- Email: [email protected]
The influence of pentoxifylline (Trental)* on the antifertility effect of intrauterine devices in rats†‡
FERTILITY AND STERILITY
Vol. 62. No.1. July 1994
Printed on acid-free paper in U. S. A.
Copyright c 1994 The American Fertility Society
The influence of pentoxifylline (Trental)* on the antifertility effect of intrauterine devices in ratst=l=
Jacques W. Ramey, M.D., Ph.D. Michelle E. Starke, M.D. William E. Gibbons, M.D. David F. Archer, M.D.§ The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia
Objective: To test the hypothesis that the inflammatory response stimulated by intrauterine devices (IUDs) plays a role in the antifertility action of IUDs. We treated rats with pentoxifylline (Trental; Hoechst-Roussel Pharmaceuticals, Inc., Somerville, NJ) and evaluated its effect on the anti -implantation action of IUDs. The number of embryos in treated compared with untreated rats was determined. Design: Breeder female Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were randomized into one of five test groups (n = 20 per group). A monofilament nylon IUD was inserted transcervically into one horn of the bicornuate uterus in two groups. The IUD-bearing groups received either intraperitoneal (IP) injections of pentoxifylline (45 mg/kg per every 12 hours) or normal saline (NS). The two non-IUD-bearing groups received IP injections of pentoxifylline or NS. The non-IUD group was not injected. All injections were administered daily for 21 days and the animals then mated. Successful mating was determined by the presence of spermatozoa in vaginal washings. The injections ofpentoxifylline or NS were continued until day 12 of pregnancy when the rats were killed. The total number of embryos in each uterine horn was determined. Setting: University research laboratory. Results: Embryo numbers (1.0 ± 0.6 [mean ± SEM]) were reduced in the IUD horn compared with the contralateral non-IUD horn (6.4 ± 1.0) and with the uterine horns from each of the four other test groups. The number of embryos were increased in the IUD horn (3.5 ± 0.9) of the pentoxifylline-treated rats. Conclusion: Pentoxifylline appeared to reduce the contraceptive effectiveness of the IUDs in this model. Fertil Steril1994;62:181-5 Key Words: IUD, pentoxifylline, leukocytes
The exact mechanism by which inert intrauterine devices (IUDs) exert their antifertility effect reReceived August 2, 1993; revised and accepted February 21, 1994. * Trental; Hoechst-Roussel Pharmaceuticals, Inc., Somerville, New Jersey. t Supported in part by a basic research grant from Eastern Virginia Medical School, Norfolk, Virginia; a grant from the Southern Medical Association, Birmingham, Alabama; and an award from The American College of Obstetricians and Gynecology/Mead Johnson Laboratories, Washington, D.C. Presented at the 39th Annual Meeting of the Society for
*
Vol. 62, No.1, July 1994
mains unclear. Intrauterine devices are known to produce a "sterile" foreign-body, inflammatory reaction in the uterus. It is hypothesized that this foreign-body response is responsible for producing a hostile uterine environment, unfavorable for embryo development.
Gynecologic Investigation, San Antonio, Texas, March 18 to 21, 1992. § Reprint requests: David F. Archer, M.D., The Jones Institute for Reproductive Medicine, 601 Colley Avenue, Norfolk, Virginia 23507 (FAX: 804-446-8998). Ramey et al.
Effect of pentoxifylline on IUD action
181
Increased numbers of intrauterine inflammatory cells, both polymorphonuclear and mononuclear leukocytes have been reported with IUD use in humans and animals (1). A dose-response effect between the numbers of intrauterine leukocytes and the antifertility effect produced by IUDs has been reported in the rabbit (2). Products of activated macrophages have been shown to block the development of preimplantation mouse embryos (3). Takeuchi et al. (4) also have demonstrated the presence of embryotoxic factor(s) found in IUD-bearing secretions from mice uteri. The etiology of the proposed cytotoxic substance(s) is unknown but may be related to substances released from inflammatory cells into the uterine fluid. For example, tumor necrosis factor (TNF), a cytokine released from activated macrophages, has been demonstrated to reduce significantly sperm motility, as well as to decrease the ability of sperm to penetrate zona-free hamster eggs (5-7). Certainly, other local factors cannot be excluded as contributory to the IUD mechanism of action; however, it does appear that leukocytes may play an important role in IUD-induced infertility via the release of cytotoxic factors and/or enhanced phagocytic events that may be directed against embryo formation. Pentoxifylline (Trental; Hoechst- Roussel Pharmaceuticals, Inc., Somerville, NJ), a dimethylxanthine analogue, has been shown to have significant inhibitory effects on leukocyte/macrophage function. Currently, pentoxifylline is approved for clinical use in the treatment of intermittent claudication. It acts by increasing red blood cell deformability, thus enhancing blood flow by decreasing blood viscosity (8). Recent studies have shown that it also has profound direct effects on neutrophil cell membrane function and structure, as well as the ability to inhibit the phagocytosis of latex particles by macrophages in a dose-dependent manner (9, 10). This compound is capable of blocking the production of neutrophil-induced TNF and the action of inflammatory cytokines on neutrophils (11). The rat has been widely used for IUD research (12-15). The insertion of a segment of monofilament nylon by a nonsurgical technique, through the cervical os into one horn of the bicornuate uterus, produces effective sterilization in that horn without affecting the estrus cycle (14). The uterine horn not containing the IUD can act as each animal's own control. Although the antifertility action of IUDs in rats may be due, in part, to inhibition of the decidualizing process, leukocytes also appear to play a role (15). Thus, the objective of the present study 182
Ramey et al.
Effect of pentoxifylline on IUD action
was to examine what effect pentoxifylline may have on the antifertility action of IUDs using a rat model.
MATERIALS AND METHODS Animals
Adult female Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 250 to 300 g were supplied with Purina Laboratory Chow and water ad libitum. The animals were maintained for the duration of the experiment (5 weeks) in a temperature- and light-controlled room (22°C lights on from 5:00 A.M. to 7:00 P.M.). Monofilament nylon IUDs were placed via the vagina and through the cervix in one of the two uterine horns of 40 rats. These animals were then divided into two groups, i.e., control and experimental groups (n = 20 each). The IUD control group received daily injections of normal saline (IUD + NS), whereas the IUD experimental group (IUD + pentoxifylline) received daily injections of pentoxifylline 45 mg/kg per day. All injections were intraperitoneally every 12 hours beginning 3 days before the placement of the IUDs and then for an additional 18 days. At this time, the female rats were mated with proven fertile males. Successful mating was determined by the presence of spermatozoa in vaginal washings. This was considered day 1 of pregnancy. Whole blood was collected via cardiac puncture at the time the rats were killed. Once the rats were killed, the abdomens were opened and the uterine horns examined for the number of embryos. The intraperitoneal (IP) administration of NS or pentoxifylline was continued throughout the pregnancy until the animals were killed on day 12 of pregnancy. Three additional control groups were developed, each with 20 rats. This group of 60 rats did not receive IUDs. One group received daily injections of pentoxifylline; another group received NS; and the final group received no injections (control). Otherwise, these control animals were handled as previously described. Overall, the study consisted of five test groups consisting of a total of 100 animals, i.e., IUD + NS; IUD + pentoxifylline; NS; pentoxifylline; and control. The dose of pentoxifylline used was 45 mg/kg IP per day given every 12 hours (16). Crystalline pentoxifylline (Hoechst-Roussel Pharmaceuticals, Inc.) was solubilized in sterile normal saline immediately before injection. The control groups reFertility and Sterility
<0.05 was considered significant. The data are expressed as mean ± SEM. RESULTS Success of IUD Placement
Figure 1 Rat IUD device made from monofilament nylon (see text for description).
ceived IP injections of sterile NS of equal volume (200 ~L).
Seventy-five percent of the IUDs inserted were recovered in the uterus at the conclusion of the study, i.e., 15 from the rats receiving NS and 15 from animals receiving pentoxifylline. Generally, the IUDs were located low in the uterine horn, 0.5 to 1.0 cm from the internal os of the cervix. Rats that were not found to have an IUD in place were excluded. In 2 of these animals, the IUDs were recovered from within the abdomen. In the other 8 rats, the IUDs were thought to have been expelled spontaneously.
Intrauterine Devices
Effect of the IUDs
The construction and insertion of the IUDs into the rats were performed by the method described by Wrenn et al. (14). The IUDs were made of mono filament nylon 0.28 mm in diameter (81b fishing line). To form a double "5" shape (similar configuration as that of the Lippes loop used in humans), the nylon was wound into a figure-eight configuration between two headless nails, fixed 2 mm apart. This was then immersed into boiling water for 2 minutes and cooled. Double "5" -shaped segments were cut from the filament, resulting in a 20 to 25-mm-Iong, 3-mm-wide device, possessing "memory" when loaded into an inserter for placement in the uterus. Figure 1 shows the IUD device used. Before placement of the IUDs, the rats were lightly anesthetized with ketamine and xylozine at 60 mg/kg and 8 mg/kg IP, respectively. The cervix was visualized through an otoscope using a 5-mm aural speculum placed in the vagina. The cervix was cleaned with betadine on a cotton swab before placement of the IUD. The inserter consisted of a 12.7cm, 20-gauge spinal needle with a blunted end. This was passed through either of the two cervical canals and into the uterine horn for a distance of 35 to 40 mm. The nylon IUD was expelled into the uterus by pushing on the stylet plunger of the special needle.
Group variability for the IUD-containing rats are shown as a raw data plot in Figure 2. In the IUD + NS group, the average number of embryos observed in the IUD-bearing uterine horn was 1.0 ± 0.6 (mean ± SEM). This was significantly less (P < 0.05) than the number of embryos found in the non -IUD bearing contralateral horn 6.4 ± 1.0, as well as the number of embryos observed in the uterine horns of each of the other four test groups. The number of embryos was lower in the uterine horn that contained the IUD in the IUD + pentoxifylline group (3.5 ± 0.9) as compared with the contralateral horn (7.2 ± 0.9), but this difference was not a statistically significant reduction.
14.----------------, 12
e
10
~
8.
8
~
.s Ji
6
C
... NS
PTX
IUD(-) + NS
Data Analysis
The data were analyzed statistically by a two-way analysis of variance. Differences between treatment means were further analyzed by least significant difference using the T method. A P value of Vol. 62, No.1, July 1994
.. I.... IUD(+) + NS
IUD(-) + PTX
...
IUD(+) + PTX
Figure 2 A comparison of the number of embryos per horn a$ represented using a raw data plot for the five test groups are shown in Figure 2. IUD (+ ) represents the number of embryos in the IUD-containing uterine horn, whereas IUD (-) is the number in the horn without an IUD in the two test groups receiving IUDs. The three test groups not receiving IUDs are designated control (C), pentoxifylline (PTX), and normal saline (NS). Ramey et al.
Effect of pentoxifylline on IUD action
183
The Effect of Pentoxifylline
Comparisons between the two groups with IUDbearing horns and the three groups without IUDs can also be seen in Figure 2. Pentoxifylline significantly increased the number of embryos found in the IUD-bearing uterine horn as compared with the IUD-bearing horn of the NS + IUD group. In the three control groups without IUDs (control, NS, and pentoxifylline) the number of embryos per uterine horn in the NS-injected group (5.0 ± 0.6) was significantly less than the control (no injections) (6.8 ± 0.7) or pentoxifylline groups (7.0 ± 0.4). Within the non-IUD bearing groups receiving injections, pentoxifylline significantly increased the number of embryos comparable with those numbers found in the control group. The number of embryos per horn in the NS group was not significantly different compared with the number of embryos in the IUD horn of the IUD + pentoxifylline group. The number of embryos per horn in the control and pentoxifylline groups, however, were significantly greater than those observed for embryos found in the IUD horn of the pentoxifylline + IUD group.
DISCUSSION
The sterile inflammatory response stimulated by IUDs produces an elevation in uterine leukocytes, which are subsequently hypothesized to playa role in the contraceptive effectiveness of the IUD. The objective of the present study was to further elucidate what role leukocytes may play in IUD-induced contraception by inhibiting leukocyte function using pentoxifylline. Few studies have investigated the ability of anti-inflammatory drugs to reverse the action of IUDs. Naproxen (Syntex Laboratories, Inc., Palo Alto, CA) and other similar nonsteroidal, anti-inflammatory drugs can reduce edema and inflammation in IUD endometrium, but these drugs do not appear to interfere with the antifertility effect of IUDs in the rat (12, 17). In contrast, chlorambucil, a carcinogen and potent suppressor of bone marrow function, has been shown to partially overcome the antifertility effect of the IUDbearing horn in rats, which would suggest that its effect may be due to the suppression of leukocyte production (13). However, this drug also significantly reduced the number of embryos found in the non -IUD bearing horn, suggesting that chlorambucil alone has an antifertility effect (13). Using less toxic drugs that inhibit leukocyte 184
Ramey et al.
Effect of pentoxifylline on IUD action
function directly, such as pentoxifylline, may prove to be more informative. Pentoxifylline has been reported to enhance fertility. In a novel study, Steinleitner et al. (18) demonstrated that transferring heterologous activated peritoneal inflammatory cells from an inbred strain of mice intraperitoneally into separate mice of the same strain resulted in the reduction of fertility in those animals. This effect was partially reversed by pretreating the mice with pentoxifylline. A relationship between infertility and endometriosis is well documented in the medical literature (19). Endometriosis produces a localized peritoneal inflammatory response, and this has been implicated as playing a role in endometriosis-induced infertility (19). Consequently, in a separate study, Steinleitner et al. (20), using a murine animal model for endometriosis, also demonstrated that periovulatory administration of pentoxifylline could reduce the adverse effects of surgically induced endometriosis on fertilization. The positive influence pentoxifylline had on reproductive performance in these two studies was concluded to be the result of the immunomodulatory actions of pentoxifylline (18, 20). In the present study, the contraceptive action of the IUD appeared to be reversed after treatment with pentoxifylline. This response may be the result of the immunomodulatory properties associated with pentoxifylline. Future studies are required to elucidate the mechanism(s) by which pentoxifylline appears to enhance fertility. A surprising observation was that IP injections of NS significantly reduced fertility in these animals. The reason for the reduction in embryo numbers is not known. No gross IP abnormalities, such as increased adhesions, were noted in these animals at the time of autopsy. The IP injections every 12 hours could have produced a mild IP inflammatory response that, in turn, may have altered fertility. The administration ofpentoxifylline appears to abrogate the observed adverse influence of the IP injection of NS on the number of embryos. This enhancement may be due to the immunomodulatory actions of pentoxifylline; however, other activities of pentoxifylline cannot be ruled out. Pentoxifylline has been reported to augment motility in dysfunctional human sperm (21). Consequently, the enhancement of sperm performance may be an alternate mechanism by which pentoxifylline may contribute to increased fertility (20). An additional experiment group consisting of IUD-bearing rats not receiving any IP injections may have been helpFertility and Sterility
ful in interpreting the specific effect(s) of pentoxifylline in this model. The results of the present study suggest that pentoxifylline can reverse the antifertility action of IUDs in rats. This effect may be due to the inhibition of the inflammatory response stimulated by the IUDs. Other enhancing actions of pentoxifylline on fertility cannot be ruled out. Pentoxifylline is currently marketed for the treatment of peripheral vascular disease, but recent data propose that it may also have applications in the treatment of pathological conditions involving the overproduction of inflammatory cytokines, i.e., sepsis and hem0rrhagic shock (22). This study and others also suggest that pentoxifylline, as well as related more active analogues, may have a new therapeutic role in the treatment and/or prevention of certain forms of infertility (18, 20, 23).
8.
9.
10.
11.
12.
13.
Acknowledgments. The authors express their appreciation to Ms. Pauline M. Clynes of the Jones Institute for Reproductive Medicine, Norfolk, Virginia, for her critical editorial review and preparation of this manuscript and to Phile Koury, M.D., and Thang Tran, M.D., for their helpful technical assistance in the undertaking of this study. The authors also thank HoechstRoussel Pharmaceuticals, Inc., Somerville, New Jersey, for providing the crystalline pentoxifylline.
14.
REFERENCES
18.
1. W orid Health Organization. Mechanism of action, safety and efficacy of intrauterine devices. WHO Tech Rep Ser 1987;753:1-91. 2. Sahwi SE, Moyer DL. The leukocytic response to an intrauterine foreign body in the rabbit. Fertil Steril1971;22:398408. 3. Hill JA, Haimovici F, Anderson DJ. Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro. J Immunol 1987;139:2250-4. 4. Takeuchi K, Mori A, Yamamoto S, Sonoda T, Nagata Y. Effect of the secretions from the IUD-bearing uterus on peri-implantation mouse embryos. Contraception 1990;41: 655-62. 5. Papadimitriou JM, Ashman RB. Macrophages: current views on their differentiation, structure and function. UItrastruct Pathol 1989;13:343-72. 6. Hill JA, Haimovici F, Politch JA, Anderson DJ. Effects of soluble products of activated lymphocytes and macrophages (lymphokines and monokines) on human sperm motion parameters. Fertil Steril1987;47:460-5. 7. Hill JA, Cohen J, Anderson DJ. The effects oflymphokines
Vol. 62, No.1, July 1994
15.
16.
17.
19.
20.
21.
22. 23.
and monokines on human sperm fertilizing ability in the zona-free hamster egg penetration test. Am J Obstet Gynecol 1989;160:1154-9. Ward A, Clissold SP. Pentoxifylline, a review of its pharmacodynamic and pharmacokinetic properties and its therapeutic efficacy. Drugs 1987;34:50-97. Currie MS, Rao KMK, Padmanabham J, Jones A, Crawford J, Cohen HS. Stimulus-specific effects of pentoxifylline on neutrophil CR3 expression, degranulation and superoxide production. J Leukoc BioI 1990;47:244-50. Bessler H, Gilgal R, Djaldetti M, Zahavi 1. Effect of pentox ifylline on the phagocytic activity, cAMP levels and superoxide anion production by monocytes and polymorphonuclear cells. J Leukoc BioI 1986;40:747-54. Sullivan GW, Carper HT, Novick WJ Jr, Mandell GL. Inhibition of the inflammatory action of interleukin-1 and tumor necrosis factor (alpha) on neutrophil function by pentoxifylline. Infect Immun 1988;568:1722-9. Dachung T, Xirui W, Biren Z, Yanggiang L, Yu L. Influence of naproxen on uterine PGF2a and antifertility effect of IUDs in rats. Eicosanoids 1989;2:47-9. Holub WR. Immunoglobulin G levels and use of immunosuppressives with an intrauterine device in the rat. Am J Obstet Gynecol 1972;114:492-9. Wrenn TR, Wood JR, Bitman J. A new technique for introducing IUDs into rat uteri. J Reprod FertiI1968;16:515-7. Wood JC, Kirby DRS. The effect of uterine anastomosis on the action of IUCD in rats. Am J Obstet Gynecol 1968;102:1041-2. Brunner LJ, Vadici K, Lyer LV, Luke DR. Prevention of cyclosporine-induced nephrotoxicity with pentoxifylline. Ren Fail Hi89;11:97-104. Barthwal \1:, Srivastava K. Histologic studies on endometrium of menstruating monkeys wearing IUDs: comparative evaluation of drugs. Adv Contracept 1990;6:113-24. Steinleitner A, Lambert H, Roy S. Immunomodulation with pentoxifylline abrogates macrophage-mediated infertility in an in vivo model: a paradigm for a novel approach to the treatment of endometriosis-associated subfertility. Fertil Steril 1991;55:26-31. Halme J, Surrey ES. Endometriosis and infertility: the mechanisms involved. In: Chadha DR, Buttram VC, editors. Current concepts in endometriosis. N ew York: Alan R. Liss, Inc., 1990:157-78. Steinleitner A, Lambert H, Suarez M, Serpa N, Roy S. Immunomodulation in the treatment of endometriosis-associated subfertility: use ofpentoxifylline to reverse the inhibition of fertilization by surgically induced endometriosis in a rodent model. Fertil Steril 1991;56:975-9. Yovich JM, Edirisinghe WR, Cummins JM, Yovich JL. Influence of pentoxifylline in severe male factor infertility. Fertil Steril 1990;53:715-22. Novick WJ Jr. Pentoxifylline and leukocyte function. Crit Care Rep 1991;2:236-40. Steinleitner A, Lambert H, Kazensky C, Danks P, Roy S. Pentoxifylline, a methylxanthine derivative, prevents postsurgical adhesion reformation in rabbits. Obstet Gynecol 1990;75:926-8.
Ramey et al.
Effect of pentoxifylline on IUD action
185
Vol. 62. No.1. July 1994
Printed on acid-free paper in U. S. A.
Copyright c 1994 The American Fertility Society
The influence of pentoxifylline (Trental)* on the antifertility effect of intrauterine devices in ratst=l=
Jacques W. Ramey, M.D., Ph.D. Michelle E. Starke, M.D. William E. Gibbons, M.D. David F. Archer, M.D.§ The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia
Objective: To test the hypothesis that the inflammatory response stimulated by intrauterine devices (IUDs) plays a role in the antifertility action of IUDs. We treated rats with pentoxifylline (Trental; Hoechst-Roussel Pharmaceuticals, Inc., Somerville, NJ) and evaluated its effect on the anti -implantation action of IUDs. The number of embryos in treated compared with untreated rats was determined. Design: Breeder female Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were randomized into one of five test groups (n = 20 per group). A monofilament nylon IUD was inserted transcervically into one horn of the bicornuate uterus in two groups. The IUD-bearing groups received either intraperitoneal (IP) injections of pentoxifylline (45 mg/kg per every 12 hours) or normal saline (NS). The two non-IUD-bearing groups received IP injections of pentoxifylline or NS. The non-IUD group was not injected. All injections were administered daily for 21 days and the animals then mated. Successful mating was determined by the presence of spermatozoa in vaginal washings. The injections ofpentoxifylline or NS were continued until day 12 of pregnancy when the rats were killed. The total number of embryos in each uterine horn was determined. Setting: University research laboratory. Results: Embryo numbers (1.0 ± 0.6 [mean ± SEM]) were reduced in the IUD horn compared with the contralateral non-IUD horn (6.4 ± 1.0) and with the uterine horns from each of the four other test groups. The number of embryos were increased in the IUD horn (3.5 ± 0.9) of the pentoxifylline-treated rats. Conclusion: Pentoxifylline appeared to reduce the contraceptive effectiveness of the IUDs in this model. Fertil Steril1994;62:181-5 Key Words: IUD, pentoxifylline, leukocytes
The exact mechanism by which inert intrauterine devices (IUDs) exert their antifertility effect reReceived August 2, 1993; revised and accepted February 21, 1994. * Trental; Hoechst-Roussel Pharmaceuticals, Inc., Somerville, New Jersey. t Supported in part by a basic research grant from Eastern Virginia Medical School, Norfolk, Virginia; a grant from the Southern Medical Association, Birmingham, Alabama; and an award from The American College of Obstetricians and Gynecology/Mead Johnson Laboratories, Washington, D.C. Presented at the 39th Annual Meeting of the Society for
*
Vol. 62, No.1, July 1994
mains unclear. Intrauterine devices are known to produce a "sterile" foreign-body, inflammatory reaction in the uterus. It is hypothesized that this foreign-body response is responsible for producing a hostile uterine environment, unfavorable for embryo development.
Gynecologic Investigation, San Antonio, Texas, March 18 to 21, 1992. § Reprint requests: David F. Archer, M.D., The Jones Institute for Reproductive Medicine, 601 Colley Avenue, Norfolk, Virginia 23507 (FAX: 804-446-8998). Ramey et al.
Effect of pentoxifylline on IUD action
181
Increased numbers of intrauterine inflammatory cells, both polymorphonuclear and mononuclear leukocytes have been reported with IUD use in humans and animals (1). A dose-response effect between the numbers of intrauterine leukocytes and the antifertility effect produced by IUDs has been reported in the rabbit (2). Products of activated macrophages have been shown to block the development of preimplantation mouse embryos (3). Takeuchi et al. (4) also have demonstrated the presence of embryotoxic factor(s) found in IUD-bearing secretions from mice uteri. The etiology of the proposed cytotoxic substance(s) is unknown but may be related to substances released from inflammatory cells into the uterine fluid. For example, tumor necrosis factor (TNF), a cytokine released from activated macrophages, has been demonstrated to reduce significantly sperm motility, as well as to decrease the ability of sperm to penetrate zona-free hamster eggs (5-7). Certainly, other local factors cannot be excluded as contributory to the IUD mechanism of action; however, it does appear that leukocytes may play an important role in IUD-induced infertility via the release of cytotoxic factors and/or enhanced phagocytic events that may be directed against embryo formation. Pentoxifylline (Trental; Hoechst- Roussel Pharmaceuticals, Inc., Somerville, NJ), a dimethylxanthine analogue, has been shown to have significant inhibitory effects on leukocyte/macrophage function. Currently, pentoxifylline is approved for clinical use in the treatment of intermittent claudication. It acts by increasing red blood cell deformability, thus enhancing blood flow by decreasing blood viscosity (8). Recent studies have shown that it also has profound direct effects on neutrophil cell membrane function and structure, as well as the ability to inhibit the phagocytosis of latex particles by macrophages in a dose-dependent manner (9, 10). This compound is capable of blocking the production of neutrophil-induced TNF and the action of inflammatory cytokines on neutrophils (11). The rat has been widely used for IUD research (12-15). The insertion of a segment of monofilament nylon by a nonsurgical technique, through the cervical os into one horn of the bicornuate uterus, produces effective sterilization in that horn without affecting the estrus cycle (14). The uterine horn not containing the IUD can act as each animal's own control. Although the antifertility action of IUDs in rats may be due, in part, to inhibition of the decidualizing process, leukocytes also appear to play a role (15). Thus, the objective of the present study 182
Ramey et al.
Effect of pentoxifylline on IUD action
was to examine what effect pentoxifylline may have on the antifertility action of IUDs using a rat model.
MATERIALS AND METHODS Animals
Adult female Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 250 to 300 g were supplied with Purina Laboratory Chow and water ad libitum. The animals were maintained for the duration of the experiment (5 weeks) in a temperature- and light-controlled room (22°C lights on from 5:00 A.M. to 7:00 P.M.). Monofilament nylon IUDs were placed via the vagina and through the cervix in one of the two uterine horns of 40 rats. These animals were then divided into two groups, i.e., control and experimental groups (n = 20 each). The IUD control group received daily injections of normal saline (IUD + NS), whereas the IUD experimental group (IUD + pentoxifylline) received daily injections of pentoxifylline 45 mg/kg per day. All injections were intraperitoneally every 12 hours beginning 3 days before the placement of the IUDs and then for an additional 18 days. At this time, the female rats were mated with proven fertile males. Successful mating was determined by the presence of spermatozoa in vaginal washings. This was considered day 1 of pregnancy. Whole blood was collected via cardiac puncture at the time the rats were killed. Once the rats were killed, the abdomens were opened and the uterine horns examined for the number of embryos. The intraperitoneal (IP) administration of NS or pentoxifylline was continued throughout the pregnancy until the animals were killed on day 12 of pregnancy. Three additional control groups were developed, each with 20 rats. This group of 60 rats did not receive IUDs. One group received daily injections of pentoxifylline; another group received NS; and the final group received no injections (control). Otherwise, these control animals were handled as previously described. Overall, the study consisted of five test groups consisting of a total of 100 animals, i.e., IUD + NS; IUD + pentoxifylline; NS; pentoxifylline; and control. The dose of pentoxifylline used was 45 mg/kg IP per day given every 12 hours (16). Crystalline pentoxifylline (Hoechst-Roussel Pharmaceuticals, Inc.) was solubilized in sterile normal saline immediately before injection. The control groups reFertility and Sterility
<0.05 was considered significant. The data are expressed as mean ± SEM. RESULTS Success of IUD Placement
Figure 1 Rat IUD device made from monofilament nylon (see text for description).
ceived IP injections of sterile NS of equal volume (200 ~L).
Seventy-five percent of the IUDs inserted were recovered in the uterus at the conclusion of the study, i.e., 15 from the rats receiving NS and 15 from animals receiving pentoxifylline. Generally, the IUDs were located low in the uterine horn, 0.5 to 1.0 cm from the internal os of the cervix. Rats that were not found to have an IUD in place were excluded. In 2 of these animals, the IUDs were recovered from within the abdomen. In the other 8 rats, the IUDs were thought to have been expelled spontaneously.
Intrauterine Devices
Effect of the IUDs
The construction and insertion of the IUDs into the rats were performed by the method described by Wrenn et al. (14). The IUDs were made of mono filament nylon 0.28 mm in diameter (81b fishing line). To form a double "5" shape (similar configuration as that of the Lippes loop used in humans), the nylon was wound into a figure-eight configuration between two headless nails, fixed 2 mm apart. This was then immersed into boiling water for 2 minutes and cooled. Double "5" -shaped segments were cut from the filament, resulting in a 20 to 25-mm-Iong, 3-mm-wide device, possessing "memory" when loaded into an inserter for placement in the uterus. Figure 1 shows the IUD device used. Before placement of the IUDs, the rats were lightly anesthetized with ketamine and xylozine at 60 mg/kg and 8 mg/kg IP, respectively. The cervix was visualized through an otoscope using a 5-mm aural speculum placed in the vagina. The cervix was cleaned with betadine on a cotton swab before placement of the IUD. The inserter consisted of a 12.7cm, 20-gauge spinal needle with a blunted end. This was passed through either of the two cervical canals and into the uterine horn for a distance of 35 to 40 mm. The nylon IUD was expelled into the uterus by pushing on the stylet plunger of the special needle.
Group variability for the IUD-containing rats are shown as a raw data plot in Figure 2. In the IUD + NS group, the average number of embryos observed in the IUD-bearing uterine horn was 1.0 ± 0.6 (mean ± SEM). This was significantly less (P < 0.05) than the number of embryos found in the non -IUD bearing contralateral horn 6.4 ± 1.0, as well as the number of embryos observed in the uterine horns of each of the other four test groups. The number of embryos was lower in the uterine horn that contained the IUD in the IUD + pentoxifylline group (3.5 ± 0.9) as compared with the contralateral horn (7.2 ± 0.9), but this difference was not a statistically significant reduction.
14.----------------, 12
e
10
~
8.
8
~
.s Ji
6
C
... NS
PTX
IUD(-) + NS
Data Analysis
The data were analyzed statistically by a two-way analysis of variance. Differences between treatment means were further analyzed by least significant difference using the T method. A P value of Vol. 62, No.1, July 1994
.. I.... IUD(+) + NS
IUD(-) + PTX
...
IUD(+) + PTX
Figure 2 A comparison of the number of embryos per horn a$ represented using a raw data plot for the five test groups are shown in Figure 2. IUD (+ ) represents the number of embryos in the IUD-containing uterine horn, whereas IUD (-) is the number in the horn without an IUD in the two test groups receiving IUDs. The three test groups not receiving IUDs are designated control (C), pentoxifylline (PTX), and normal saline (NS). Ramey et al.
Effect of pentoxifylline on IUD action
183
The Effect of Pentoxifylline
Comparisons between the two groups with IUDbearing horns and the three groups without IUDs can also be seen in Figure 2. Pentoxifylline significantly increased the number of embryos found in the IUD-bearing uterine horn as compared with the IUD-bearing horn of the NS + IUD group. In the three control groups without IUDs (control, NS, and pentoxifylline) the number of embryos per uterine horn in the NS-injected group (5.0 ± 0.6) was significantly less than the control (no injections) (6.8 ± 0.7) or pentoxifylline groups (7.0 ± 0.4). Within the non-IUD bearing groups receiving injections, pentoxifylline significantly increased the number of embryos comparable with those numbers found in the control group. The number of embryos per horn in the NS group was not significantly different compared with the number of embryos in the IUD horn of the IUD + pentoxifylline group. The number of embryos per horn in the control and pentoxifylline groups, however, were significantly greater than those observed for embryos found in the IUD horn of the pentoxifylline + IUD group.
DISCUSSION
The sterile inflammatory response stimulated by IUDs produces an elevation in uterine leukocytes, which are subsequently hypothesized to playa role in the contraceptive effectiveness of the IUD. The objective of the present study was to further elucidate what role leukocytes may play in IUD-induced contraception by inhibiting leukocyte function using pentoxifylline. Few studies have investigated the ability of anti-inflammatory drugs to reverse the action of IUDs. Naproxen (Syntex Laboratories, Inc., Palo Alto, CA) and other similar nonsteroidal, anti-inflammatory drugs can reduce edema and inflammation in IUD endometrium, but these drugs do not appear to interfere with the antifertility effect of IUDs in the rat (12, 17). In contrast, chlorambucil, a carcinogen and potent suppressor of bone marrow function, has been shown to partially overcome the antifertility effect of the IUDbearing horn in rats, which would suggest that its effect may be due to the suppression of leukocyte production (13). However, this drug also significantly reduced the number of embryos found in the non -IUD bearing horn, suggesting that chlorambucil alone has an antifertility effect (13). Using less toxic drugs that inhibit leukocyte 184
Ramey et al.
Effect of pentoxifylline on IUD action
function directly, such as pentoxifylline, may prove to be more informative. Pentoxifylline has been reported to enhance fertility. In a novel study, Steinleitner et al. (18) demonstrated that transferring heterologous activated peritoneal inflammatory cells from an inbred strain of mice intraperitoneally into separate mice of the same strain resulted in the reduction of fertility in those animals. This effect was partially reversed by pretreating the mice with pentoxifylline. A relationship between infertility and endometriosis is well documented in the medical literature (19). Endometriosis produces a localized peritoneal inflammatory response, and this has been implicated as playing a role in endometriosis-induced infertility (19). Consequently, in a separate study, Steinleitner et al. (20), using a murine animal model for endometriosis, also demonstrated that periovulatory administration of pentoxifylline could reduce the adverse effects of surgically induced endometriosis on fertilization. The positive influence pentoxifylline had on reproductive performance in these two studies was concluded to be the result of the immunomodulatory actions of pentoxifylline (18, 20). In the present study, the contraceptive action of the IUD appeared to be reversed after treatment with pentoxifylline. This response may be the result of the immunomodulatory properties associated with pentoxifylline. Future studies are required to elucidate the mechanism(s) by which pentoxifylline appears to enhance fertility. A surprising observation was that IP injections of NS significantly reduced fertility in these animals. The reason for the reduction in embryo numbers is not known. No gross IP abnormalities, such as increased adhesions, were noted in these animals at the time of autopsy. The IP injections every 12 hours could have produced a mild IP inflammatory response that, in turn, may have altered fertility. The administration ofpentoxifylline appears to abrogate the observed adverse influence of the IP injection of NS on the number of embryos. This enhancement may be due to the immunomodulatory actions of pentoxifylline; however, other activities of pentoxifylline cannot be ruled out. Pentoxifylline has been reported to augment motility in dysfunctional human sperm (21). Consequently, the enhancement of sperm performance may be an alternate mechanism by which pentoxifylline may contribute to increased fertility (20). An additional experiment group consisting of IUD-bearing rats not receiving any IP injections may have been helpFertility and Sterility
ful in interpreting the specific effect(s) of pentoxifylline in this model. The results of the present study suggest that pentoxifylline can reverse the antifertility action of IUDs in rats. This effect may be due to the inhibition of the inflammatory response stimulated by the IUDs. Other enhancing actions of pentoxifylline on fertility cannot be ruled out. Pentoxifylline is currently marketed for the treatment of peripheral vascular disease, but recent data propose that it may also have applications in the treatment of pathological conditions involving the overproduction of inflammatory cytokines, i.e., sepsis and hem0rrhagic shock (22). This study and others also suggest that pentoxifylline, as well as related more active analogues, may have a new therapeutic role in the treatment and/or prevention of certain forms of infertility (18, 20, 23).
8.
9.
10.
11.
12.
13.
Acknowledgments. The authors express their appreciation to Ms. Pauline M. Clynes of the Jones Institute for Reproductive Medicine, Norfolk, Virginia, for her critical editorial review and preparation of this manuscript and to Phile Koury, M.D., and Thang Tran, M.D., for their helpful technical assistance in the undertaking of this study. The authors also thank HoechstRoussel Pharmaceuticals, Inc., Somerville, New Jersey, for providing the crystalline pentoxifylline.
14.
REFERENCES
18.
1. W orid Health Organization. Mechanism of action, safety and efficacy of intrauterine devices. WHO Tech Rep Ser 1987;753:1-91. 2. Sahwi SE, Moyer DL. The leukocytic response to an intrauterine foreign body in the rabbit. Fertil Steril1971;22:398408. 3. Hill JA, Haimovici F, Anderson DJ. Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro. J Immunol 1987;139:2250-4. 4. Takeuchi K, Mori A, Yamamoto S, Sonoda T, Nagata Y. Effect of the secretions from the IUD-bearing uterus on peri-implantation mouse embryos. Contraception 1990;41: 655-62. 5. Papadimitriou JM, Ashman RB. Macrophages: current views on their differentiation, structure and function. UItrastruct Pathol 1989;13:343-72. 6. Hill JA, Haimovici F, Politch JA, Anderson DJ. Effects of soluble products of activated lymphocytes and macrophages (lymphokines and monokines) on human sperm motion parameters. Fertil Steril1987;47:460-5. 7. Hill JA, Cohen J, Anderson DJ. The effects oflymphokines
Vol. 62, No.1, July 1994
15.
16.
17.
19.
20.
21.
22. 23.
and monokines on human sperm fertilizing ability in the zona-free hamster egg penetration test. Am J Obstet Gynecol 1989;160:1154-9. Ward A, Clissold SP. Pentoxifylline, a review of its pharmacodynamic and pharmacokinetic properties and its therapeutic efficacy. Drugs 1987;34:50-97. Currie MS, Rao KMK, Padmanabham J, Jones A, Crawford J, Cohen HS. Stimulus-specific effects of pentoxifylline on neutrophil CR3 expression, degranulation and superoxide production. J Leukoc BioI 1990;47:244-50. Bessler H, Gilgal R, Djaldetti M, Zahavi 1. Effect of pentox ifylline on the phagocytic activity, cAMP levels and superoxide anion production by monocytes and polymorphonuclear cells. J Leukoc BioI 1986;40:747-54. Sullivan GW, Carper HT, Novick WJ Jr, Mandell GL. Inhibition of the inflammatory action of interleukin-1 and tumor necrosis factor (alpha) on neutrophil function by pentoxifylline. Infect Immun 1988;568:1722-9. Dachung T, Xirui W, Biren Z, Yanggiang L, Yu L. Influence of naproxen on uterine PGF2a and antifertility effect of IUDs in rats. Eicosanoids 1989;2:47-9. Holub WR. Immunoglobulin G levels and use of immunosuppressives with an intrauterine device in the rat. Am J Obstet Gynecol 1972;114:492-9. Wrenn TR, Wood JR, Bitman J. A new technique for introducing IUDs into rat uteri. J Reprod FertiI1968;16:515-7. Wood JC, Kirby DRS. The effect of uterine anastomosis on the action of IUCD in rats. Am J Obstet Gynecol 1968;102:1041-2. Brunner LJ, Vadici K, Lyer LV, Luke DR. Prevention of cyclosporine-induced nephrotoxicity with pentoxifylline. Ren Fail Hi89;11:97-104. Barthwal \1:, Srivastava K. Histologic studies on endometrium of menstruating monkeys wearing IUDs: comparative evaluation of drugs. Adv Contracept 1990;6:113-24. Steinleitner A, Lambert H, Roy S. Immunomodulation with pentoxifylline abrogates macrophage-mediated infertility in an in vivo model: a paradigm for a novel approach to the treatment of endometriosis-associated subfertility. Fertil Steril 1991;55:26-31. Halme J, Surrey ES. Endometriosis and infertility: the mechanisms involved. In: Chadha DR, Buttram VC, editors. Current concepts in endometriosis. N ew York: Alan R. Liss, Inc., 1990:157-78. Steinleitner A, Lambert H, Suarez M, Serpa N, Roy S. Immunomodulation in the treatment of endometriosis-associated subfertility: use ofpentoxifylline to reverse the inhibition of fertilization by surgically induced endometriosis in a rodent model. Fertil Steril 1991;56:975-9. Yovich JM, Edirisinghe WR, Cummins JM, Yovich JL. Influence of pentoxifylline in severe male factor infertility. Fertil Steril 1990;53:715-22. Novick WJ Jr. Pentoxifylline and leukocyte function. Crit Care Rep 1991;2:236-40. Steinleitner A, Lambert H, Kazensky C, Danks P, Roy S. Pentoxifylline, a methylxanthine derivative, prevents postsurgical adhesion reformation in rabbits. Obstet Gynecol 1990;75:926-8.
Ramey et al.
Effect of pentoxifylline on IUD action
185