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The rat as an animal model for endometriosis to examine recurrence of ectopic endometrial tissue after regression*†
FERTILITY AND STERILITY
Vol. 53, No.5, May 1990
Copyright " 1990 The American Fertility Society
Printed on acid-free paper in U.S.A.
The rat as an animal model for endometriosis to examine recurrence of ectopic endometrial tissue after regression*t
Kadaba Rajkumar, Ph.D.:j: Philipp W. Schott, B.S. Charles W. Simpson, M.D. Reproductive Biology Research Unit, Department of Obstetrics and Gynecology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
The effects of pregnancy and ovariectomy on the growth of endometrial implants were examined in rats with experimentally induced endometriosis. Ectopic implants regressed completely after ovariectomy and we were unable to detect any viable endometrial cells in histological examination of the implant sites. Administration of 17!3-estradiol cypionate to ovariectomized rats with regressed endometrial implants led to recurrence of the regressed implants. Animals with experimentally induced endometriosis were fertile and the number of embryos on day 10 of pregnancy was not different from the control group. Ectopic implants which regressed during pregnancy recurred 1 month after parturition. It is concluded that endometrial cells may survive at the implanted site even after apparent complete morphological regression, which has the potential to grow into an implant. Fertil Steril53:921, 1990
Endometriosis is a disease characterized by the presence and proliferation of endometrial tissue outside the uterus. Its incidence ranges from 7% to 50% of menstruating women. 1•2 In mild to moderate forms of endometriosis, endocrine treatments are commonly used. The beneficial aspects of danazol in the treatment of endometriosis are counterbalanced by recurrence rates of 20% to 50%.3 Although the highest recurrence rates (51%) were reported when symptoms served as the diagnostic parameter,4 implants were noted at repeat laparoscopy in 23% of patients 6 months after completing therapy. 5 Gonadotropin-releasing hormone agonists (GnRH-a), which allow temporary suppres-
Received June 19, 1989; revised and accepted January 2, 1990. * Supported by the Saskatchewan Health Research Board. t Presented at the Second International Symposia on Endometriosis, Houston, Texas, May 2 to 4, 1989. t Reprint requests: Kadaba Rajkumar, Ph.D., Department of Obstetrics and Gynecology, University of Saskatchewan College of Medicine, Reproductive Biology Research Unit, Saskatoon, Saskatchewan S7N OXO, Canada. Vol. 53, No.5, May 1990
sion of ovarian function, are the most recent hormonal therapeutic agents. Complete suppression of endometriosis has been demonstrated with GnRH-a therapy. 6 However, recurrence of symptoms of endometriosis within 6 months of completion of GnRH-a treatment has been reported. 7•8 In human studies it cannot be ascertained whether recurrence after the withdrawal of therapy is due to growth of regressed implants or the presence of new implants. Animal models that do not have spontaneous endometriosis may be useful to investigate this phenomenon. In the present studies we examined the regression of ectopic implants after ovariectomy and during pregnancy. Further, the recurrences of the implants were monitored in ovariectomized animals treated with estrogen and in pregnant animals after parturition. MATERIALS AND METHODS
Mature cycling female rats (Sprague-Dawley) of 170 to 200 g were maintained on a 14- to 10-hour (light to dark) photoperiod and fed rat chow and Rajkumar et al. Endometriosis in rat models
921
water ad libitum. The rats were monitored daily for reproductive cyclicity by examination of vaginal cytological smears. Only those rats exhibiting a normal estrous cycle were used. All surgical procedures were done under aseptic conditions in a sterile flow hood. Animals were administered 0.05 to 0.1 mg of ketamine hydrochloride solution (200 mg/mL), followed by anesthesia using halothane. During laparotomy, the blood vessels supplying the right uterine horn were ligated and approximately 2 em were resected. End-to-end anastomosis of the uterus after resection was performed with 6-0 nylon sutures (Ethicon, Johnson & Johnson Company, Somerville, NJ). The resected horn was opened in isotonic sterile saline and the myometrium was peeled from the endometrium under a dissecting microscope. Two pieces of endometrium measuring 5 X 6 mm (approximate) were cut and then sutured intraperitoneally to the inside of the peritoneal membrane with the sutures (6-0 nylon; Ethicon) passing through the membrane and anchoring into the abdominal musculature on all four sides of the implant. We attempted to place the implant at a point equidistance between the ventral midline, the vertebral column, the caudal rib, and cranial pelvis (i.e., center of the flank). In addition, an effort was made to suture the implant to a site very near a larger blood vessel to ensure as rapid a vascularization as possible. After transplantation the size of the implanted tissue was measured. The criterion used to assess the viability of the graft was the formation of a cyst within 1 month after transplantation. One month after transplantation of endometrial tissue, the animals were laparotomized and endometrial implant surface area (length X width) was measured. One subgroup of animals underwent ovariectomy to remove the endogenous supply of ovarian steroids; the other subgroup served as control. After 1, 2, and 4 months of ovariectomy, the endometrial implants were examined under laparotomy. In some animals, implanted sites were removed by dissection 2 months after ovariectomy and processed for histology. Two and 4 months after ovariectomy, 17/J-estradiol (E 2 ) cypionate (60 ,ug/kg) was administered subcutaneously in oil, twice a week for 30 days. After 1 month of treatment with E 2 , the animals were again laparotomized and the endometrial implant size was measured. Some implants were processed for histology. In a second group of rats, 15 days after laparotomy (to measure the size of the endometrial implant), the animals were mated with males of proven fertility. The successful mating was as922
Rajkumar et al.
Endometriosis in rat models
Figure 1 A healthy endometrial implant on the peritoneal surface of the abdominal wall of a rat 1 month after surgical implantation.
sessed by the presence of sperm in the vaginal smear. This was designated as day 1 of pregnancy. One group of sham-operated control rats was included in the study in addition to the animals with induced endometriosis. In sham-operated control, 2 em of the right uterine horn was resected followed by end-to-end anastomosis of the resected horn. The suture without endometrium were also attached to the peritoneal cavity. On day 10 of pregnancy the animals were laparotomized and the number of embryos in the uterine horns was counted. In rats with induced endometriosis, the endometrial implant size was also measured. At term the number of fetus delivered was also recorded. In animals with induced endometriosis the endometrial implant sizes were measured within 24 hours after parturition and 2 months later. The results of this study were subject to nested analysis of variance. 9 When a significant F value was present, Fisher's least significant difference test was used for individual comparison of means. RESULTS
The ectopic endometrial implants took well to the abdominal environment and produced viable, healthy tissue (Fig. 1). All the transplanted endometrial sections exhibited accumulation of fluid at laparotomy 4 weeks after operation. The implants were well vascularized and covered by a sheath of connective tissue. On histological section a good thickness of stromal endometrial tissue is apparent (Fig. 2). There is very little glandular development in this tissue but marked vascularity is obvious, Fertility and Sterility
Table 2 Effect of Pregnancy on the Endometrial Implant and Its Recurrence 2 Months After Parturition Implant sizea
Parameter
At the time of surgical induction (n 1 mo after induction (n = 8) On day 10 of pregnancy (n = 8) At the time of parturition (n = 6) 2 mo after parturition (n = 4)
=
8)
28.22 ± 39.22 ± 2.77 ± 8.83 ± 19.20 ±
2.08 2.05 1.10b 0.76b 6.67c
Values are means ± SEM. < 0.01, when compared with implant size at 1 month after induction. c P < 0.01, when compared with implant size at the time of parturition. a
bP
Figure 2 A histological section (63X) of endometrial implant indicating viable stromal and epithelial tissue, and lack of glandular development at 1 month after original implantation.
and a high leucocyte infiltration was noted. One month after transplantation the implant size was slightly larger than the original size (P < 0.05; Tables 1 and 2). No adhesions were present in rats with surgically induced endometriosis. When the rats were ovariectomized, the implants disappeared grossly in some animals within a month of the surgery (Fig. 3), whereas in other animals implants of smaller size were observed. The mean decrease in implant size after 1 month of ovariectomy was 90% (Table 1). The mean decreases in implant size after 1 and 2 months of ovariectomy were statistically significant (P < 0.01).
The implant size iri the control group did not appreciably change at 1, 2, and 3 months after induction of endometriosis (34.82 ± 3.20, 37.64 ± 4.24, and 33.84 ± 3.60). Two months after ovariectomy the implants regressed completely iri all animals. Histological sections of the area that once contained the implant indicated no viable endometrial tissue but only disintegrated cells of indeterminate origin (Fig. 4). These sections were investigated closely to determine whether endometrial stem cells were present, but the search was inconclusive. Figure 5 shows clearly the very robust endometrial tissue resulting after the administration of estrogens to previously ovariectomized rats where the implants had grossly disappeared within 1 month after ovariectomy. The recurrence of implants with estrogen administration at 2 and 4 months of ovariectomy was statistically significant (P < 0.01; Table 1). Each implant was well vascularized and had leucocyte infiltration. This regen-
Table 1 Effect of Ovariectomy on Regression of Endometrial Implant and Its Recurrence with Estrogen Administration Parameter
Implant size a
At the time of surgical induction (n = 8) 1 mo after induction (n = 8) 1 mo after ovariectomy (n = 8) 2 mo after ovariectomy (n = 8) 4 mo after ovariectomy (n = 8) Estrogen administration after 2 mo of ovariectomy (n = 6) Estrogen administration after 4 mo of ovariectomy (n = 8)
29.38 ± 1.12 37.12 ± 3.44 3.75 ± 2.11b o.oo ± o.oob o.oo ± o.oob 41.67 ± 8.9SC 26.85 ± 4.50d
Values are means ± SEM. P < 0.01, when compared with implant size at 1 month after induction of endometriosis. c P < 0.01, when compared with implant size at 2 months after ovariectomy. d P < 0.01, when compared with implant size at 4 months after ovariectomy. a
b
Vol. 53, No.5, May 1990
Figure 3 The total apparent regression of an endometrial implant 1 month after ovariectomy. Rajkumar et al.
Endometriosis in rat models
923
.....
.
Figure 4 A histological section (63X} of the site of the original ectopic endometrium 2 months after ovariectomy indicating the absence of any viable endometrial tissue.
erated tissue had both epithelial and stromal cells but no glandular formation was visible. Two months after induction of endometriosis, rats were successfully mated with fertile males. The mean number of embryos in endometriosis animals was not significantly different from control (control group 13.4 ± 0.45; endometriosis group 14.0 ± 1.36). The number of fetus at term was slightly lower in animals with endometriosis as compared with control (control group 12.6 ± 0.54; endometriosis group 10.8 ± 1.03); however, the difference was not significantly different. The effect of pregnancy on the size of the endometrial implants is depicted in Table 2. The mean size of the implants was significantly reduced (P < 0.01) by day 10 of gestation. Many of the animals showed complete regression of endometrial implants at parturition. When animals were examined 1 month after parturition, visible endometrial implants were seen in animals where there was apparent complete regression during pregnancy.
ated histologically nor has the potential of the regressed implant to grow been examined. NisollePochet et al. 13 have concluded from their recent study in humans that recurrence of endometriosis after hormonal treatment is due to the persistence of active glandular epithelium in endometriotic foci at the end of therapy. In the present study in rats, 2 months after ovariectomy, we were unable to detect histologically any viable endometrial tissue; however, estrogen administration at 2 and 4 months of ovariectomy to ovariectomized animals led to recurrence of the implants in all the animals. As recurrence was observed in animals that had complete morphological regression of implant within 1 month of ovariectomy, we suggest that estrogen-responsive cells in the implant may survive at the implanted site for long periods of time after complete morphological regression. The human endometrium is characterized by constant regeneration and cyclic changes of cell proliferation, differentiation, and death. The stem cell concept has been proposed to explain the constant regeneration of endometrium. 14•15 The rat endometrium, unlike the human endometrium, does not slough during the estrous cycle and is primarily regenerated by mitotic division of endometrial epithelial cells in response to estrogen. 16 In the present study it is possible that estrogen-responsive endometrial cells may have persisted at the implant site after 2 and 4 months of ovariectomy, and then may have regenerated into an implant after estrogen administration. We hypothesize, based on the results of this study, that clinically reported recur-
DISCUSSION
The present study clearly demonstrates that induction of experimental endometriosis in the female rat is feasible and supports the findings of other investigators. 10•11 Removal of ovaries effectively inhibited the growth of endometrial implants in all rats. This suggests that endometrial implants depend on ovarian steroids for their continued growth. Regression of endometrial implants after ovariectomy has been demonstrated earlier in rats, 12 but neither has the regressed site been evalu924
Rajkumar et al.
Endometriosis in rat models
Figure 5 A histological section (63X) of the recurred endometrial implant after 1 month of estradiol cypionate administration to ovariectomized animal with apparent complete regression of original implant.
Fertility and Sterility
renee after withdrawal of endocrine therapy in the treatment endometriosis may be due to the growth of regressed implant. In the present study, endometriosis did notreduce the mean number of embryos at midgestation (day 10). The fertility results of this study differ from those observed in rabbits 17•18 and rats. 11 In the study of Vernon and Wilson11 in rats, a total offour endometriotic implants were attached to the mesentery and the utero-ovarian ligament and moderate to severe adhesions were present in all animals with surgically induced endometriosis, whereas· in the present study, two implants were sutured to the peritoneal wall and there were no adhesions. Recently Kaplan et al. 19 have reported that preovarian adhesions in endometriosis impair ovulation in rabbits. It is not known whether the difference in the results of this study with the study of Vernon and Wilson 11 could be attributed to the difference in adhesions. We have recently observed that increasing the amount of endometrium transplanted to the peritoneum (6 implants) did not alter fertility in rats. 20 This suggests that the difference in the fertility of animals of this study relative to previous work is not because of the amount of endometrium transplanted. Endometrial implant size was markedly reduced during pregnancy. At the-time of parturition in some animals there was a total regression of endometrial implants. However 1 month after parturition, the implant which had apparently totally regressed grew back. This corroborates the findings of Vernon and Wilson11 and suggests that pregnancy has only a temporary suppressive effect on ectopic endometrial implants. In conclusion, the results of this study demonstrate the regression of endometrial implants in rats during pregnancy and after ovariectomy. After short periods of ovariectomy, endometrial cells may survive in the regressed implant, maintaining its capability to grow. The recurrence of the endometriosis observed in clinical practice after withdrawal of endocrine therapy for endometriosis may be because of survival of endometrial cells in the regressed implant at the completion of therapy. The ovariectomized animal model described in this study may be useful in the future to determine whether it is possible to completely destroy the ectopic tissue with longer periods of endocrine therapy and abolish recurrence after withdrawal of therapy. Acknowledgments. The authors thank MF. Hoa Ly for his ex• cellent technical assistance and Mr. DennisDyck for the preparation of plates.
Vol. 53, No.5, May 1990
REFERENCES 1. Williams T J, Pratt JH: Endometriosis in 1000 consecutive celiotomies: incidence and management. Am J Obstet Gynecol129:245, 1977 2. Gray LA: Surgical treatment of endometriosis. Clin Obstet Gynaecol3:472, 1960 3. Barbieri RL, Evans S, Kistner RW: Danazol in the treatment of endometriosis: analysis of 100 cases with a 4-year follow up. Fertil Steril37:737, 1982 4. Moore EE, Harger JH, Rock JA, Archer DF: Management of pelvic endometriosis with low-dose danazol. Fertil Steril 36:15,1985 5. Daniell JF, Christianson C: Combined laparoscopic surgery and danazol therapy for pelvic endometriosis. Fertil Steril 35:521, 1981 6. Steingold KA, Cedars M, Lu JKH, Randle D, Judd HL, Meldrum DR: Treatment of endometriosis with a long-acting gonadotropin-releasing hormone agonist. Obstet Gynecol69:403, 1987 7. Lemay A, Quesnel G: Potential new treatment of endometriosis: reversible inhibition of pituitary-ovarian function by chronic intranasal administration of a luteinizing hormone releasing hormone (LHRH) agonist. Fertil Steril38:376, 1982 8. Schriock E, Monroe SE, Henzl M, Jaffe RB: Treatment of endometriosis with a potent agonist of gonadotropin-releasing hormone (Nafarelin). Fertil Steril44:583, 1985 9. Sokal RR, RohlfFJ: Nested Analysis of Variance. In Biometry, Edited by RR Sokal, FJ Rohlf. New York, W.H. Freeman and Company, 1981, p 271 10. Golan A, Dargenio R, ·Winston RML: The effect of treatment on experimentally produced endometrial peritoneal implants. Fertil Steril 46:954, 1986 11. Vernon MW, Wilson EA: Studies on the surgical induction of endometriosis in the rat. Fertil Steril44:684, 1985 12. Jones RC: The effect of luteinizing hormone releasing hormone (LRH) agonist (WY -40,972), Levonorgestsel, danazol, and ovariectomy on experimental endometriosis in the rat. Acta Endocrinol (Copenh) 106:282, 1984 13. Nisolle-Pochet M, Casanas-Roux F, Donnez J: Histological study of ovarian endometriosis after hormonal therapy. Fertil Steril49:423, 1988 14. Prianishnikov VA: On the concept of stem cell and a model of function-morphological structure of the endometrium. Contraception 18:213, 1978 15. Satyaswaroop PG, Mortel R: Endometrial carcinoma: an aberration of endometrial cell differentiation. Am J Obstet Gynecol140:62, 1981 16. Clark BF: The effects of oestrogen and progesterone on uterine cell division and epithelial morphology in spayed, adrenolactomized rats. J Endocrinol50:527, 1971 17. Hahn DW, Carraher RP, Foldesy RG, McGuire JL: Experimental evidence for failure to implant as a mechanism of infertility associated with endometriosis. Am J Obstet Gynecol155:1109, 1986 18. Schenken RS, Asch RH: Surgical induction of endometriosis in the rabbit: effects on fertility and concentration of peritoneal fluid prostaglandins. Fertil Steril 34:581, 1980 19. Kaplan CR, Eddy CA, Olive DL, Schenken RS: Effect of ovarian endometriosis on ovulation in rabbits. Am J Obstet Gynecol160:40, 1989 20. Rajkumar K, Simpson CW: Unpublished data
Rajkumar et al.
Endometriosis in rat models
925
Vol. 53, No.5, May 1990
Copyright " 1990 The American Fertility Society
Printed on acid-free paper in U.S.A.
The rat as an animal model for endometriosis to examine recurrence of ectopic endometrial tissue after regression*t
Kadaba Rajkumar, Ph.D.:j: Philipp W. Schott, B.S. Charles W. Simpson, M.D. Reproductive Biology Research Unit, Department of Obstetrics and Gynecology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
The effects of pregnancy and ovariectomy on the growth of endometrial implants were examined in rats with experimentally induced endometriosis. Ectopic implants regressed completely after ovariectomy and we were unable to detect any viable endometrial cells in histological examination of the implant sites. Administration of 17!3-estradiol cypionate to ovariectomized rats with regressed endometrial implants led to recurrence of the regressed implants. Animals with experimentally induced endometriosis were fertile and the number of embryos on day 10 of pregnancy was not different from the control group. Ectopic implants which regressed during pregnancy recurred 1 month after parturition. It is concluded that endometrial cells may survive at the implanted site even after apparent complete morphological regression, which has the potential to grow into an implant. Fertil Steril53:921, 1990
Endometriosis is a disease characterized by the presence and proliferation of endometrial tissue outside the uterus. Its incidence ranges from 7% to 50% of menstruating women. 1•2 In mild to moderate forms of endometriosis, endocrine treatments are commonly used. The beneficial aspects of danazol in the treatment of endometriosis are counterbalanced by recurrence rates of 20% to 50%.3 Although the highest recurrence rates (51%) were reported when symptoms served as the diagnostic parameter,4 implants were noted at repeat laparoscopy in 23% of patients 6 months after completing therapy. 5 Gonadotropin-releasing hormone agonists (GnRH-a), which allow temporary suppres-
Received June 19, 1989; revised and accepted January 2, 1990. * Supported by the Saskatchewan Health Research Board. t Presented at the Second International Symposia on Endometriosis, Houston, Texas, May 2 to 4, 1989. t Reprint requests: Kadaba Rajkumar, Ph.D., Department of Obstetrics and Gynecology, University of Saskatchewan College of Medicine, Reproductive Biology Research Unit, Saskatoon, Saskatchewan S7N OXO, Canada. Vol. 53, No.5, May 1990
sion of ovarian function, are the most recent hormonal therapeutic agents. Complete suppression of endometriosis has been demonstrated with GnRH-a therapy. 6 However, recurrence of symptoms of endometriosis within 6 months of completion of GnRH-a treatment has been reported. 7•8 In human studies it cannot be ascertained whether recurrence after the withdrawal of therapy is due to growth of regressed implants or the presence of new implants. Animal models that do not have spontaneous endometriosis may be useful to investigate this phenomenon. In the present studies we examined the regression of ectopic implants after ovariectomy and during pregnancy. Further, the recurrences of the implants were monitored in ovariectomized animals treated with estrogen and in pregnant animals after parturition. MATERIALS AND METHODS
Mature cycling female rats (Sprague-Dawley) of 170 to 200 g were maintained on a 14- to 10-hour (light to dark) photoperiod and fed rat chow and Rajkumar et al. Endometriosis in rat models
921
water ad libitum. The rats were monitored daily for reproductive cyclicity by examination of vaginal cytological smears. Only those rats exhibiting a normal estrous cycle were used. All surgical procedures were done under aseptic conditions in a sterile flow hood. Animals were administered 0.05 to 0.1 mg of ketamine hydrochloride solution (200 mg/mL), followed by anesthesia using halothane. During laparotomy, the blood vessels supplying the right uterine horn were ligated and approximately 2 em were resected. End-to-end anastomosis of the uterus after resection was performed with 6-0 nylon sutures (Ethicon, Johnson & Johnson Company, Somerville, NJ). The resected horn was opened in isotonic sterile saline and the myometrium was peeled from the endometrium under a dissecting microscope. Two pieces of endometrium measuring 5 X 6 mm (approximate) were cut and then sutured intraperitoneally to the inside of the peritoneal membrane with the sutures (6-0 nylon; Ethicon) passing through the membrane and anchoring into the abdominal musculature on all four sides of the implant. We attempted to place the implant at a point equidistance between the ventral midline, the vertebral column, the caudal rib, and cranial pelvis (i.e., center of the flank). In addition, an effort was made to suture the implant to a site very near a larger blood vessel to ensure as rapid a vascularization as possible. After transplantation the size of the implanted tissue was measured. The criterion used to assess the viability of the graft was the formation of a cyst within 1 month after transplantation. One month after transplantation of endometrial tissue, the animals were laparotomized and endometrial implant surface area (length X width) was measured. One subgroup of animals underwent ovariectomy to remove the endogenous supply of ovarian steroids; the other subgroup served as control. After 1, 2, and 4 months of ovariectomy, the endometrial implants were examined under laparotomy. In some animals, implanted sites were removed by dissection 2 months after ovariectomy and processed for histology. Two and 4 months after ovariectomy, 17/J-estradiol (E 2 ) cypionate (60 ,ug/kg) was administered subcutaneously in oil, twice a week for 30 days. After 1 month of treatment with E 2 , the animals were again laparotomized and the endometrial implant size was measured. Some implants were processed for histology. In a second group of rats, 15 days after laparotomy (to measure the size of the endometrial implant), the animals were mated with males of proven fertility. The successful mating was as922
Rajkumar et al.
Endometriosis in rat models
Figure 1 A healthy endometrial implant on the peritoneal surface of the abdominal wall of a rat 1 month after surgical implantation.
sessed by the presence of sperm in the vaginal smear. This was designated as day 1 of pregnancy. One group of sham-operated control rats was included in the study in addition to the animals with induced endometriosis. In sham-operated control, 2 em of the right uterine horn was resected followed by end-to-end anastomosis of the resected horn. The suture without endometrium were also attached to the peritoneal cavity. On day 10 of pregnancy the animals were laparotomized and the number of embryos in the uterine horns was counted. In rats with induced endometriosis, the endometrial implant size was also measured. At term the number of fetus delivered was also recorded. In animals with induced endometriosis the endometrial implant sizes were measured within 24 hours after parturition and 2 months later. The results of this study were subject to nested analysis of variance. 9 When a significant F value was present, Fisher's least significant difference test was used for individual comparison of means. RESULTS
The ectopic endometrial implants took well to the abdominal environment and produced viable, healthy tissue (Fig. 1). All the transplanted endometrial sections exhibited accumulation of fluid at laparotomy 4 weeks after operation. The implants were well vascularized and covered by a sheath of connective tissue. On histological section a good thickness of stromal endometrial tissue is apparent (Fig. 2). There is very little glandular development in this tissue but marked vascularity is obvious, Fertility and Sterility
Table 2 Effect of Pregnancy on the Endometrial Implant and Its Recurrence 2 Months After Parturition Implant sizea
Parameter
At the time of surgical induction (n 1 mo after induction (n = 8) On day 10 of pregnancy (n = 8) At the time of parturition (n = 6) 2 mo after parturition (n = 4)
=
8)
28.22 ± 39.22 ± 2.77 ± 8.83 ± 19.20 ±
2.08 2.05 1.10b 0.76b 6.67c
Values are means ± SEM. < 0.01, when compared with implant size at 1 month after induction. c P < 0.01, when compared with implant size at the time of parturition. a
bP
Figure 2 A histological section (63X) of endometrial implant indicating viable stromal and epithelial tissue, and lack of glandular development at 1 month after original implantation.
and a high leucocyte infiltration was noted. One month after transplantation the implant size was slightly larger than the original size (P < 0.05; Tables 1 and 2). No adhesions were present in rats with surgically induced endometriosis. When the rats were ovariectomized, the implants disappeared grossly in some animals within a month of the surgery (Fig. 3), whereas in other animals implants of smaller size were observed. The mean decrease in implant size after 1 month of ovariectomy was 90% (Table 1). The mean decreases in implant size after 1 and 2 months of ovariectomy were statistically significant (P < 0.01).
The implant size iri the control group did not appreciably change at 1, 2, and 3 months after induction of endometriosis (34.82 ± 3.20, 37.64 ± 4.24, and 33.84 ± 3.60). Two months after ovariectomy the implants regressed completely iri all animals. Histological sections of the area that once contained the implant indicated no viable endometrial tissue but only disintegrated cells of indeterminate origin (Fig. 4). These sections were investigated closely to determine whether endometrial stem cells were present, but the search was inconclusive. Figure 5 shows clearly the very robust endometrial tissue resulting after the administration of estrogens to previously ovariectomized rats where the implants had grossly disappeared within 1 month after ovariectomy. The recurrence of implants with estrogen administration at 2 and 4 months of ovariectomy was statistically significant (P < 0.01; Table 1). Each implant was well vascularized and had leucocyte infiltration. This regen-
Table 1 Effect of Ovariectomy on Regression of Endometrial Implant and Its Recurrence with Estrogen Administration Parameter
Implant size a
At the time of surgical induction (n = 8) 1 mo after induction (n = 8) 1 mo after ovariectomy (n = 8) 2 mo after ovariectomy (n = 8) 4 mo after ovariectomy (n = 8) Estrogen administration after 2 mo of ovariectomy (n = 6) Estrogen administration after 4 mo of ovariectomy (n = 8)
29.38 ± 1.12 37.12 ± 3.44 3.75 ± 2.11b o.oo ± o.oob o.oo ± o.oob 41.67 ± 8.9SC 26.85 ± 4.50d
Values are means ± SEM. P < 0.01, when compared with implant size at 1 month after induction of endometriosis. c P < 0.01, when compared with implant size at 2 months after ovariectomy. d P < 0.01, when compared with implant size at 4 months after ovariectomy. a
b
Vol. 53, No.5, May 1990
Figure 3 The total apparent regression of an endometrial implant 1 month after ovariectomy. Rajkumar et al.
Endometriosis in rat models
923
.....
.
Figure 4 A histological section (63X} of the site of the original ectopic endometrium 2 months after ovariectomy indicating the absence of any viable endometrial tissue.
erated tissue had both epithelial and stromal cells but no glandular formation was visible. Two months after induction of endometriosis, rats were successfully mated with fertile males. The mean number of embryos in endometriosis animals was not significantly different from control (control group 13.4 ± 0.45; endometriosis group 14.0 ± 1.36). The number of fetus at term was slightly lower in animals with endometriosis as compared with control (control group 12.6 ± 0.54; endometriosis group 10.8 ± 1.03); however, the difference was not significantly different. The effect of pregnancy on the size of the endometrial implants is depicted in Table 2. The mean size of the implants was significantly reduced (P < 0.01) by day 10 of gestation. Many of the animals showed complete regression of endometrial implants at parturition. When animals were examined 1 month after parturition, visible endometrial implants were seen in animals where there was apparent complete regression during pregnancy.
ated histologically nor has the potential of the regressed implant to grow been examined. NisollePochet et al. 13 have concluded from their recent study in humans that recurrence of endometriosis after hormonal treatment is due to the persistence of active glandular epithelium in endometriotic foci at the end of therapy. In the present study in rats, 2 months after ovariectomy, we were unable to detect histologically any viable endometrial tissue; however, estrogen administration at 2 and 4 months of ovariectomy to ovariectomized animals led to recurrence of the implants in all the animals. As recurrence was observed in animals that had complete morphological regression of implant within 1 month of ovariectomy, we suggest that estrogen-responsive cells in the implant may survive at the implanted site for long periods of time after complete morphological regression. The human endometrium is characterized by constant regeneration and cyclic changes of cell proliferation, differentiation, and death. The stem cell concept has been proposed to explain the constant regeneration of endometrium. 14•15 The rat endometrium, unlike the human endometrium, does not slough during the estrous cycle and is primarily regenerated by mitotic division of endometrial epithelial cells in response to estrogen. 16 In the present study it is possible that estrogen-responsive endometrial cells may have persisted at the implant site after 2 and 4 months of ovariectomy, and then may have regenerated into an implant after estrogen administration. We hypothesize, based on the results of this study, that clinically reported recur-
DISCUSSION
The present study clearly demonstrates that induction of experimental endometriosis in the female rat is feasible and supports the findings of other investigators. 10•11 Removal of ovaries effectively inhibited the growth of endometrial implants in all rats. This suggests that endometrial implants depend on ovarian steroids for their continued growth. Regression of endometrial implants after ovariectomy has been demonstrated earlier in rats, 12 but neither has the regressed site been evalu924
Rajkumar et al.
Endometriosis in rat models
Figure 5 A histological section (63X) of the recurred endometrial implant after 1 month of estradiol cypionate administration to ovariectomized animal with apparent complete regression of original implant.
Fertility and Sterility
renee after withdrawal of endocrine therapy in the treatment endometriosis may be due to the growth of regressed implant. In the present study, endometriosis did notreduce the mean number of embryos at midgestation (day 10). The fertility results of this study differ from those observed in rabbits 17•18 and rats. 11 In the study of Vernon and Wilson11 in rats, a total offour endometriotic implants were attached to the mesentery and the utero-ovarian ligament and moderate to severe adhesions were present in all animals with surgically induced endometriosis, whereas· in the present study, two implants were sutured to the peritoneal wall and there were no adhesions. Recently Kaplan et al. 19 have reported that preovarian adhesions in endometriosis impair ovulation in rabbits. It is not known whether the difference in the results of this study with the study of Vernon and Wilson 11 could be attributed to the difference in adhesions. We have recently observed that increasing the amount of endometrium transplanted to the peritoneum (6 implants) did not alter fertility in rats. 20 This suggests that the difference in the fertility of animals of this study relative to previous work is not because of the amount of endometrium transplanted. Endometrial implant size was markedly reduced during pregnancy. At the-time of parturition in some animals there was a total regression of endometrial implants. However 1 month after parturition, the implant which had apparently totally regressed grew back. This corroborates the findings of Vernon and Wilson11 and suggests that pregnancy has only a temporary suppressive effect on ectopic endometrial implants. In conclusion, the results of this study demonstrate the regression of endometrial implants in rats during pregnancy and after ovariectomy. After short periods of ovariectomy, endometrial cells may survive in the regressed implant, maintaining its capability to grow. The recurrence of the endometriosis observed in clinical practice after withdrawal of endocrine therapy for endometriosis may be because of survival of endometrial cells in the regressed implant at the completion of therapy. The ovariectomized animal model described in this study may be useful in the future to determine whether it is possible to completely destroy the ectopic tissue with longer periods of endocrine therapy and abolish recurrence after withdrawal of therapy. Acknowledgments. The authors thank MF. Hoa Ly for his ex• cellent technical assistance and Mr. DennisDyck for the preparation of plates.
Vol. 53, No.5, May 1990
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Endometriosis in rat models
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