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The effect of hormonal state on the dissolution of copper in the rabbit uterus
THE EFFECT OF HORMONAL STATE ON THE DISSOLUTION OF COPPER IN THE RABBIT UTERUS
J.A. Tobert
Department of Pharmacology, The School of Pharmacy, University of London, Brunswick Square, London WClN 1AX. United Kingdom
ABSTRACT
Since the antifertility effect of the copper IUD is probably related to the release of copper into the uterus, it is important to understand the factors governing this release. In this study the hormonal state of the rabbit is shown to influence the dissolution rate of copper from copper IUDsl the loss of copper during the first 14 days in situ is much greater when the rabbit is oestrous than when it is pseudopregnant or ovariectomised. To obtain information on the effect of the surface area of metal exposed on release rates, devices bearing different areas of copper were inserted into oestrous and pseudopregnant rabbits. In both cases copper loss was approximately proportional to surface area. Deciduomata were observed in the pseudopregnant animals in the vicinity of the device.
Accepted
AUGUST
for publication
1974
VOL.
June 28,
10 NO. 2
1974
203
CONTRACEPTION
INTRODUCTION The addition of copper wire to a plastic IUD results in increased effectiveness (1,2); the copper exerts an antifertility action permitting design of the carrier to be aimed only at reducing expulsions and side effects. It is likely (though not yet proved) that this antifertility action is mediated by copper released into uterine fluid and/or tissues (probably as complexed Cu(I1) It is therefore important to know something of ions). the factors governing the dissolution of the metal. One factor that might be expected to influence the dissolution rate of copper is the environment in which it is placed. To test this hypothesis, copper IUDs were inserted into the uteri of oestrous, pseudopregnant and ovariectomised rabbits and the copper lost from them In addition, to obtain information on determined. the effect of surface area on copper loss, devices bearing two different areas of copper were used. METHODS
AND MATERIALS
Animals New Zealand White virgin female rabbits, Twelve 3-4-S kg, caged singly, were used in this study. Four rabbits were rendered pseudopregnant by intravenous injection of 50 I.U. HCG on the day prior to insertion of the devices. A further four were bilaterally ovariectomised when the devices were inserted, and four more, judged to be oestrous by the condition of the vulva, received no The oestrous and pseudotreatment prior to insertion. pregnant states were confirmed by inspection of the ovaries at laparotomy and at termination of the experiments. IUDS The copper consisted of preweighed rectangular pieces of 99.9% pure 6.5pm foil (Goodfellow Metals), either 2x7mm or 6x7mm, affixed by means of an epoxy resin adhesive (Araldite, Ciba) in a shallow recession in an acrylic rod (Perspex, ICI) 21mm long and 2.4rmn in diameter. The copper then formed a sleeve 2 or 6nun long and the area of metal exposed was 14 or 42mm2. The sleeve in both A hole cases terminated lmm from one end of the rod. was drilled in the other end and a 4/O silk thread was passThe security of the knot was ed through it and knotted. ensured by a sleeve of medical grade silicone tubing (Silastic, Dow Corning), (1Omm long, 1.5mm I.D. x 2mm 0-D.) The devices were disinl pulled over this end of the rod. fected prior to insertion by immersion in 0.2% chlorhexidine solution.
204
AUGUST
1974
VOL. 10 NO. 2
CONTRACEPTION
Surqery Laparotomy was performed in aseptic conditions under pentobarbitone/halothane anaesthesia. The IUDs were inserted via a small antimesometrial incision into the caudal third of the uterine lumen, and secured there by knotting the IUD thread to the suture closing the incision. The device had minimal mobility within the lumen. A 42nun2 device was placed in one horn (right and left alternating in successive experiments) and a 14mm2 device in the other horn, with the exce tion of the ovariectomised group in which only a 42mm Iz device was used, the other horn bearing a similar perspex device without copper. Determination
of copper loss
Fourteen days after surgery the animals were killed and the IUDs removed. The copper remaining on them was dissolved in nitric acid, which was then buffered and diluted with sodium acetate to give solutions containing 2-6pg/ml Cu at pH 4.6. The copper concentration was found by measuring the absorbance at 4321-m of the complex formed with diethanolamine bis(2-hydroxyethyl)dithiocarbamate (3). The copper loss during the fourteen days in situ was obtained by subtraction of the mass remaining from the mass originally present. Histoloqy Some of the uteri were fixed in formalin, embedded in paraffin wax, sectioned and stained by haematoxylin and eosin. Statistical
analysis
The significance of differences tested by Student's t test.
between
means was
RESULTS The copper losses during the 14 days in situ are shown in Table I. The mean loss in the oestrous animals was significantly greater than that in the pseudopregnant group ( p < 0.001 for both types of device) and the ovariectomised group (p < 0.01). The difference between the pseudopregnant and ovariectomised rabbits did not attain statistical significance (0.05< p < 0.1). In the oestrous animals, about half the metal originally present was dissolved away, and dissolution must have been uneven since in some cases the foil was perforated in places. Thus with thicker foil,the loss would probably have been slightly greater still.
AUGUST 1974 VOL. 10 NO. 2
205
CONTRACEPTION
Polypoid deciduomata in a 15-day Fig-l. pseudopregnant rabbit uterus induced by a 42 mm2 copper IUD. Transverse section X 12, haematoxylin and eosin.
Same specimen as above, showing ulcerated Fig.2. mucosa and decidual tissue. Transverse section X 45, haematoxylin and eosin.
206
AUGUST
1974
VOL. 10 NO. 2
CONTRACEPTION
Table I. Copper
losses
in rabbits
in different
hormonal
states
VARIECTOMISED -2 -1 Calculation of the results in terms of loss cm day (Table 1) shows that the dissolution rate from the devices used is roughly proportional to the surface area of metal exposed in both oestrous and pseudopregnant rabbits. Preliminary analysis of the histological data showed polypoid deciduomata in the endometrium adjacent to the device in the pseudopregnant rabbits (Fig.1 ). An inflammatory reaction was seen in the vicinity of the device in all tissues examined but this was very marked in the pseudopregnant rabbits in which ulcers were visible in the mucosa and decidual tissue, and frank pus was generally present in These findings are currently the uterine lumen (Fig.2 ). the subject of further investigation. DISCUSSION These results show that the hormonal state of the rabbit has a marked effect on the dissolution rate of This may be due to changes in the intrauterine copper. fluid surrounding the device (4,5). Copper is oxidised by protein solutions,perhaps by a mechanism involving the disulphide bridges (6). The relative abundance of proteins in rabbit uterine fluid is hormone dependent (7): uterine fluid in theoestrous rabbit is less viscous than in the pseudopregnant state and may be better mixed by the more motileoestrousmyometrium. The composition of uterine fluid may be further modified by the reaction to the devices (7), especially in the pseudopregnant animals where inflammatory exudate is most evident and decidualisation occurs. Any of these factors could influence the dissolution rate of intrauterine copper.
AUGUST 1974 VOL. 10 NO. 2
207
CONTRACEPTION
If copper loss in the human uterus is similarly hormone dependent, this could be one explanation of the difference in copper levels of uterine washings obtained in the proliferative and secretory phases of the cycle in copper IUD users (8). Average copper loss over several months in women using copper IUDs has been estimated to be 29Fg/day from Cu-T devices bearing 120 and 135 mm2 of copper and about 50pg/day from the 200mm2 device (9). The initial rate of release from the 200mm2 Cu-7 device is estimated to be 55pg/day, falling to 20pg/day at 12 months (10). The average release rates observed in this study in the pseudopregnant andoestrousrabbit from a device bearing only 42mm2 of copper are 40 and 95pg/day, respectively. Direct comparison of rates in women and rabbits is difficult because of such factors as the different form and area of the and the short length of time over copper in thedevicesused, which copper loss was measured in the rabbits. However, the results presented do indicate the possibility that the metal may be attacked at different rates in different species: this should be considered when selecting animal models for studies on the copper IUD. Hagenfeldt (9) has provided data showing a considerable variation in dissolution rates in copper IUD It is possible that some fraction of the pregnant .es users. and perhaps side effects seen with the device (1,2) could be attributed to unusually low or high dissolution rates in certain patients.
ACKNOWLEDGEMENTS
The advice and comments of Dr. I.H.M. Main and Dr. P.B. Marley, and the technical assistance of Mr. G.S. Crouch and Miss R.A. Hearn, are gratefully acknowledged.
208
AUGUST
1974 VOL. 10 NO. 2
CONTRACEPTION
REFERENCES 1.
Metallic Tatum, H.J. contraceptive agent. 117: 602-18 (1973).
2.
Intrauterine devices. Orlans, F.B. Report Series B No 1 (1973).
3.
Woelfel, W.C. Calorimetric determination of copper with carbon disulfide and diethanolamine. Anal Chem 20 : 722-724 (1948).
4.
Heap, R.B. and Lamming, G.E. The influence of ovarian hormones on some chemical constituents of the uterine washing of the rat and rabbit. J Endocrinol 25:57-68 (1962).
5.
Volume and protein concentration Kulangara, A.C. of rabbit uterine fluid. J Reprod Fert 28:419-425 (1972).
6.
Oster, G.K. Chemical intrauterine device. (1972).
7.
Johnson, M.H. The protein composition of secretions from pregnant and pseudopregnant rabbit uteri with and without a copper intrauterine device. Fertil Steril 23 : 123-130 (1972).
8.
Hagenfeldt, K. Intrauterine contraception with the Copper-T device,4. Influence on protein and copper concentrations and enzyme activities in uterine washings. Contraception 6 : 219-230 (1972).
9.
Hagenfeldt, K. Intrauterine contraception with the Copper-T device.l.Effects on trace elements in the endometrium, cervical mucus and plasma. Contraception 6 : 37-54 (1972).
10.
copper as an intrauterine Amer J Obstet Gynecol
Population
reaction to the copper Fertil Steril 23: 18-23
Gibor, Y., Nissen, C.H. andBrunsfe1.d PI. (1972) Unpublished data quoted by Orlans (2).
AUGUST
1974
VOL. 10 NO. 2
209
J.A. Tobert
Department of Pharmacology, The School of Pharmacy, University of London, Brunswick Square, London WClN 1AX. United Kingdom
ABSTRACT
Since the antifertility effect of the copper IUD is probably related to the release of copper into the uterus, it is important to understand the factors governing this release. In this study the hormonal state of the rabbit is shown to influence the dissolution rate of copper from copper IUDsl the loss of copper during the first 14 days in situ is much greater when the rabbit is oestrous than when it is pseudopregnant or ovariectomised. To obtain information on the effect of the surface area of metal exposed on release rates, devices bearing different areas of copper were inserted into oestrous and pseudopregnant rabbits. In both cases copper loss was approximately proportional to surface area. Deciduomata were observed in the pseudopregnant animals in the vicinity of the device.
Accepted
AUGUST
for publication
1974
VOL.
June 28,
10 NO. 2
1974
203
CONTRACEPTION
INTRODUCTION The addition of copper wire to a plastic IUD results in increased effectiveness (1,2); the copper exerts an antifertility action permitting design of the carrier to be aimed only at reducing expulsions and side effects. It is likely (though not yet proved) that this antifertility action is mediated by copper released into uterine fluid and/or tissues (probably as complexed Cu(I1) It is therefore important to know something of ions). the factors governing the dissolution of the metal. One factor that might be expected to influence the dissolution rate of copper is the environment in which it is placed. To test this hypothesis, copper IUDs were inserted into the uteri of oestrous, pseudopregnant and ovariectomised rabbits and the copper lost from them In addition, to obtain information on determined. the effect of surface area on copper loss, devices bearing two different areas of copper were used. METHODS
AND MATERIALS
Animals New Zealand White virgin female rabbits, Twelve 3-4-S kg, caged singly, were used in this study. Four rabbits were rendered pseudopregnant by intravenous injection of 50 I.U. HCG on the day prior to insertion of the devices. A further four were bilaterally ovariectomised when the devices were inserted, and four more, judged to be oestrous by the condition of the vulva, received no The oestrous and pseudotreatment prior to insertion. pregnant states were confirmed by inspection of the ovaries at laparotomy and at termination of the experiments. IUDS The copper consisted of preweighed rectangular pieces of 99.9% pure 6.5pm foil (Goodfellow Metals), either 2x7mm or 6x7mm, affixed by means of an epoxy resin adhesive (Araldite, Ciba) in a shallow recession in an acrylic rod (Perspex, ICI) 21mm long and 2.4rmn in diameter. The copper then formed a sleeve 2 or 6nun long and the area of metal exposed was 14 or 42mm2. The sleeve in both A hole cases terminated lmm from one end of the rod. was drilled in the other end and a 4/O silk thread was passThe security of the knot was ed through it and knotted. ensured by a sleeve of medical grade silicone tubing (Silastic, Dow Corning), (1Omm long, 1.5mm I.D. x 2mm 0-D.) The devices were disinl pulled over this end of the rod. fected prior to insertion by immersion in 0.2% chlorhexidine solution.
204
AUGUST
1974
VOL. 10 NO. 2
CONTRACEPTION
Surqery Laparotomy was performed in aseptic conditions under pentobarbitone/halothane anaesthesia. The IUDs were inserted via a small antimesometrial incision into the caudal third of the uterine lumen, and secured there by knotting the IUD thread to the suture closing the incision. The device had minimal mobility within the lumen. A 42nun2 device was placed in one horn (right and left alternating in successive experiments) and a 14mm2 device in the other horn, with the exce tion of the ovariectomised group in which only a 42mm Iz device was used, the other horn bearing a similar perspex device without copper. Determination
of copper loss
Fourteen days after surgery the animals were killed and the IUDs removed. The copper remaining on them was dissolved in nitric acid, which was then buffered and diluted with sodium acetate to give solutions containing 2-6pg/ml Cu at pH 4.6. The copper concentration was found by measuring the absorbance at 4321-m of the complex formed with diethanolamine bis(2-hydroxyethyl)dithiocarbamate (3). The copper loss during the fourteen days in situ was obtained by subtraction of the mass remaining from the mass originally present. Histoloqy Some of the uteri were fixed in formalin, embedded in paraffin wax, sectioned and stained by haematoxylin and eosin. Statistical
analysis
The significance of differences tested by Student's t test.
between
means was
RESULTS The copper losses during the 14 days in situ are shown in Table I. The mean loss in the oestrous animals was significantly greater than that in the pseudopregnant group ( p < 0.001 for both types of device) and the ovariectomised group (p < 0.01). The difference between the pseudopregnant and ovariectomised rabbits did not attain statistical significance (0.05< p < 0.1). In the oestrous animals, about half the metal originally present was dissolved away, and dissolution must have been uneven since in some cases the foil was perforated in places. Thus with thicker foil,the loss would probably have been slightly greater still.
AUGUST 1974 VOL. 10 NO. 2
205
CONTRACEPTION
Polypoid deciduomata in a 15-day Fig-l. pseudopregnant rabbit uterus induced by a 42 mm2 copper IUD. Transverse section X 12, haematoxylin and eosin.
Same specimen as above, showing ulcerated Fig.2. mucosa and decidual tissue. Transverse section X 45, haematoxylin and eosin.
206
AUGUST
1974
VOL. 10 NO. 2
CONTRACEPTION
Table I. Copper
losses
in rabbits
in different
hormonal
states
VARIECTOMISED -2 -1 Calculation of the results in terms of loss cm day (Table 1) shows that the dissolution rate from the devices used is roughly proportional to the surface area of metal exposed in both oestrous and pseudopregnant rabbits. Preliminary analysis of the histological data showed polypoid deciduomata in the endometrium adjacent to the device in the pseudopregnant rabbits (Fig.1 ). An inflammatory reaction was seen in the vicinity of the device in all tissues examined but this was very marked in the pseudopregnant rabbits in which ulcers were visible in the mucosa and decidual tissue, and frank pus was generally present in These findings are currently the uterine lumen (Fig.2 ). the subject of further investigation. DISCUSSION These results show that the hormonal state of the rabbit has a marked effect on the dissolution rate of This may be due to changes in the intrauterine copper. fluid surrounding the device (4,5). Copper is oxidised by protein solutions,perhaps by a mechanism involving the disulphide bridges (6). The relative abundance of proteins in rabbit uterine fluid is hormone dependent (7): uterine fluid in theoestrous rabbit is less viscous than in the pseudopregnant state and may be better mixed by the more motileoestrousmyometrium. The composition of uterine fluid may be further modified by the reaction to the devices (7), especially in the pseudopregnant animals where inflammatory exudate is most evident and decidualisation occurs. Any of these factors could influence the dissolution rate of intrauterine copper.
AUGUST 1974 VOL. 10 NO. 2
207
CONTRACEPTION
If copper loss in the human uterus is similarly hormone dependent, this could be one explanation of the difference in copper levels of uterine washings obtained in the proliferative and secretory phases of the cycle in copper IUD users (8). Average copper loss over several months in women using copper IUDs has been estimated to be 29Fg/day from Cu-T devices bearing 120 and 135 mm2 of copper and about 50pg/day from the 200mm2 device (9). The initial rate of release from the 200mm2 Cu-7 device is estimated to be 55pg/day, falling to 20pg/day at 12 months (10). The average release rates observed in this study in the pseudopregnant andoestrousrabbit from a device bearing only 42mm2 of copper are 40 and 95pg/day, respectively. Direct comparison of rates in women and rabbits is difficult because of such factors as the different form and area of the and the short length of time over copper in thedevicesused, which copper loss was measured in the rabbits. However, the results presented do indicate the possibility that the metal may be attacked at different rates in different species: this should be considered when selecting animal models for studies on the copper IUD. Hagenfeldt (9) has provided data showing a considerable variation in dissolution rates in copper IUD It is possible that some fraction of the pregnant .es users. and perhaps side effects seen with the device (1,2) could be attributed to unusually low or high dissolution rates in certain patients.
ACKNOWLEDGEMENTS
The advice and comments of Dr. I.H.M. Main and Dr. P.B. Marley, and the technical assistance of Mr. G.S. Crouch and Miss R.A. Hearn, are gratefully acknowledged.
208
AUGUST
1974 VOL. 10 NO. 2
CONTRACEPTION
REFERENCES 1.
Metallic Tatum, H.J. contraceptive agent. 117: 602-18 (1973).
2.
Intrauterine devices. Orlans, F.B. Report Series B No 1 (1973).
3.
Woelfel, W.C. Calorimetric determination of copper with carbon disulfide and diethanolamine. Anal Chem 20 : 722-724 (1948).
4.
Heap, R.B. and Lamming, G.E. The influence of ovarian hormones on some chemical constituents of the uterine washing of the rat and rabbit. J Endocrinol 25:57-68 (1962).
5.
Volume and protein concentration Kulangara, A.C. of rabbit uterine fluid. J Reprod Fert 28:419-425 (1972).
6.
Oster, G.K. Chemical intrauterine device. (1972).
7.
Johnson, M.H. The protein composition of secretions from pregnant and pseudopregnant rabbit uteri with and without a copper intrauterine device. Fertil Steril 23 : 123-130 (1972).
8.
Hagenfeldt, K. Intrauterine contraception with the Copper-T device,4. Influence on protein and copper concentrations and enzyme activities in uterine washings. Contraception 6 : 219-230 (1972).
9.
Hagenfeldt, K. Intrauterine contraception with the Copper-T device.l.Effects on trace elements in the endometrium, cervical mucus and plasma. Contraception 6 : 37-54 (1972).
10.
copper as an intrauterine Amer J Obstet Gynecol
Population
reaction to the copper Fertil Steril 23: 18-23
Gibor, Y., Nissen, C.H. andBrunsfe1.d PI. (1972) Unpublished data quoted by Orlans (2).
AUGUST
1974
VOL. 10 NO. 2
209