FERTILITY AND STERILITY Copyright ~ 1975 The American Fertility Society
Vol. 26, No. 8, August 1975 Printed in U.S A.
THE EFFECT OF A COPPER INTRAUTERINE DEVICE DURING THE EARLY STAGES OF PREGNANCY IN THE RABBIT EHARD F. NU'ITING, PH.D.,
MARGUERITE R. MUELLER
AND
Department of Biological Research, Searle Laboratories, Chicago, Illinois 60680
Clinical studies by Zipper et al. 1 and Tatum2 have shown that the addition of 200 sq mm of copper to the plastic frame of an intrauterine contraceptive device decreases the pregnancy rate and enhances the use continuation rate significantly. Although studies have been made in rats, 3 • 5 hamsters, 4 and rabbits 6 • 7 to determine the mode of the antifertility activity of copper, the mechanism of action is still undecided. In the rabbit, Zipper and co-workers 6 have reported that a copper intrauterine device completely inhibits fertility 10 days after insemination. Polidoro and Black7 confirmed these results and determined that the inhibition was not the result of fertilization failure. Before more definitive studies can be undertaken to elucidate the mechanism of action of copper in the rabbit, the specific stage of gestation affected by the device must be more precisely delineated. The present study was undertaken to observe the temporal effects of copper associated with preimplantation and early implantation stages of pregnancy and to obtain a better understanding of the mechanism of action of copper. MATERIALS AND METHODS
A copper wire intrauterine device (CuiUD) was made from 32-gauge pure virgin electrolytic copper wire. A gold wire intrauterine device (AuiUD) made Received August 1, 1974.
from 32-gauge 0.999 pure gold wire was used as a control. The wire was formed into a device with a loop at one end and two parallel projecting arms with loops on the terminal ends. One arm was slightly longer than the other. The device was weighed and an anchor thread was attached to the device. This was done by passing one end of a length of monofilament polyethylene 5-0 suture thread (Davis and Geck Co., Pearl River, N. Y.) through the central loop of the device. Heat was applied to one end of the thread with a hot instrument to form a smooth, rounded bead, preventing the thread from slipping through the loop. The device, with the attached anchor thread, was inserted into an envelope and gas-sterilized prior to insertion (Fig. 1). The animals were anesthetized with Somnopentyl and surgery was performed under sterile conditions. The uterine horn was visualized through a dorsolateral incision. The unbeaded end of the anchor thread, attached to the device, was threaded into a straight surgical needle. The needle was inserted through the posterior antimesometrial border of the uterine horn into the lumen, directed toward the oviduct for a distance of approximately 2 em, and brought back to the exterior of the uterus. The device was pulled into the lumen through the puncture in the uterine wall by means of the anchor thread and drawn anteriorly through the lumen to the point of emergence of the thread. The needle and
838
Vol. 26, No.8
EFFECT OF A COPPER IUD DURING EARLY PREGNANCY
839
FIG. 1. Photographs of gold wire intrauterine device (left) and copper wire intrauterine device with attached anchor suture thread (right).
thread were then drawn through a 0.5cm length of medical grade Silastic tubing, passing through one wall and emerging through the lumen of the tube. The free end of the thread was trimmed so that a length of approximately 1 em projected from the piece of Silastic tubing. The tip of the thread was melted with a warm instrument to form a bead at the end and secure the thread to the tubing. The piece of Silastic functioned as an anchor for the device, while the projecting length of thread ensured that there would be some slack between the anchor and the device. The abdominal incision was closed with 4-0 silk suture and 9-mm skin clips. Crysticillin (150,000 U) and Bicillin (100,000 U) were administered intramuscularly after surgery. Intact, mature female rabbits (Dutch strain) weighing 1.5 to 3.0 kg were randomly assigned to one or three experimental groups of three to five animals each for each time period. In one group,
a CuiUD was inserted into the right horn and a sham operation was performed on the left horn (CuiUD/sham). In a second group, an AuiUD was inserted into the right horn and a sham operation was performed on the left horn (AuiUD/ sham). In a third group, the CuiUD was inserted into one horn and the AuiUD was inserted into the contralateral horn (CuiUD/AuiUD). This last group (CuiUD/AuiUD) was the only experimental group employed in later experiments. The weight of each device was recorded at insertion. Two weeks after insertion of the devices, the animals were artificially inseminated with rabbit semen as described by Nutting and Mares. 8 Semen was collected from three or more mature bucks with the aid of an artificial vagina, pooled to reduce the chance occurrence of an infertile sample, and diluted with physiologic saline. A semen dilution of 1:9 and an insemination volume of 0.5 ml
840
NUTTING AND MUELLER
were employed as a standard practice. Immediately preceding artificial insemination, each doe received an intravenous injection of luteinizing hormone (1 mg/0.2 ml) to induce ovulation. Animals were killed at various intervals between 48 and 192 hours after insemination. In rabbits autopsied 48 to 144 hours postinsemination, the reproductive tract was removed, and the oviducts and uterus were flushed with physiologic saline. The washings from the CuiUDbearing horn, AuiUD-bearing horn, and sham-operated horn, as well as from the respective oviducts, were collected separately in a watch glass and viewed immediately beneath a dissecting microscope at a magnification of x 80. The stage and condition of development of ova were noted, and the external diameters of the mucin layer and zona pellucida were measured with an eyepiece reticle. Ova were then transferred to a hanging-drop preparation in a depression slide to facilitate further examination at higher magnification, using the light microscope and, in some cases, a phase-contrast microscope. In the groups of animals autopsied 192 hours postinsemination, the reproductive tract was visualized, and the number, location, and condition of the implantation sites were recorded. The ovaries were removed, and the number of corpora lutea were counted. Data from animals with corpora albicantia or with corpora lutea that were obviously larger than corpora lutea in control animals were excluded from experimental results. The devices were removed carefully from the uterus to minimize tissue damage. They were cleaned, air-dried, and weighed to determine the loss of copper from the device during the experiment. Student's t-test was used to determine the significance of differences between weights taken before and after the experiment.
August 1975
For ease of comparison, the percentage of ova recovered (100 x number of ova recovered + number of corpora lutea) was calculated from the total in each group. The number of ova recovered and the number of ova fertilized were compared by means of X2 analysis using original rather than percentage values. The method of analysis of variance was used to determine the significance of differences between groups in the number of corpora lutea and the diameter of the mucin layer. RESULTS
A significant (P < 0.05) loss of copper occurred during the 17 to 22 days the devices were in utero (Table 1). The estimated daily loss of copper ranged from 51 to 73 J.tg/day. There was no significant weight change in the A uiUD devices during the same period. The results of autopsies performed 48 to 144 hours after artificial insemination are shown in Table 2. Forty-eight hours after insemination, there was no significant difference in the fertilization rate of ova recovered from the site of the reproductive tract containing an AuiUD and the contralateral sham-operated side. The incidence of fertilized ova in the side of the tract containing a CuiUD was significantly (p < 0.01) less than that in the contralateral side bearing either an AuiUD or sham-operated. All of the fertilized ova recovered at 48 hours were normal 8- to 16-cell embryos. The presence of a CuiUD or AuiUD had no effect on the recovery rate or the distribution of ova within the reproductive tract 48 hours after insemination. Conflicting results were observed between the two groups of rabbits bearing a CuiUD which were autopsied at 72 hours. The fertilization rate was significantly (p < 0.05) lower in the presence of the CuiUD in the group of animals in which the contralateral control side was
Vol. 26, No.8
841
EFFECT OF A COPPER IUD DURING EARLY PREGNANCY
TABLE 1. The Loss of Copper and Gold in Utero from Copper Wire and Gold Wire Inserted into Uteri of Adult Rabbits14 Days before Insemination No. of hours from insemination to autopsy
Total no. of days in situ
Total no. of devices
48 72 96 120 144 192
17 18 19 20 21 22
8 9 4 5 2 7
CuiUD
AulUD
Initial wt. ± SE
Average copper loBS'
mg
l'g/day
8.80 8.86 8.98 8.90 8.97 8.89
± ± ± ± ± ±
Total no. of devices
-57 -51 -54 -73 -52 -53
0.08 0.04 0.14 0.04 0.03 0.10
9 12 4 5 4 7
Initial wt. ± SE
Average gold loas
mg
l'g/day
18.88 18.86 18.68 18.68 18.56 18.97
± ± ± ± ± ±
-6.0 -7.0 +4.5 +4.5 -3.6 -7.0
0.09 0.09 0.04 0.07 0.04 0.22
•Final weights were significantly different from initial weights!}' < 0.05).
sham-operated. However, in the group containing a CuiUD and a contralateral AuiUD, fertilization was not affected by the presence of either device. The AuiUD also had no effect on fertilization in animals with contralateral sham-operated control horns. Various stages of embryonic development, from 16 cells to early blastocysts, were observed, but the morphologic development of all of the
embryos recovered at 72 hours was normal. The diameter of the mucin layer surrounding the embryos was variable within each treatment group, and no consistent effect of the CuiUD or AuiUD on the thickness of this layer was observed at 48 or 72 hours after insemination. The recovery rate and the distribution of ova within the reproductive tract 72 hours after insemination were not
TABLE 2. The Effect of the Insertion of Copper Wire, Gold Wire, and Sham Operation 14 Days before Insemination on Fertilization of Ova and Recovery of Embryos at Various Intervals after Insemination of Adult Rabbits Treatment of
ri\~~~~ne uterine horn
Condition No. of hours of from in· uterine semination horn to autopsy
No. of uterine horns treated
No. of ova fertilized Diameter Recovered ova No. of - - - - - - - - - - - - - - - - - - - - - - - - - - - - of corpora No. in No. in % Total mucin lutea oviduct (%) uterus (%) recovered Total layer %
mm
CuiUD/sham AuiUD/sham CuiUD/AuiUD CuiUD/sham AuiUD/sham CuiUD/AuiUD CuiUD/AuiUD CuiUD/AuiUD CuiUD/AuiUD
CuiUD Sham AuiUD Sham CuiUD AuiUD CuiUD Sham AuiUD Sham CuiUD AuiUD CuiUD AuiUD CuiUD AuiUD CuiUD AuiUOC
48 48 48 48 48 48 72 72 72 72 72 72 96 96 120 120 144 144
4 4 5 5 4
4 3 3 6 6 6 6 4 4 5 5 4
3d
19 11 23 26 12 16 16 12 36 24 22 21 20 17 25 17 16 14
18 (100) 8 (100) 20 (100) 22 (100) 12 (100) 15 (100) 16 (100) 9 (100) 36 (100) 23 (100) 22 (100) 14 (62) 0 (0) 0 (0) 0 0 0 0
(0) (0) (0) (0)
0 0 0 0
(0) (0) (0) (0)
0 (0) 0 (0)
0 (0) 0 (0)
0 (0) 0 (0)
0 (0) 5 (38) 16(100) 15(100) 20(100) 15(100) 1 (lOO) 14(100)
95 73 87 85 100 94 100 75 100 96 100 90 80 88 80 88 6• 100
3 7 19 22 5 15 8 9 35 23 18 17 14 15 13 12 1
14
17" 88 95 100 42"
100 5Qb 100 97 100 82 89
0.248 0.234 0.220 0.237 0.274 0.256 0.297 0.327 0.362 0.342 0.392 0.355
88 100 65 80 100 100
"Significantly different from value for contralateral horn!}' < 0.01). bSignificantly different from value for contralateral horn !}' < 0.05). c AuiUD perforated uterine horn in one animal and was in the peritoneal cavity at time of autopsy. dData for one uterine horn were excluded because ovulation did not occur in the adjacent ovary.
I
NUTTING AND MUELLER
842
August 1975
TABLE 3. The Effect of Insertion of Copper Wire, Gold Wire, and Sham Operation 14 Days before Insemination on Implantation in Adult Rabbits 192 Hours after Insemination Treatment of right uterine hom/left uterine horn
CuiUD/sham AuiUD/sham CuiUD/AuiUD
Condition of uterine horn
No. of uterine horns treated
No. of corpora lutea
No. of sites
CuiUD Sham AuiUD Sham CuiUD AuiUD
4 4 5 5 4 4
14 18 30 22 20 16
0 14 28 19 0 12
Implantation rate
Average size of sites
%
em
(}"
78 93 86
0.86 0.98 0.94
(}"
75
1.00
"Significantly different from value for contralateral horn(]> < 0.01).
affected by the presence of either device. The fertilization rate of ova recovered at 96 and 120 hours after insemination was not affected by the presence of either a CuiUD or AuiUD. There was no effect on the recovery rate or the distribution of embryos in the uterine horn containing a CuiUD in comparison with the contralateral horn containing an AuiUD. All of the blastocysts recovered from uterine horns bearing an AuiUD were normal, while the incidence of normal blastocysts recovered from uterine horns containing a CuiUD was 72% at 96 hours and 62% at 120 hours, but the differences were not significant. At 144 hours after insemination, the presence of an AuiUD had no effect on the recovery rate or the distribution of the blastocysts within the reproductive tract. In comparison, only one blastocyst was recovered from uterine horns containing a CuiUD. This low recovery rate suggests that the unrecovered ova were expelled from the uterus prior to autopsy at 144 hours or were lysed beyond recognition. All of the embryos recovered at this time were normal, denuded blastocysts. The effects of a CuiUD or AuiUD on implantation in rabbits autopsied at 192 hours are shown in Table 3. No implantation sites were observed in uterine horns containing a CuiUD, while normal implantation did occur in contralateral control horns bearing an AuiUD or sham-
operated. There was no significant difference in implantation rates or the size of implantation sites in the uterine horn bearing an AuiUD and the contralateral sham-operated control horn. Four of the fourty implantation sites in the AuiUD horn were observed in positions in contact with the AuiUD. DISCUSSION
The present study was designed to determine the specific time during early gestation when a copper intrauterine device interrupts pregnancy in the rabbit. In addition, a gold, chemically inert, intrauterine device with physical characteristics identical with those of the copper device was employed as a control to separate the physical effects of the CuiUD on fertility from the chemical effects. As expected, the CuiUD was chemically reactive, as shown in these studies by the significant quantities of copper which were eluted from the CuiUD while in the uterine environment. The AuiUD had no effect on any of the parameters offertility measured in this study. The device was so innocuous that several implantations occurred immediately adjacent to the device. In comparison, the CuiUD apparently interrupted pregnancy between 120 and 144 hours after insemination, or just prior to the time of implantation,. Thus, the copper eluting from the device and not the physical characteristics of the metallic
Vol. 26, No.8
EFFECT OF A COPPER IUD DURING EARLY PREGNANCY
IUD was associated with the contraceptive effect of the CuiUD. The presence of the CuiUD had no effect on the ovulation process, as indicated by the finding that normal number of corpora lutea occurred in all groups, regardless of treatment. Although the fertilization rate was low in the presence of a CuiUD 48 hours after insemination the normal rates observed at 72, 96, and' 120 hours suggest that this effect was not associated with the presence of the CuiUD. This interpretation is consistent with that of Polidoro and Black, 7 who concluded from observations made in rabbits 48 hours after insemination that the presence of a copper IUD did not interfer with the fertilization of ova. The CuiUD also had no significant effect on embryonic development or on the rate of transport of the ova through the reproductive tract until just prior to implantation. In summary, all physiologic and embryologic processes of reproduction were essentially normal 120 hours after insemination. At the 144-hour stage, only 1 of 16 ovulated ova was found in the uterine horn containing a CuiUD, and at 192 hours no implantations occurred in the CuiUD-bearing horn. The contraceptive effect of the CuiUD was clearly not systemic. The complete absence of implantation in the CuiUD-containing horn shows that the local effect of the metallic copper device extends beyond the area of the tissue immediately adjacent to the device. In this respect, the metallic copper device differs from a plastic IUD in which implantation is prevented mainly in the area of the device. These studies do not show whether the implantation-inhibiting effect of copper is directly on the blastocyst or is mediated indirectly by more subtle alterations in the uterine tissue. Nutting and Mueller, 9 using tissues obtained from studies in this report as well as from studies in immature rab-
843
bits treated with progesterone, have observed the effects of a CuiUD on the histology and carbonic anhydrase concentration of the uterine tissue and concluded that copper antagonizes the action of progesterone on the uterus. These effects were apparent 48 hours before any demonstrable effect on the blastocyst. This suggests that in the presence of a CuiUD the temporal developmental sequence of tissue changes or production of secretory products under the influence of progesterone are suppressed or delayed during the early stages of pregnancy, creating a uterine milieu which is not favorable for the developing blastocyst. This concept of copper-induced progesterone antagonism is supported by the studies of Eich and Nutting 10 in rabbits which show that blastokinin, a progesterone-dependent uterine protein, is decreased by the presence of copper. Prager11 has reported evidence indicating that protein synthesis is reduced in rats bearing a copper IUD. Chang and Tatum3 used blastocyst transfer procedures and showed that rat blastocysts developed normally when they were transferred from a copper IUDbearing horn to a normal horn. Thus, implantation failure in the presence of copper appears to be caused by an alteration of the uterine environment and not to be a direct effect of copper on the developing blastocyst. SUMMARY
Copper wire intrauterine devices (CuiUD) were surgically inserted into mature female rabbits 14 days prior to artificial insemination. Gold wire intrauterine devices and sham surgery served as controls. Animals were autopsied 48, 72, 96, 120, 144, and 192 hours after artificial insemination. At autopsies performed 48 to 144 hours after insemination, the reproductive tract was flushed
Ntrl'TING AND MUELLER
844
and the embryos were examined. At 192 hours, implantation sites were counted and measured. A significant (}' < 0.05) loss of copper from the devices was observed when the devices were weighed after autopsy. The CuiUD had no effect on ovulation, fertilization, or embryo transport up to 120 hours after artificial insemination. The presence of a CuiUD resulted in a slight, but not statistically significant, increase in embryonic degeneration by 120 hours, almost total absence of blastocysts from the uterus by 144 hours, and complete absence of implantation sites 192 hours after insemination. The antifertility effect of a copper intrauterine device appears to occur in the late preimplantation stage of pregnancy in the rabbit. Acknowledgments. We would especially like to thank M. Cole, J. Pautsch, and M. Dal Corobbo for technical assistance. REFERENCES 1. Zipper JA, Tatum HJ, Medel M, Pastene L, Rivera M: Contraception through the use of intrauterine metals. 1. Copper as an adjunct to the "T" device. Am J Obstet Gynecol 109:771, 1970
August 1975
2. Tatum HJ: Copper-bearing intrauterine devices. Clin Obstet Gynecol 17(1):93, 1974 3. Chang CC, Tatum HJ: A study of the antifertility effect of intrauterine copper. Contraception 1:265, 1970 4. Chang CC, Tatum HJ, Kincl FA: The effect of intrauterine copper and other metals on implantation in rats and hamster. Fertil Steril21:274, 1970 5. Chang CC, Tatum HJ: Some temporal relationships between intrauterine copper wire and its contraceptive action in the rat. Fertil Steril 23:191, 1972 6. Zipper J, Medel M, Prager R: Suppression of fertility by intrauterine copper and zinc in rabbits. Am J Obstet Gynecol 105:529, 1969 7. Polidoro JP, Black DL: The failure of the copper IUD to inhibit fertilization in the rabbit. J Reprod Fertil 23:151, 1970 8. Nutting EF, Mares SE: Inhibition of fertilization in rabbits during treatment with progesterone. Bioi Reprod 2:230, 1970 9. Nutting EF, Mueller MR: The effect of a copper intrauterine device on the uterine histology and progestational response in pregnant and immature rabbits. Fertil Steril .26:845, 1975 10. Eich E, Nutting EF: Unpublished data 11. Prager R: Effect of various types of intrauterine foreign bodies on the incorporation of 35S into microprotein and of thymidine 2-'4C into DNA of rat's endometrium. Fertil Steril 20:944, 1969
Vol. 26, No. 8, August 1975 Printed in U.S A.
THE EFFECT OF A COPPER INTRAUTERINE DEVICE DURING THE EARLY STAGES OF PREGNANCY IN THE RABBIT EHARD F. NU'ITING, PH.D.,
MARGUERITE R. MUELLER
AND
Department of Biological Research, Searle Laboratories, Chicago, Illinois 60680
Clinical studies by Zipper et al. 1 and Tatum2 have shown that the addition of 200 sq mm of copper to the plastic frame of an intrauterine contraceptive device decreases the pregnancy rate and enhances the use continuation rate significantly. Although studies have been made in rats, 3 • 5 hamsters, 4 and rabbits 6 • 7 to determine the mode of the antifertility activity of copper, the mechanism of action is still undecided. In the rabbit, Zipper and co-workers 6 have reported that a copper intrauterine device completely inhibits fertility 10 days after insemination. Polidoro and Black7 confirmed these results and determined that the inhibition was not the result of fertilization failure. Before more definitive studies can be undertaken to elucidate the mechanism of action of copper in the rabbit, the specific stage of gestation affected by the device must be more precisely delineated. The present study was undertaken to observe the temporal effects of copper associated with preimplantation and early implantation stages of pregnancy and to obtain a better understanding of the mechanism of action of copper. MATERIALS AND METHODS
A copper wire intrauterine device (CuiUD) was made from 32-gauge pure virgin electrolytic copper wire. A gold wire intrauterine device (AuiUD) made Received August 1, 1974.
from 32-gauge 0.999 pure gold wire was used as a control. The wire was formed into a device with a loop at one end and two parallel projecting arms with loops on the terminal ends. One arm was slightly longer than the other. The device was weighed and an anchor thread was attached to the device. This was done by passing one end of a length of monofilament polyethylene 5-0 suture thread (Davis and Geck Co., Pearl River, N. Y.) through the central loop of the device. Heat was applied to one end of the thread with a hot instrument to form a smooth, rounded bead, preventing the thread from slipping through the loop. The device, with the attached anchor thread, was inserted into an envelope and gas-sterilized prior to insertion (Fig. 1). The animals were anesthetized with Somnopentyl and surgery was performed under sterile conditions. The uterine horn was visualized through a dorsolateral incision. The unbeaded end of the anchor thread, attached to the device, was threaded into a straight surgical needle. The needle was inserted through the posterior antimesometrial border of the uterine horn into the lumen, directed toward the oviduct for a distance of approximately 2 em, and brought back to the exterior of the uterus. The device was pulled into the lumen through the puncture in the uterine wall by means of the anchor thread and drawn anteriorly through the lumen to the point of emergence of the thread. The needle and
838
Vol. 26, No.8
EFFECT OF A COPPER IUD DURING EARLY PREGNANCY
839
FIG. 1. Photographs of gold wire intrauterine device (left) and copper wire intrauterine device with attached anchor suture thread (right).
thread were then drawn through a 0.5cm length of medical grade Silastic tubing, passing through one wall and emerging through the lumen of the tube. The free end of the thread was trimmed so that a length of approximately 1 em projected from the piece of Silastic tubing. The tip of the thread was melted with a warm instrument to form a bead at the end and secure the thread to the tubing. The piece of Silastic functioned as an anchor for the device, while the projecting length of thread ensured that there would be some slack between the anchor and the device. The abdominal incision was closed with 4-0 silk suture and 9-mm skin clips. Crysticillin (150,000 U) and Bicillin (100,000 U) were administered intramuscularly after surgery. Intact, mature female rabbits (Dutch strain) weighing 1.5 to 3.0 kg were randomly assigned to one or three experimental groups of three to five animals each for each time period. In one group,
a CuiUD was inserted into the right horn and a sham operation was performed on the left horn (CuiUD/sham). In a second group, an AuiUD was inserted into the right horn and a sham operation was performed on the left horn (AuiUD/ sham). In a third group, the CuiUD was inserted into one horn and the AuiUD was inserted into the contralateral horn (CuiUD/AuiUD). This last group (CuiUD/AuiUD) was the only experimental group employed in later experiments. The weight of each device was recorded at insertion. Two weeks after insertion of the devices, the animals were artificially inseminated with rabbit semen as described by Nutting and Mares. 8 Semen was collected from three or more mature bucks with the aid of an artificial vagina, pooled to reduce the chance occurrence of an infertile sample, and diluted with physiologic saline. A semen dilution of 1:9 and an insemination volume of 0.5 ml
840
NUTTING AND MUELLER
were employed as a standard practice. Immediately preceding artificial insemination, each doe received an intravenous injection of luteinizing hormone (1 mg/0.2 ml) to induce ovulation. Animals were killed at various intervals between 48 and 192 hours after insemination. In rabbits autopsied 48 to 144 hours postinsemination, the reproductive tract was removed, and the oviducts and uterus were flushed with physiologic saline. The washings from the CuiUDbearing horn, AuiUD-bearing horn, and sham-operated horn, as well as from the respective oviducts, were collected separately in a watch glass and viewed immediately beneath a dissecting microscope at a magnification of x 80. The stage and condition of development of ova were noted, and the external diameters of the mucin layer and zona pellucida were measured with an eyepiece reticle. Ova were then transferred to a hanging-drop preparation in a depression slide to facilitate further examination at higher magnification, using the light microscope and, in some cases, a phase-contrast microscope. In the groups of animals autopsied 192 hours postinsemination, the reproductive tract was visualized, and the number, location, and condition of the implantation sites were recorded. The ovaries were removed, and the number of corpora lutea were counted. Data from animals with corpora albicantia or with corpora lutea that were obviously larger than corpora lutea in control animals were excluded from experimental results. The devices were removed carefully from the uterus to minimize tissue damage. They were cleaned, air-dried, and weighed to determine the loss of copper from the device during the experiment. Student's t-test was used to determine the significance of differences between weights taken before and after the experiment.
August 1975
For ease of comparison, the percentage of ova recovered (100 x number of ova recovered + number of corpora lutea) was calculated from the total in each group. The number of ova recovered and the number of ova fertilized were compared by means of X2 analysis using original rather than percentage values. The method of analysis of variance was used to determine the significance of differences between groups in the number of corpora lutea and the diameter of the mucin layer. RESULTS
A significant (P < 0.05) loss of copper occurred during the 17 to 22 days the devices were in utero (Table 1). The estimated daily loss of copper ranged from 51 to 73 J.tg/day. There was no significant weight change in the A uiUD devices during the same period. The results of autopsies performed 48 to 144 hours after artificial insemination are shown in Table 2. Forty-eight hours after insemination, there was no significant difference in the fertilization rate of ova recovered from the site of the reproductive tract containing an AuiUD and the contralateral sham-operated side. The incidence of fertilized ova in the side of the tract containing a CuiUD was significantly (p < 0.01) less than that in the contralateral side bearing either an AuiUD or sham-operated. All of the fertilized ova recovered at 48 hours were normal 8- to 16-cell embryos. The presence of a CuiUD or AuiUD had no effect on the recovery rate or the distribution of ova within the reproductive tract 48 hours after insemination. Conflicting results were observed between the two groups of rabbits bearing a CuiUD which were autopsied at 72 hours. The fertilization rate was significantly (p < 0.05) lower in the presence of the CuiUD in the group of animals in which the contralateral control side was
Vol. 26, No.8
841
EFFECT OF A COPPER IUD DURING EARLY PREGNANCY
TABLE 1. The Loss of Copper and Gold in Utero from Copper Wire and Gold Wire Inserted into Uteri of Adult Rabbits14 Days before Insemination No. of hours from insemination to autopsy
Total no. of days in situ
Total no. of devices
48 72 96 120 144 192
17 18 19 20 21 22
8 9 4 5 2 7
CuiUD
AulUD
Initial wt. ± SE
Average copper loBS'
mg
l'g/day
8.80 8.86 8.98 8.90 8.97 8.89
± ± ± ± ± ±
Total no. of devices
-57 -51 -54 -73 -52 -53
0.08 0.04 0.14 0.04 0.03 0.10
9 12 4 5 4 7
Initial wt. ± SE
Average gold loas
mg
l'g/day
18.88 18.86 18.68 18.68 18.56 18.97
± ± ± ± ± ±
-6.0 -7.0 +4.5 +4.5 -3.6 -7.0
0.09 0.09 0.04 0.07 0.04 0.22
•Final weights were significantly different from initial weights!}' < 0.05).
sham-operated. However, in the group containing a CuiUD and a contralateral AuiUD, fertilization was not affected by the presence of either device. The AuiUD also had no effect on fertilization in animals with contralateral sham-operated control horns. Various stages of embryonic development, from 16 cells to early blastocysts, were observed, but the morphologic development of all of the
embryos recovered at 72 hours was normal. The diameter of the mucin layer surrounding the embryos was variable within each treatment group, and no consistent effect of the CuiUD or AuiUD on the thickness of this layer was observed at 48 or 72 hours after insemination. The recovery rate and the distribution of ova within the reproductive tract 72 hours after insemination were not
TABLE 2. The Effect of the Insertion of Copper Wire, Gold Wire, and Sham Operation 14 Days before Insemination on Fertilization of Ova and Recovery of Embryos at Various Intervals after Insemination of Adult Rabbits Treatment of
ri\~~~~ne uterine horn
Condition No. of hours of from in· uterine semination horn to autopsy
No. of uterine horns treated
No. of ova fertilized Diameter Recovered ova No. of - - - - - - - - - - - - - - - - - - - - - - - - - - - - of corpora No. in No. in % Total mucin lutea oviduct (%) uterus (%) recovered Total layer %
mm
CuiUD/sham AuiUD/sham CuiUD/AuiUD CuiUD/sham AuiUD/sham CuiUD/AuiUD CuiUD/AuiUD CuiUD/AuiUD CuiUD/AuiUD
CuiUD Sham AuiUD Sham CuiUD AuiUD CuiUD Sham AuiUD Sham CuiUD AuiUD CuiUD AuiUD CuiUD AuiUD CuiUD AuiUOC
48 48 48 48 48 48 72 72 72 72 72 72 96 96 120 120 144 144
4 4 5 5 4
4 3 3 6 6 6 6 4 4 5 5 4
3d
19 11 23 26 12 16 16 12 36 24 22 21 20 17 25 17 16 14
18 (100) 8 (100) 20 (100) 22 (100) 12 (100) 15 (100) 16 (100) 9 (100) 36 (100) 23 (100) 22 (100) 14 (62) 0 (0) 0 (0) 0 0 0 0
(0) (0) (0) (0)
0 0 0 0
(0) (0) (0) (0)
0 (0) 0 (0)
0 (0) 0 (0)
0 (0) 0 (0)
0 (0) 5 (38) 16(100) 15(100) 20(100) 15(100) 1 (lOO) 14(100)
95 73 87 85 100 94 100 75 100 96 100 90 80 88 80 88 6• 100
3 7 19 22 5 15 8 9 35 23 18 17 14 15 13 12 1
14
17" 88 95 100 42"
100 5Qb 100 97 100 82 89
0.248 0.234 0.220 0.237 0.274 0.256 0.297 0.327 0.362 0.342 0.392 0.355
88 100 65 80 100 100
"Significantly different from value for contralateral horn!}' < 0.01). bSignificantly different from value for contralateral horn !}' < 0.05). c AuiUD perforated uterine horn in one animal and was in the peritoneal cavity at time of autopsy. dData for one uterine horn were excluded because ovulation did not occur in the adjacent ovary.
I
NUTTING AND MUELLER
842
August 1975
TABLE 3. The Effect of Insertion of Copper Wire, Gold Wire, and Sham Operation 14 Days before Insemination on Implantation in Adult Rabbits 192 Hours after Insemination Treatment of right uterine hom/left uterine horn
CuiUD/sham AuiUD/sham CuiUD/AuiUD
Condition of uterine horn
No. of uterine horns treated
No. of corpora lutea
No. of sites
CuiUD Sham AuiUD Sham CuiUD AuiUD
4 4 5 5 4 4
14 18 30 22 20 16
0 14 28 19 0 12
Implantation rate
Average size of sites
%
em
(}"
78 93 86
0.86 0.98 0.94
(}"
75
1.00
"Significantly different from value for contralateral horn(]> < 0.01).
affected by the presence of either device. The fertilization rate of ova recovered at 96 and 120 hours after insemination was not affected by the presence of either a CuiUD or AuiUD. There was no effect on the recovery rate or the distribution of embryos in the uterine horn containing a CuiUD in comparison with the contralateral horn containing an AuiUD. All of the blastocysts recovered from uterine horns bearing an AuiUD were normal, while the incidence of normal blastocysts recovered from uterine horns containing a CuiUD was 72% at 96 hours and 62% at 120 hours, but the differences were not significant. At 144 hours after insemination, the presence of an AuiUD had no effect on the recovery rate or the distribution of the blastocysts within the reproductive tract. In comparison, only one blastocyst was recovered from uterine horns containing a CuiUD. This low recovery rate suggests that the unrecovered ova were expelled from the uterus prior to autopsy at 144 hours or were lysed beyond recognition. All of the embryos recovered at this time were normal, denuded blastocysts. The effects of a CuiUD or AuiUD on implantation in rabbits autopsied at 192 hours are shown in Table 3. No implantation sites were observed in uterine horns containing a CuiUD, while normal implantation did occur in contralateral control horns bearing an AuiUD or sham-
operated. There was no significant difference in implantation rates or the size of implantation sites in the uterine horn bearing an AuiUD and the contralateral sham-operated control horn. Four of the fourty implantation sites in the AuiUD horn were observed in positions in contact with the AuiUD. DISCUSSION
The present study was designed to determine the specific time during early gestation when a copper intrauterine device interrupts pregnancy in the rabbit. In addition, a gold, chemically inert, intrauterine device with physical characteristics identical with those of the copper device was employed as a control to separate the physical effects of the CuiUD on fertility from the chemical effects. As expected, the CuiUD was chemically reactive, as shown in these studies by the significant quantities of copper which were eluted from the CuiUD while in the uterine environment. The AuiUD had no effect on any of the parameters offertility measured in this study. The device was so innocuous that several implantations occurred immediately adjacent to the device. In comparison, the CuiUD apparently interrupted pregnancy between 120 and 144 hours after insemination, or just prior to the time of implantation,. Thus, the copper eluting from the device and not the physical characteristics of the metallic
Vol. 26, No.8
EFFECT OF A COPPER IUD DURING EARLY PREGNANCY
IUD was associated with the contraceptive effect of the CuiUD. The presence of the CuiUD had no effect on the ovulation process, as indicated by the finding that normal number of corpora lutea occurred in all groups, regardless of treatment. Although the fertilization rate was low in the presence of a CuiUD 48 hours after insemination the normal rates observed at 72, 96, and' 120 hours suggest that this effect was not associated with the presence of the CuiUD. This interpretation is consistent with that of Polidoro and Black, 7 who concluded from observations made in rabbits 48 hours after insemination that the presence of a copper IUD did not interfer with the fertilization of ova. The CuiUD also had no significant effect on embryonic development or on the rate of transport of the ova through the reproductive tract until just prior to implantation. In summary, all physiologic and embryologic processes of reproduction were essentially normal 120 hours after insemination. At the 144-hour stage, only 1 of 16 ovulated ova was found in the uterine horn containing a CuiUD, and at 192 hours no implantations occurred in the CuiUD-bearing horn. The contraceptive effect of the CuiUD was clearly not systemic. The complete absence of implantation in the CuiUD-containing horn shows that the local effect of the metallic copper device extends beyond the area of the tissue immediately adjacent to the device. In this respect, the metallic copper device differs from a plastic IUD in which implantation is prevented mainly in the area of the device. These studies do not show whether the implantation-inhibiting effect of copper is directly on the blastocyst or is mediated indirectly by more subtle alterations in the uterine tissue. Nutting and Mueller, 9 using tissues obtained from studies in this report as well as from studies in immature rab-
843
bits treated with progesterone, have observed the effects of a CuiUD on the histology and carbonic anhydrase concentration of the uterine tissue and concluded that copper antagonizes the action of progesterone on the uterus. These effects were apparent 48 hours before any demonstrable effect on the blastocyst. This suggests that in the presence of a CuiUD the temporal developmental sequence of tissue changes or production of secretory products under the influence of progesterone are suppressed or delayed during the early stages of pregnancy, creating a uterine milieu which is not favorable for the developing blastocyst. This concept of copper-induced progesterone antagonism is supported by the studies of Eich and Nutting 10 in rabbits which show that blastokinin, a progesterone-dependent uterine protein, is decreased by the presence of copper. Prager11 has reported evidence indicating that protein synthesis is reduced in rats bearing a copper IUD. Chang and Tatum3 used blastocyst transfer procedures and showed that rat blastocysts developed normally when they were transferred from a copper IUDbearing horn to a normal horn. Thus, implantation failure in the presence of copper appears to be caused by an alteration of the uterine environment and not to be a direct effect of copper on the developing blastocyst. SUMMARY
Copper wire intrauterine devices (CuiUD) were surgically inserted into mature female rabbits 14 days prior to artificial insemination. Gold wire intrauterine devices and sham surgery served as controls. Animals were autopsied 48, 72, 96, 120, 144, and 192 hours after artificial insemination. At autopsies performed 48 to 144 hours after insemination, the reproductive tract was flushed
Ntrl'TING AND MUELLER
844
and the embryos were examined. At 192 hours, implantation sites were counted and measured. A significant (}' < 0.05) loss of copper from the devices was observed when the devices were weighed after autopsy. The CuiUD had no effect on ovulation, fertilization, or embryo transport up to 120 hours after artificial insemination. The presence of a CuiUD resulted in a slight, but not statistically significant, increase in embryonic degeneration by 120 hours, almost total absence of blastocysts from the uterus by 144 hours, and complete absence of implantation sites 192 hours after insemination. The antifertility effect of a copper intrauterine device appears to occur in the late preimplantation stage of pregnancy in the rabbit. Acknowledgments. We would especially like to thank M. Cole, J. Pautsch, and M. Dal Corobbo for technical assistance. REFERENCES 1. Zipper JA, Tatum HJ, Medel M, Pastene L, Rivera M: Contraception through the use of intrauterine metals. 1. Copper as an adjunct to the "T" device. Am J Obstet Gynecol 109:771, 1970
August 1975
2. Tatum HJ: Copper-bearing intrauterine devices. Clin Obstet Gynecol 17(1):93, 1974 3. Chang CC, Tatum HJ: A study of the antifertility effect of intrauterine copper. Contraception 1:265, 1970 4. Chang CC, Tatum HJ, Kincl FA: The effect of intrauterine copper and other metals on implantation in rats and hamster. Fertil Steril21:274, 1970 5. Chang CC, Tatum HJ: Some temporal relationships between intrauterine copper wire and its contraceptive action in the rat. Fertil Steril 23:191, 1972 6. Zipper J, Medel M, Prager R: Suppression of fertility by intrauterine copper and zinc in rabbits. Am J Obstet Gynecol 105:529, 1969 7. Polidoro JP, Black DL: The failure of the copper IUD to inhibit fertilization in the rabbit. J Reprod Fertil 23:151, 1970 8. Nutting EF, Mares SE: Inhibition of fertilization in rabbits during treatment with progesterone. Bioi Reprod 2:230, 1970 9. Nutting EF, Mueller MR: The effect of a copper intrauterine device on the uterine histology and progestational response in pregnant and immature rabbits. Fertil Steril .26:845, 1975 10. Eich E, Nutting EF: Unpublished data 11. Prager R: Effect of various types of intrauterine foreign bodies on the incorporation of 35S into microprotein and of thymidine 2-'4C into DNA of rat's endometrium. Fertil Steril 20:944, 1969
