09 i. 0.38 2.98 + 0.19
105.0 f 6.0*
65.3 f 4.1 50.0 k 2.4 71.7 i 1.3
Estrus (9)+
D-4 Pregnancy (S)+
D-5 Pregnancy (8)+
T/T = N.S. =
=
P=O.OS
D-4 Pregnancy/ D-5 Pregnancy
c/c
P co.05
P=O.OS
Diestrus/Estrus
Control horn vs control horn IUD horn vs IUD horn Not significant
N.S.
T/T
C/C
STATISTICAL ANALYSIS:
N.S.
P qo.02
C/C
* P x0.05 Control horn compared with the IUD horn. + Number of animals shown in parentheses.
96.0 + 4.5*
N.S.
P 10.05
T/T
2.80? 0.35*
1.54 k 0.16
62.9 i. 4.3*
47.5 ? 4.1
Diestrus (S)+
80.0 + 6.4*
Uterine Protein/Horn/100 g Body weight(mg) Control horn IUD horn
GROUP
Uterine weight/Horn/100 g Body weight(mg) Control horn IUD horn
EFFECT OF COPPER AS AN IUD ON UTERINE WEIGHT AND PROTEIN CONTENT OF CONTROL & IUD HORN OF CYCLING & PREGNANT RATS
TABLE 2
= = =
c/c
T'/T N.S.
P < 0.05 N.S. N.S.
c/c
N.S. N.S. N.S.
T/T
Controlhorn vs Controlhorn IUD horn vs IUD horn Not significant
Control/Estrogen Control/Progesterone Control/Estrogen + Progesterone
STATISTICAL ANALYSIS: P co.02 N.S. P co.05
C/C
P < 0.02 - Controlhorn ccmrpared with the ILJDhorn
*
80.7 + 3.2
Numberof animalsshown in parenthesis
92.8 t 9.0
Estrogen+ Progesterone(9)+
al.9 f 3.9
100.0 f 1.9
88.4 f.3.8
+
85.1 + 5.3
109.1 + 4.9
84.5 + 4.3
Lacticdehydrogenase activity/ Horn/100g Body weight Controlhorn IuDhom
Progesterone (a)+
Estrogen(9)+
Ovariectomised Control(9)+
GROUP
P co.01 N.S. N.S.
T/T
171.7f 13.5
101.0+ 16.3
284.9+ 30.0
116.9+ 12.4
245.0+ 17.0*
218.8f 19.4*
654.1+ 22.4*
242.8+ 13.7*
Beta-Glucuronidase activity/ Horn/100g Body weight Controlhorn IUDhom
EFFECIOF COPPERAS AN IUD ON UTERINELACTICDEHYDRCXXNASE AND BETA-GLLJCURONIDASE IN THE PRESENCEOF EXOGENOUSESTROGENAND PROGESTERONE IN RAT
TABLE 3A
50.2
12.2 ?r 3.1 22.7 + 7.0
Progesterone (8)+
Estrogen + Progesterone (9)+
P x0.02
c/c
=
T/T = N.S. =
P = 0.05 N.S. P = 0.05
P co.02 N.S. N.S.
Control horn vs Control horn IUD horn vs IUD horn Not significant
T/T
C/C
Control horn vs IUD horn
Control/Estrogen Control/Progesterone Control/Estrogen+Progesterone
-
k 8.7*
f 6.6* * i16.0
C/C
+_ 1.8 * 2.1 f 5.9
9.3 22.0 15.4
N.S. P
T/T
+ 0.6
6.1
9.4*
1.2*
o.s*
45.2 + 10.4*
44.2 +
19.1 +
25.9 f
Acid phosphatase activity/ Horn/100 g Body weight Control horn IUD horn
P = 0.05 P x0.02 P co.02
&16.1*
Number of animals shown in parenthesis
STATISTICAL ANALYSIS:
*
+
100.3
38.5 + 5.6
Estrogen (9)+
84.9
64.7
19.5 f 3.2
Alkaline phosphatase activity/ Horn/100 g Body weight Control horn IUD horn
Ovariectomised Control (9)+
GROUP
EFFECT OF COPPER AS AN IUD ON RAT UTERINE ALKALINE AND ACIP PHOSPHATASES IN THE PRESENCE OF EXOGENOUS ESTROGEN AND PROGESTERONE
TABLE 3B
3
8
= = =
244.4 -t 4.6* 958.2 + 41.0** 519.9 ? 23.0** 265.4 k 15.9**
178.0 + 7.6 441.3 + 7.3 215.4 k 5.4 168.4 ? 5.7
Beta-Glucuronidase activity/ Horn/100g Body weight IUDhom Controlhorn
N.S.
P <0.05
T/T
P co.01 P co.02 P <0.05
T/T P co.01
c/c
P co.05 - Controlhorn comparedwith the IUD horn
Controlhorn vs Controlhorn IUD horn vs IUD horn Not significant
N.S.
Day-4 Pregnancy/ Day-5 Pregnancy
C/C T/T N.S.
P co.02
c/c
Diestrus/Estrus
STATISTICAlANALYSIS:
P co.02
*
523.3 ? 35.0
**
541.4 + 13.0
Day-5 Pregnancy(8)+
456.8 i-26.5
Number of animals shown in parenthesis
537.7 + 12.1
Day-4 Pregnancy(S)+
7.4
269.2 2 42.0*
175.6 t
+
308.7 + 35.6
Estrus (9)+
7.6
160.5 +
Lactic dehydrogenaseactivity/ Horn/100g Body weight Controlhorn IUDhom
Diestms (8)+
GROUP
EFFECT OF COPPER AS AN IUDONUTERINE LACTICDEHYDRCKXNASE AND BETA-GLUCURONIDASE OF INTACT CYCLINGAND PREGNANTRAT
TABLE4A
393.2+ 15.8**
245.5f 16.5**
c/c
=
Controlhorn vs Controlhorn IUD horn vs IUD horn Not significant
P co.02
P 10.05
Day-4 Pregnancy/ Day-5Pregnancy
T/T = N.S. =
75.1 k 1.3
70.0 f 4.5
81.9 k 4.6
32.9 + 2.7
110.5 f 5.4*
123.1f 5.2**
218.9f 6.6**
102.8 k 6.9**
Acid phosphataseactivity/ Horn/l00g Body weight Controlhorn IuDhom
N.S.
P co.01
N.S.
P co.01
Controlhorn comparedwith IUD horn
P co.01
T/T
P co.05
P
*
Diestrus/Estrus
STATISTICAL ANALYSIS:
P co.02
7.6**
329.5 2 8.4**
135.9+
of animalsshown in parenthesis
**
132.3f 2.8
Day-5Pregnancy(S)+
mber
98.8 of 5.3
Day-4Pregnancy(S)+
+
99.1 f
Estrus (9)+ 3.9
39.3 f 4.9
Diestrus(8)+
Alkalinephosphataseactivity/ Horn/100g Body weight Controlhorn IUDhom
EFFECTOF COPPERAS AN IUD ON WERINE ALKALINEAND ACID PHOSPHATASES OF INTAm CYCLINGAND PREGNANTRAT
TABLE 4B
CONTRACEPTION
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1974
VOL. 9 NO. I
75
CONTRACEPTION
Intact cycling and pregnant animals: The activity of B-glucuronidase was significantly higher in the IUD horn compared with the control horn of animals in diestrus, esirus, day 4 and day 5 of pregnancy. The enzyme activity was higher in both control and IUD horns of the animals in estrus compared with the corresponding horns of the animals in diestrus and pregnancy (Table 4A). The alkaline and acid phosphatasesshowed significantly higher activitiesin the IUD horn compared with the control horn of animals in diestrus, estrus, day 4 and 5 of pregnancy (Table 4B). Both control and IUD horns showed higher activity of alkaline phosphatase in the estrus than in the diestrus. Alkaline phosphatase activity was higher in both the horns on day 5 of pregnancy compared with the corresponding horns on day 4 of pregnancy. The activity of acid phosphatase on the day of estrus was higher than that in the animals in diestrus and pregnancy. The LDH activity was lower in the IUD horn compared with the control horn in estrus, day 4 and day 5 of pregnancy (Table 4A). In diestrus the activity of LDH was higher in the IUD horn than in the control horn; however,it was not significant. The activity of LDH in the IUD horn of animals on day 4 and day 5 was higher compared with the corresponding horns of the animals in estrus and diestrus. Incorporation incornoration significantly control horn (Table 5 ) .
of dl-leucine-l-14C into proteins: The of dl-leucine-l-14, into nroteins was higher in IUD hornLas compared with the of animals both in estrus and diestrus
DISCUSSION This investigation shows that the presence of a copper IUD in the rat uterus increased the uterine weight, protein content,acid and alkaline phosphatases The effect of the and 6-glucuronidase activities. copper IUD in increasing the weight and protein content of the horn is similar to what has been observed previously with a silk thread as an IUD (1,8,11,12,13). Chang -et al.(5) have suggested that copper acts in the rat uterus by inhibiting growth and development of morulae into blastocysts and,furthermore,copper reduces the response of the endometrium to a decidual reaction. Metallic copper is known to dissolve from a copper IUD
76
JANUARY 1974
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CONTRACEPTION
in vivo andin the presence of uterine secretions in vitro --in weight and protein content of the (14) * The increase copper IUD uterus which was comparable to that of a silk thread IUD suggested that the dissolved couner from the copper IUD may-not be producing a generalised effect in the rat and its increased effectiveness in preventing pregnancy may be due to an increased local effect of the copper IUD on the uterus. Oster (14) has found that copper causes a scission of disulphide bonds resulting in a modification of S - S containing proteins, however, the S - S scission will only occur in molecules which are in intimate and direct contact with the copper surface suggestive of an increased local effect. Manning et al.(lS) have shown that 6-glucuronidase and alkaline phGpKtase are known to be under the influence of estrogens. The enzyme,alkaline phosphatase, seems to be involved in the process of growth and differentiation of new tissue and membrane transport (16). The effect of silk thread as an IUD has been reoorted to increase alkaline phosphatase and B-glucuronidase in the IUD horn compared with the control (1). The present finding of increased activity of B-glucuronidase, alkaline phosphatase and acid phosphatase in the copper IUD horn of the ovariectomised rats and also after treatment with estrogen indicated that it is under some sort of estrogenThis contention is further like effect 11.17.18.19.20). substantiated by the-results of higher 8-glucuronidase and alkaline and acid phosphatase activities in the IUD horn compared with the control horn of animals in diestrus, estrus, day 4 and day 5 of pregnancy. Compared with the rat, the human endometrium in the presence of Cu-T was shown to have decreased activity of alkaline phosphatase and B-glucuronidase during the secretory phase,while in the proliferative phase an increase in protein concentration and an increased acid phosphatase activity was observed (21,22). The results in the secretory phase of the cycle in the human thus appeared at variance with the rat which may be due to species variation. Considerable evidence has accumulated which points to the effect of an IUD at the endometrial level that discourages implantation. Addition of copper to the simple inert IUD enhances this antifertility effect in animals and in humans (2,23). The dissolution of copper from the copper IUD has been suggested to cause nrecinitation of albumin which sticks to-the uterine walls preventing implantation,loss of stickiness of the uterine secretions which may also prevent implantation, the formation of cupric ions which may suppress myometrial contractions and inactivation of enzymes believed to be crucial in reproductive physiology (14). The alterations in the uterine enzyme activities of acid phosphatase alkaline phosphatase and fi-glucuronidase may further contribute to the antiimplantation effect of the copper IUD.
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CONTRACEPTION
ACKNOWLEDCXNT Thiswork was supported by grantsfrom IndianCouncilof MedicalResearch, New Delhi,the Ford Foundation, New York,and the WorldHealthOrganization, Geneva.
REFERENCES 1.
Chatterjee, S. and Laumas, K.R.: Studies on some uterine enzymes in the presence of an intrauterine contraceptive device (IUD) in the rat. Biol. Reprod., 7 : 307-313 (1972).
2.
Zipper, J., Medel, M. and Prager, R.: Toxic action of copper and zinc on implantation rates in rabbits. 1n:Abstract of the 6th World Congress on Sterility and Fertility, Tel Aviv, May 20-27, 1968, p.154 (1968).
3.
Zipper, J., Medel, M. and Prager, R.; Suppression of fertility by intrauterine copper and zinc in rabbits. Amer. J. Obstet. 6 Gynec., 105 : 529-534 (1969).
4.
Chang, C.C. and Tatum, H.J.: A study of the antifertility effect of intrauterine copper. Contraception, 1 : 265-270 (1970).
5.
Chang, C.C., Tatum, H.J. and Kincl, F.A.: The effect of intrauterine copper and other metals on implantation in rats and hamsters. Fertil. Steril., 21 : 274 -278 (1970).
6.
Cabaud, P.G. and Wroblewski, F.W.: Calorimetric measurement of lactic dehydrogenase activity. Amer. J. Clin. Path., 30 : 234-236 (1958).
7.
Fishman, W.H. and Bernfeld, P.: Methods in Enzymology. Edited by Colowick,S.P. and Kaplan, M.O., Academic Press Inc., publishers, New York, Vol.1 (Sec. 31): p.262-269 (1962).
8.
King, E.J. and Wootton, I.D.P.: Alkaline and acid phosphatase estimation by determination of phenol with 4-amino-antipyrine. In: Micro analysis in Medical Biochemistry, 3rd Ed., p. 85-88 (1959).
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9.
Laumas, glucose uterine 389-400
K.R. and Yadava, H.S.: Metabolism of(U-14C) in the rat uterus in the presence of an intracontraceptive device, J. Reprod. Fertil., 20 : (1969).
10. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J.: Protein measurement with Folin-phenol reagent. J. Biol. Chem., 193 : 265-275 (1951). 11. Parr, E.L. and Segal, S.J.: The effect of an intrauterine contraceptive device on the weight of the uterus. Fertil. Steril., 17 : 648-653 (1966). 12. Joshi, S.G. and Sujan-Tejuja, S.: Biochemistry of the human endometrium in users of the intrauterine contraceptive device. Fertil. Steril., 20 : 98-110 (1969). 13.
El Sahwi, S. and Moyer, D.L.: Antifertility effects of the intrauterine foreign body. Contraception, 2 : l-28 (1970).
14.
Oster, G.K.: Chemical reactions of the copper intrauterine devices, Fertil. Steril., 23 : 16-23 (1972).
15.
Manning,J.P., Meli, A. and Steinetz, B.G.: Alkaline phosphatase and B-glucuronidase activity in the rat uterus during early pregnancy. J. Endocrinol., 35 : 385-391 (1966).
16
Hester, L.L., Kellett, W.W., Spicer, S.S., Williamson, H.O. and Pratt-Thomas, H.R.: Effect of the intrauterine contraceptive device on endometrial enzyme and carbohydrate histochemistry. Amer. J. Obstet. 8 Gynec., 106 : 1144-1154 (1970).
17
Pincus, G.: In discussion : Chapter on mechanism of contraceptive action of IUCD. Proceedings of the second international conference on intrauterine contraception. Oct. 2-3, 1964, New York, Excerpta Medica Foundation, International Congress, Series No.86, p.211 (1965).
18.
Kar, A.B., Goswami, A., Kamboj, V.P. and Chowdhury,S.R.: Effect of foreign body on the response of the uterus of ovariectomised rats to estrogen. Steroids, 4 : 159-174 (1964).
19.
Kar, A.B.: Mechanism of action of intrauterine contraceptive device. J. Sci. Industr. Res., 27 : 70-77 (1968).
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20.
Laumas, K.R.; Biochemical changes in the uterus in the presence of an intrauterine contraceptive device. Proceedings of the seminar on Review of Research work in India on Intrauterine Contraceptive Devices (K.R. Laumas, Editor). Published by the Indian Council of Medical Research, New Delhi, p. 3-11 (1969).
21
Hagenfeldt, K.: Intrauterine contraception with the Copper-T device 2. Influence on endometrial acid and alkaline phosphatase, 8-glucuronidase and lactic dehydrogenase activities, Contraception, 6, 191 (1972).
22
Hagenfeldt, K: Intrauterine contraception with Copper-T device.4. Influence on protein and copper concentration and enzyme activities in uterine washings. Contraception 6, 219 (1972).
23
Zipper, J., Tatum, H.J., Pastene, L., Medel, M. and Rivera, M.: Metalic copper as an intrauterine contraceptive adjunct to the 'T' device. Amer. J. Obst. Gynec. 105 : 1274-1278, 1969
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