Clinical relevance of the baboon as a model for the study of endometriosis

FERTILITYAND STERILITY@ Copyright

o 1997 American

Published

by Elsevier

Vol. 68, No. 4, October

Society for Reproductive

Science

Medicine

Printed

on acid-free

1997

paper in U. S. A.

Inc.

Clinical relevance of the baboon as a model for the study of endometriosis

Thomas M. D’Hooghe, M.D., Ph.D.*t$ Institute

of Primate

Research,

Nairobi, Kenya, and University Hospital

Gasthuisberg,

Leuven, Belgium

Objective: To review the value of the baboon as a model for the study of endometriosis. Data Identification and Selection: Studies performed at the Institute of Primate Research in Nairobi, Kenya (1990- 19941, and published in peer-reviewed journals. Result(s): Spontaneous endometriosis was found in about 25% of the baboons, and its prevalence increased with the duration of captivity. The laparoscopic appearance, pelvic localization, and microscopic aspects of the disease were similar to endometriosis in women. Microscopic endometriosis in macroscopically normal peritoneum was rare. Sampson’s hypothesis (i.e., retrograde menstruation causes endometriosis) was supported by the increased incidence of retrograde menstruation in baboons with spontaneous endometriosis, the observation that cervical occlusion could cause retrograde menstruation and endometriosis, and the finding that intrapelvie injection of menstrual endometrium caused experimental moderate to severe endometriosis similar to the spontaneous disease. During follow-up of more than 2 years, endometriosis in baboons appeared to be a progressive disease, with active remodeling between several types of lesions. Progression was stimulated by high-dose immunosuppression. Fertility was normal in baboons with minimal disease but was reduced in baboons with mild, moderate, or severe endometriosis, possibly related to an increased incidence and recurrence of the luteinized unruptured follicle syndrome. (Fertil Steril@ Conclusion(s): The baboon is a good model for the study of endometriosis. 1997;68:613-25. 0 1997 by American Society for Reproductive Medicine.) Key Words: Baboon, Pupio, primates, laparoscopy

endometriosis, evolution, pathogenesis, infertility, immunology,

Endometriosis is an important benign gynecologic disease that is pathologically defined by the ectopic presence of both endometrial glands and stroma. It is associated with pelvic pain and infertil-

Received December 24, 1997; revised and accepted June 12, 1997. Supported by the Commission of the European Communities (DG VIII Development and DG XII Science, Research and Development); the Vlaamse Interuniversitaire Raad (Flemish Interuniversity Council), Brussels, Belgium; the Collen Research Foundation/Faculty of Medicine, University of Leuven, Leuven, Belgium; and the Fearing Research Laboratory Endowment, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts. * Department of Reproduction, Institute of Primate Research. t Leuven University Fertility Center, Department of Obstetrics and Gynecology, University Hospital Gasthuisberg. $ Reprint requests: Thomas M. D’Hooghe, M.D., Ph.D., Leuven University Fertility Center, Department of Obstetrics and Gynecology, University Hospital Gasthuisberg, B-3000 Leuven, Belgium (FAX: 32-16-343607). 0015-0262/97/$17.00 PI1 s0015-0282(97)00277-x

Endometriosis has been well described and known for more than 50 years, yet our current knowledge of its pathogenesis, of the pathophysiology of related infertility, and of spontaneous evolution is still unclear. Several reasons contribute to this state. First, at the time of diagnosis, most patients with endometriosis have had the disease for an extended period, making it difficult to initiate any clinical experiments that would determine definitely the etiology or progression of the disease (1). Second, it is difficult to carry out randomized studies with sufficient numbers of patients and meaningful controls, which obviates the need for multicenter-oriented research (2). An important reason for the lack of progress in endometriosis research is study design; very few studies have been carried out so far using adequate control groups. When symptomatic patients with endometriosis are compared with women with a nor-

ity.

613

ma1 pelvis, adenomyosis, leiomyomas, adhesions, or other pelvic pathology, two factors usually are studied in a combined way: the pelvic condition (presence of endometriosis or other pathology) and symptoms (none, infertility, pain, and other symptoms). To study the effect of endometriosis itself, it would be necessary to exclude patients with other possible causes of infertility or pain and to compare patients with endometriosis and infertility with women with a normal pelvis and unexplained infertility or to compare patients with endometriosis and pain with women with a normal pelvis and pain. To study the effect of endometriosis on infertility, the study group should include infertile patients with endometriosis and women with unexplained infertility, whereas the control group should include fertile women with endometriosis and with a normal pelvis (the population that is available at interval tubal sterilization). Similarly, to study the effect of endometriosis on pain, the study group should include patients with endometriosis and pain and women with unexplained pain, whereas the control group should include asymptomatic and pain-free women with endometriosis and with a normal pelvis (the population that is available at interval tubal sterilization). Third, endometriosis occurs naturally in humans and nonhuman primates only. Because of ethical and practical considerations, properly controlled studies are very difficult and invasive experiments cannot be performed in humans. It follows from these considerations that there is an obvious need for the development of a good animal model with spontaneous endometriosis. ANIMAL MODELS

FOR THE STUDY

OF ENDOMETRIOSIS

The main advantage of rodent (rat and rabbit) models is their low cost relative to the monkey, but the disadvantages are numerous. Both rodents lack a menstrual cycle and do not have spontaneous endometriosis. Although the rat is a spontaneous ovulator, it has a shorter luteal phase than humans. The reproductive pattern of the rabbit lacks a luteal phase. In addition, there is a wide phylogenetic gap between these two species and humans. In both rodent models, induction is performed through the autotransplantation of endometrial fragments or uterine squares (3), which is not physiologic, damages the uterus, and causes adhesions that interfere with fertility. The resulting “endometriotic lesions” consist of cysts containing clear serous fluid in the rat, whereas vascularized hemorrhagic solid masses also can be found in the rabbit. These types of lesions in both species seem to be quite different from the variety of pigmented and nonpigmented lesions found in humans (4-6). 614

D’Hooghe

Baboon model for endometriosis

Monkeys, although difficult and expensive to maintain in captivity, offer unique advantages in endometriosis research when compared with rodents. First, they are phylogenetically much closer to humans and have a comparable menstrual cycle. Second, nonhuman primates, including the rhesus monkey (7), the pigtailed macaque (81, the cynomolgus monkey, the DeBrazza monkey (91, and the baboon (10, ll), are known to be afflicted with spontaneous endometriosis. It has been reported that irradiation is associated with an increased incidence of spontaneous endometriosis in rhesus monkeys, but only after at least 6 years (12). In the same species, a positive correlation was found between dioxin dose and the severity of endometriosis (13). Third, induced endometriosis results in macroscopic lesions that show similarity to the human disease (14-19). The great apes (chimpanzee, gorilla, and orangutan) are closest to humans in many anatomic and physiologic aspects of reproduction. However, because they all are protected, endangered species in the wild, they are not practical models for most studies. So far, most endometriosis research has been done in rhesus and cynomolgus monkeys. Baboons are very intelligent animals with a well-studied and interesting social life. Hypotheses about the early evolution of human social behavior have been developed by carefully studying the behavior of baboon troops living on the grassy plains of Africa (20). The baboon may offer clear advantages for the study of endometriosis when compared with rhesus and cynomolgus monkeys. First, it is phylogenetitally very close to humans; human (46 chromosomes) and baboon (42 chromosomes) karyotypes, evolving slowly, share many ancestral characters (21). Second, detailed accounts of baboon reproductive anatomy and physiology, similar to that of humans, are available, including menstrual cycle characteristics, embryo implantation, and fetal development (22). Perineal skin inflation and deflation correspond with relative precision to the follicular and luteal phases, respectively, offering external follow-up of the menstrual cycle without the need for serial blood samples to determine Ez and P levels. Third, the baboon is a proven model for research in cardiovascular and endoscopic surgery (23), endocrinology, teratology, toxicology, testing of contraceptive agents (241, and placental development (25). Fourth, the baboon is a continuous breeder that has menstrual cycles throughout the year, which continue in captivity. Fifth, the baboon is a larger and stronger primate than the rhesus or cynomolgus monkey, allowing for repetitive blood sampling and complex experimental surgery (24). Sixth, specific advantages of the baboon model in gynecologic reFertility and Sterility@

search include the spontaneous presence of peritoneal fluid (PF) and the accessibility of the uterine cavity through the cervix, allowing endometrial sampling without hysterotomy (26). Seventh, spontaneous endometriosis in the baboon has been found to be both minimal (11) and disseminated (lo), similar to the different disease stages in women. For all these reasons, the baboon is considered to be a good model for research in reproduction (24). In summary, the baboon seems to offer interesting advantages and merits to be studied as a possible model for the study of endometriosis. The aim of this review is to summarize the clinical value of the baboon as a model for the study of aspects of endometriosis (prevalence, incidence, pathogenesis, spontaneous evolution, and fertility) that cannot be studied in women for ethical reasons. The studies reviewed were carried out at the Institute of Primate Research in Nairobi, Kenya, from 1990 to 1994. PREVALENCE

OF ENDOMETRIOSIS

The prevalence of endometriosis in the general population is not known exactly. In women with pelvic pain and/or infertility, high prevalences, ranging between 20% and 80-90%, have been reported (1, 27). In asymptomatic women undergoing tubal ligation (mostly women of proven fertility), the prevalence of endometriosis ranges between 3% and 43% (28-34), including mostly (80%) minimal or mild disease. This high variability in reported endometriosis prevalence can be explained by several factors. First, the prevalence may vary with the diagnostic method used; laparoscopy generally is considered to be a better method for the diagnosis of minimal to mild endometriosis than laparotomy, and this technique has become widely available only during the last 15-20 years. Second, the interest of the surgeon may play a significant role; minimal or mild endometriosis may be looked for more eagerly in a symptomatic patient under general anesthesia than in an asymptomatic patient during tubal sterilization. Even when pelvic inspection is performed carefully in the latter case, the local anesthesia that is used most commonly today limits surgery time and pelvic access. Third, the experience of the surgeon in diagnosing the wide variability in appearance of especially subtle endometriosis implants, cysts, and adhesions is important. The concept of subtle endometrial lesions (4,5) and deep endometriosis (35) has been described only recently. The importance of vascularization, mitotic activity, and the three-dimensional structure of endometriosis lesions also has been recognized in the recent past (6,361. These data suggest that endoVol. 68, No. 4, October 1997

metriosis may be a heterogeneous disease. Fourth, most studies evaluating the prevalence of endometriosis in women of reproductive age lack systematic histologic confirmation of the macroscopic findings (28, 29, 31, 34). However, pathologic confirmation of the laparoscopic impression is essential for the diagnosis of endometriosis (37), notably for subtle lesions, but also for typical lesions, which have been reported to be histologically negative in 24% of cases (38). In baboons, spontaneous minimal endometriosis has been reported in animals of proven fertility, with a prevalence of 25% and a laparoscopic appearance, pelvic localization (391, and microscopic aspects (40) similar to the human disease. Although a significant association has been observed between the prevalence of endometriosis and a previous hysterotomy (39), the prevalence of endometriosis in baboons without previous hysterotomy was 8% in an initial study (39), comparable to the 7.5% prevalence of endometriosis in asymptomatic women undergoing tubal ligation (30). The prevalence increased to 27% in animals that had been living in captivity for more than 2 years (41). This trend could be explained, as it is in women (29), by the occurrence of more menstrual cycles uninterrupted by pregnancy in captive than in wild baboons, and/or by captivity-associated stress (41). Endometriotic lesions were not missed often during systematic laparoscopic inspection, as demonstrated by the observation that microscopic endometriosis could be found only rarely (7%) in serial sections of large flaps of macroscopically normal peritoneum from female baboons (42). The low prevalence of microscopic endometriosis in macroscopically normal peritoneum in both women (43) and baboons (42) suggests that the significance of microscopic endometriosis as a cause of disease recurrence after treatment remains to be established. PATHOGENESIS

Retrograde

OF ENDOMETRIOSIS

Menstruation

In 1927, Sampson (44) hypothesized that the pathogenesis of endometriosis could be explained by intrapelvic transplantation of endometrium shed during retrograde menstruation. Three important clinical questions need to be addressed: Is the prevalence of retrograde menstruation increased in women or nonhuman primates with endometriosis? Can experimental obstruction of the cervix lead to the development of endometriosis? Can menstrual endometrium implant on pelvic peritoneum and develop into endometriotic lesions? D’Hooghe

Baboon

model for endometriosis

615

Prevalence

of Spontaneous

Retrograde Menstruation

Macroscopically bloodstained PF, suggesting retrograde menstruation, has been reported in 76% (28), 82% (45), and 90% (46) of women during menstruation. It is not clear whether the prevalence and recurrence of retrograde menstruation is increased in patients with endometriosis when compared with women with a normal pelvis. One study (28) reported a significantly higher prevalence of retrograde menstruation in patients with endometriosis (97%) than in women with a normal pelvis (60%), but other investigators have reported a similar prevalence of retrograde menstruation (90%) in both groups (46). In baboons, the hypothesis was tested that the incidence and recurrence of retrograde menstruation is higher in baboons with than in those without spontaneous endometriosis (47). Retrograde menstruation was defined by the presence of bloodstained (red or dark brown) PF during menses. Peritoneal fluid was 10 times more frequently bloodstained during menses (62%) than during nonmenstrual phases (6%). Retrograde menstruation was observed more frequently in animals with spontaneous endometriosis (83%) than in primates with a normal pelvis (51%). Recurrence of retrograde menstruation was observed more frequently in baboons with G/5) than in those without (3/B) spontaneous endometriosis. The results of this study demonstrated that retrograde menstruation is common in baboons, with a higher prevalence and recurrence in animals with than in those without spontaneous endometriosis (47). Experimental

Retrograde

Menstruation

It is not known whether iatrogenically increased retrograde menstruation results in the development of endometriosis. Obstructed menstrual outflow, which may increase retrograde menstruation, has been associated with endometriosis in 77% of patients with a functioning endometrium and patent tubes and in up to 89% of those with hematocolpos/ hematometra (48). On the other hand, women with iatrogenic cervical stenosis (OS<2 mm in diameter) had endometriosis and hematometra in only 15% and 7% of cases, respectively (49). In rhesus monkeys, surgical diversion of the cervix into the abdomen resulted in massive adhesions, but endometriosis was found in only 50% of the animals, even after 10 months (15). In five female baboons, cervical occlusion was attempted to develop a primate model for the study of retrograde menstruation and endometriosis (50). Silicone instillation into the cervix (n = 1) or combined electrocoagulation and suturing of the ostium (n = 4) was ineffective in causing either partial outflow obstruction or increased retrograde men616

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struation and endometriosis. Supracervical ligation during laparotomy (n = 2) resulted in impeded uterine outflow, as shown by a decreased duration of antegrade menstruation and increased retrograde menstruation. Endometriosis was observed in both animals as early as 3 months after supracervical ligation (50). Intraperitoneal Transplantation Endometrium

of Menstrual

Endometrial cells shed during menses are viable (51) and can be found in the fallopian tubes and in the PF (52). The ability of endometrial cells shed during menses to implant and grow on visceral and parietal peritoneum has not yet been demonstrated conclusively. In women, endometrial cells from menstrual effluent could be transplanted to abdominal wall fascia, but the development of endometriosis occurred in only 12% of cases, even after 6 months of shedding (53). In rhesus monkeys, surgical cervical diversion resulted in massive adhesions, but intra-abdominal menstruation was not observed and endometriosis was found in only 50% of the animals after 10 months (15) or 35 months (16). Similarly, intraperitoneal seeding of menstrual endometrium has not been demonstrated successfully to induce endometriosis in other nonhuman primates (14, 16). However, nonmenstrual (luteal or follicular) endometrial cells injected retroperitoneally or intraperitoneally in monkeys have been reported to cause endometriosis in 17% (191, 75% (14, 15, 171, and 100% (18) of cases. In baboons, the Sampson hypothesis (44) was tested by comparing the effect of intrapelvic injection of menstrual versus luteal endometrium on the incidence, peritoneal involvement, stage, and evolution of endometriosis (26). Seventeen baboons were injected retroperitoneally with luteal (n = 6) or menstrual (n = 7) endometrium and intraperitoneally with menstrual (n = 4) endometrium. Laparoscopies were performed after 2 months in all animals, and after 5 and 12 months in six and five primates injected with luteal and menstrual endometrium, respectively. The peritoneal endometriosis surface area, number of implants, and incidence of typical and red subtle lesions were significantly higher after retroperitoneal injection of menstrual than of luteal endometrium. Using menstrual endometrium, intraperitoneal seeding was more successful in causing endometriosis than was retroperitoneal injection. No significant changes in the number or surface area of endometriotic lesions were observed in the six baboons induced with retroperitoneal injection of luFertility and Sterility@

teal endometrium after 5 months. At repeated laparoscopy 12 months after intrapelvic injection of menstrual endometrium, progression was recorded in three of four regularly cycling animals, whereas regression was evident in one baboon that had become amenorrheic after induction. In baboons with experimental endometriosis caused by intrapelvic injection of menstrual endometrium, the incidence of red lesions decreased and that of typical implants increased during follow-up (26). These results indicate that intrapelvic injection of menstrual endometrium can cause peritoneal endometriosis, and they offer experimental evidence supporting the Sampson hypothesis (26, 54). Fresh Corpus Luteum Without Ovulation Stigma in the Early Luteal Phase (Luteinized Unruptured Follicle Syndrome) Re-epithelialization Luteal Phase

of Ovulation Stigma in the Early

The investigation of female infertility includes a diagnostic laparoscopy in the luteal phase to assess the ovaries for the presence of a fresh ovulation stigma in a recent corpus luteum (CL). It is not exactly clear how early in the luteal phase this laparoscopy should be done, because it is unknown how fast an ovulation stigma can undergo re-epithelialization. The absence of a fresh ovulation stigma in a recent CL in the late luteal phase may be attributed to anovulation or the luteinized unruptured follicle syndrome or to the fact that the laparoscopy was carried out too late in the luteal phase, after reepithelialization of the ovulation stigma. In women, it has been reported that the opening in the CL remains visible at least during the first 1.5 days after ovulation, followed by progressive closure and complete re-epithelialization 4 to 5 days after ovulation (55). However, other investigators found that the presence of a fresh ovulation stigma in a recent CL can be found equally in the early and late luteal phases (56-58), and occasionally an ovulation stigma may be observed during the early follicular phase of the next cycle (59). These data suggest that the ovulation stigma may be visible beyond the immediate postovulatory period. The only way to resolve this controversy is to follow ovulation and subsequent closure of the ovulation stigma by serial laparoscopies in the same women or in nonhuman primates during the entire luteal phase. In baboons, serial laparoscopies were carried out to investigate the re-epithelialization of the ovulation stigma during the luteal phase (60). When a fresh ovulation stigma was observed in baboons within 5 days after ovulation, it diminished in size Vol. 68, No. 4, October 1997

but remained visible up to 8, 12, and 16 days after ovulation in 91%, 75%, and 50% of the animals, respectively (60). If the data obtained in baboons can be extrapolated to the clinical investigation of the infertile woman, it would appear that laparoscopies performed for the documentation of a fresh ovulation stigma preferably should be done as early after ovulation as possible, but can be performed safely until 4-5 days after ovulation. The results from the baboon study (60) suggest that re-epithelialization of the ovulation stigma takes time, and they explain why the ovulation stigma can be observed in the late luteal phase (56-58). Luteinized

Unruptured Follicle Syndrome

The definition of luteinized unruptured follicle syndrome remains controversial. In most studies, luteinized unruptured follicle syndrome has been defined as the visual absence of a fresh ovulation stigma on a recent CL during laparoscopy in the early luteal phase (56,611. Associated findings include low E2 and P levels in the PF (52) and normal endocrinologic presumptive signs of ovulation: biphasic basal body temperature curves, secretory endometrium, laboratory evidence of P production by elevated plasma P, or urinary pregnanediol levels (61). Using this definition, luteinized unruptured follicle syndrome was found more frequently in infertile patients with endometriosis (57) and unexplained infertility (56) than in infertile women with tubal occlusion or male factor infertility. Experimental endometriosis induced in rabbits has been reported to cause luteinized unruptured follicle syndrome in 81% (62) and 48% (18) of monkeys with moderate to severe endometriosis. Other investigators however, have failed to confirm an association between luteinized unruptured follicle syndrome and endometriosis (58, 61, 63), and have reported an incidence of 47% in infertile women with ovulatory dysfunction (63) and an incidence of 33%-47% in fertile women (58). This clinical definition of luteinized unruptured follicle syndrome has been criticized because recognition of the ovulation stigma may be related directly to the experience of the laparoscopist, the quality of the laparoscopic equipment, the presence of ovarian adhesions reducing the possibility of manipulating and thoroughly examining the ovaries, the optimum timing of laparoscopy during the luteal phase (55, 63, 64), and the fact that pregnancies have been reported in luteinized unruptured follicle cycles (63, 64). An undisputed definition of luteinized unruptured follicle syndrome involves a histologic demonstration of an entrapped oocyte within a morphologically normal-appearing CL, but this definition is impractical for clinical practice. D’Hooghe

Baboon model for endometriosis

617

An alternative definition could be the absence of egg recovery from the female uterine tract when a recent CL without ovulation stigma is found in the early luteal phase. For ethical reasons, it is difficult to perform uterine flushes for egg recovery in women. However, nonhuman primates could be useful, because nonsurgical uterine flushing has been described previously in such models (65). In baboons with and without endometriosis, the incidence and recurrence of a recent CL with ovulation stigma and its association with the uterine egg recovery rate was determined in a recently published study (66). A recent CL with ovulation stigma was found less frequently in baboons with endometriosis (67%) than in controls (85%). The incidence of a recent CL without ovulation stigma was higher in animals with stage II-IV endometriosis (32%) than in those with stage I disease (20%) or in controls (11%). The recurrence rate of a recent CL without ovulation stigma was higher in primates with stage II endometriosis (5/6) than in animals with stage I disease (l/7) or in controls (O/6). The increased incidence and recurrence of a fresh CL without ovulation stigma in baboons with mild endometriosis suggests that repetitive luteinized unruptured follicle syndrome may be a cause of endometriosis-associated subfertility (66). The egg recovery rate was lower (13%) in baboons with a recent CL without ovulation stigma than in animals with a recent CL and ovulation stigma (54%), suggesting that luteinized unruptured follicle syndrome can be diagnosed by laparoscopy with an error rate of 13% (66). In baboons, PF steroid levels were not significantly lower in cycles without fresh ovulation stigma when compared with cycles with fresh ovulation stigma (67), as reported in women by some (581, but not all (521, investigators. Immunologic Aspects of Endometriosis White Blood Cell Populations Blood

in PF and Peripheral

White blood cell (WBC) populations in both PF and peripheral blood (PB) may be important in promoting ectopic endometrial growth and diminishing fertility by their activity (681, secretion of growth factors (69, 701, and secretion of cytokines (71, 72). An increased concentration and total number of macrophages, lymphocytes, and their subsets has been reported in both PF and PB from women with endometriosis when compared with those without the disease (72, 73). However, it is not known whether changes in WBC subpopulations in PF and PB are the cause or the consequence of endometriosis. This aspect is difficult to study in women, because most 618

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Baboon model for endometriosis

patients with pain, infertility, and endometriosis have had the disease for some time at the point of diagnosis (1). In baboons, the hypothesis was tested that PB and PF WBC populations are altered in baboons with spontaneous and induced endometriosis compared with animals without the disease (74). In this study, the percentages of WBC subsets, determined by mouse anti-human monoclonal antibodies CD2, CD4, CD8, CDllB, CD20, and Leu M5, were comparable in the PB of baboons to those reported in humans, showing that WBC subsets in baboons can be analyzed with commercially available monoclonal antibodies (74). In PB, the percentage of CD4+ and interleukin-2 receptor-positive cells was increased in baboons with stage II-IV spontaneous or induced endometriosis, suggesting that alterations in PB WBC populations may be an effect of endometriosis. In PF the WBC concentration and percentages of Leu M5+ macrophages and CD8+ lymphocytes were increased only in baboons with spontaneous endometriosis and not in animals with induced disease, suggesting that alterations in PF WBC populations may lead to the development of endometriosis (74). Natural Killer Cell Activity and Antiendometrial Cytoxicity

Although retrograde menstruation has been reported in most women (28, 461, endometriosis does not develop in all of them. Accumulating evidence suggests that altered cellular immunity may be involved in the pathogenesis of endometriosis both in women and in rhesus monkeys (2, 75-77). Decreased in vitro lymphocyte proliferation in response to autologous endometrial cells has been reported in both rhesus monkeys (76) and women (78) with endometriosis. Decreased autologous antiendometrial lymphocytotoxicity and decreased natural killer (NK) cell activity has been reported in women with endometriosis when compared with women with a normal pelvis by some but not by other investigators (72). In baboons, the hypothesis was tested that antiendometrial, lymphocyte-mediated cytotoxicity and NK cell activity are reduced in animals with endometriosis when compared with controls with a normal pelvis (79). Antiendometrial cytotoxicity was significantly lower in baboons with endometriosis (mean, 5.9% 2 8.7% [*SD]; median, 0%; range, 0%-26%) than in animals with a normal pelvis (mean, 22.9% + 23% [?SD]; median, 7%; range, 0%-78%). This difference could be explained by the absence of antiendometrial cytotoxicity in baboons with moderate to severe endometriosis, probably as a result of high spontaneous release of 51Cr. Fertility and Sterility@

When stricter criteria were used and only animals with a labeling index (maximal/spontaneous release) of 1.7 or more were analyzed, the antiendometrial cytotoxicity was comparable between baboons with and without endometriosis. Natural killer cell activity also was comparable in primates with and without endometriosis. In conclusion, no difference in antiendometrial cytotoxicity and NK cell activity was observed between baboons with and without endometriosis (79). High-dose Immunosuppression Endometriosis

and the Development

of

There is evidence that immune surveillance is altered in women with endometriosis (75-77, 80), which may facilitate the implantation of retrogradely shed menstrual endometrial cells. Whether immunosuppression facilitates the development of endometriosis is unknown. Immunosuppression has known profound effects on cellular and humoral immunity: global defects of T- and B-cell populations; decreased NK cell activity; 50% suppression of phytohemagglutinin-, concavalin A-, and pokeweed mitogen-stimulated blastogenesis; decreased Thelper-T-suppressor ratio; impaired T-suppressor cell function; and reduced in vitro lymphokine-activated NK cell activity (81). In baboons, the hypothesis was tested that immunosuppression can inhibit the immune defense mechanisms that have been proposed to prevent the implantation of ectopic endometrial cells, thus allowing the development and progression of endometriosis (81). Thirty-two baboons (8 with a normal pelvis, 10 with spontaneous endometriosis, and 14 with endometriosis induced by intraperitoneal seeding of menstrual endometrium) were studied. A daily injection was given of methylprednisolone, 0.8 mg/kg, and azathioprine, 2 mg/kg, for 3 months to 16 baboons (4 with a normal pelvis, 5 with spontaneous endometriosis, and 7 with induced endometriosis). No treatment was given to the remaining 16 primates. The change in number and surface area (measured in square millimeters) of endometriotic lesions was evaluated by laparoscopy. Immunosuppressed baboons with spontaneous endometriosis had a significantly higher number and larger surface area of endometriotic lesions than did nontreated animals. One animal that had received azathioprine only had 5-cm, large, bilateral endometriotic cysts. However, immunosuppressed and nontreated primates with induced endometriosis were comparable with respect to both number and surface area of implants. A transient decrease in typical lesions was noted during immunosuppression. Immunosuppression did not cause the Vol. 68, No. 4, October 1997

development of endometriosis in baboons with a previously documented normal pelvis (81). The hypothesis that endometriosis is caused by immunosuppression was supported only partly by this study, because immunosuppression did not affect the development of induced endometriosis or cause disease in baboons with a normal pelvis (81). The dose used was high enough to cause severe immunosuppression, as demonstrated by the decrease in WBC concentration in the peripheral blood and the development of lethal pneumonia in one baboon. It is possible that in baboons with a normal pelvis, the immunosuppression period was too short to favor significantly the implantation of retrogradely shed endometrium. In a previous study in rhesus monkeys, 7 years of exposure to proton irradiation was required for the development of endometriosis (12). The temporary decrease in typical and increase in subtle endometriotic lesions in immunosuppressed baboons with either spontaneous or induced disease suggest that immunosuppression may favor the development of subtle implants and retard the physiologic transition from subtle lesions to more typical implants (81). SPONTANEOUS

EVOLUTION

The natural history of endometriosis is poorly understood. Even though endometriosis is accepted to be a progressive disease, supportive data are scarce. A cross-sectional study (82) demonstrated that the incidence of subtle lesions decreased with age. This recently was confirmed by Koninckx et al. (271, who reported in a 3-year prospective study that the incidence, pelvic area, and volume of subtle lesions decreased with age, whereas for typical lesions these parameters and the depth of infiltration increased with age. During serial observations, some investigators (83) reported deterioration (47%), improvement (29.5%), or elimination (23.5%) of disease after 6 months. However, others (84) found after 12 months that endometriosis progressed moderately in 64% of the women, improved in 27%, and remained unchanged in 9%. In baboons, the hypothesis was tested that spontaneous endometriosis is a progressive disease. Serial laparoscopic observations were performed during up to 30 months in 13 baboons with spontaneous endometriosis. During each laparoscopy, the pelvis was examined for the presence of endometriosis; the number, size, and type of endometriotic implants were noted on a pelvic map; and the endometriosis score and stage was calculated according to the revised classification of The American Fertility Society (AFS) (85). Periods of development and regression were observed, resulting in overall disease progresD’Hooghe

Baboon model for endometriosis

619

sion (861, as indicated by a significant increase in AFS score and in both the number and the surface area of lesions (86,871. Remodeling, defined by transition between typical, subtle, and suspicious implants, was observed in 23% of lesions (86). Endometriosis did not undergo regression during the first and second trimesters of pregnancy (88). Subsequently, the incidence of spontaneous endometriosis in baboons with an initially normal pelvis was determined over a period of 32 months (89). The cumulative incidence of minimal endometriosis (proven by histology) was 64% up to 32 months of follow-up. The eight baboons in which proven endometriosis developed were followed up during a longer period and had undergone more serial laparoscopies than the animals that did not get the disease (89). Remodeling of endometriotic implants also was observed in these baboons (87, 89). The baboon data suggest that endometriosis is a dynamic and moderately progressive disease with periods of development and regression, and with active remodeling between different types of lesions. It is possible that peritoneal trauma and/or irritation during repeated laparoscopies could favor the development of endometriosis, eventually through local or systemic immunologic factors. Remodeling of the lesions suggests that there is transition between subtle and typical lesions, and indicates that white plaques should not be considered to be burnt-out lesions, because they can become white vesicles at a later stage. This concept of remodeling was shown for the first time in baboons and recently has been confirmed in women with endometriosis (90). ENDOMETRIOSIS

AND

SURFERTILITY

A causal relation between endometriosis and infertility has not been established definitely. In women with moderate to severe endometriosis (851, pelvic adhesions may cause impairment of tuboovarian function and infertility. An inverse relation between pregnancy rates (PRs) and the degree of endometriosis often has been proposed, but this has not been substantiated in prospectively controlled fertility trials. Subfertility associated with minimal to mild endometriosis is even more controversial. In asymptomatic women undergoing tubal ligation (mostly women of proven fertility), a prevalence of endometriosis as high as 43% has been reported, including mostly minimal or mild disease (28, 32-34). Studies in women with minimal to mild endometriosis who are undergoing artificial insemination with donor semen have shown that the monthly fecundity rate is either reduced (91, 92) or normal (93). In baboons, two independent prospective, con620

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trolled studies (93, 94) showed that animals with minimal endometriosis have normal fertility. Subfertility was found in baboons with spontaneous or induced endometriosis of AFS stages II, III, and IV. Ovarian endometriosis was not observed in baboons with either spontaneous or induced (26) endometriosis that were participating in the fertility trials (94, 95). These results indicate that in baboons, minimal endometriosis is not associated with infertility and probably is not a disease, but rather a physiologic phenomenon caused by cyclic retrograde menstruation. More extensive peritoneal endometriosis with (AFS stages III and IV) or without (AFS stage II) adhesions is associated with subfertility, even in the absence of ovarian involvement (95). DISCUSSION

The presence of spontaneous endometriosis was demonstrated in 25% of baboons of proven fertility in the wild (39). The macroscopic appearance, pelvic localization, and microscopic aspects of baboon endometriosis were comparable in many ways to the disease known in women, suggesting that the baboon may provide an interesting model for the study of human endometriosis. In the baboons we studied, ovarian or deep endometriosis was found rarely. In our initial study, 5% of endometriotic lesions were observed on the ovarian ligament, but superficial ovarian implants or ovarian endometriotic cysts were absent (39,87,86). However, large bilateral ovarian endometriotic cysts were observed in one animal that had been immunosuppressed with azathioprine (81). Further, advanced and life-threatening stages of disseminated endometriosis have been reported in the baboon (10). The paucity of spontaneous ovarian or deep endometriosis in the baboons studied may be explained by the fact that all baboons kept at the Institute of Primate Research were of proven fertility in the wild (39,411 and often had become pregnant in captivity; therefore, they may represent a population that is different from infertile women who never have been pregnant. It is possible that a longer period without conception is required for the development of ovarian or deep endometriosis than for peritoneal endometriotic lesions. It also is possible that ovarian endometriotic cysts may have a different pathogenesis than peritoneal endometriosis. This hypothesis is supported by the observation that ovarian endometriosis could not be induced experimentally by seeding or injecting menstrual endometrium on top of or beneath the ovarian capsule, respectively (261, and that large endometriotic cysts were observed only in an immunosuppressed baboon (91). Fertility and Sterility@

The baboons we studied predominantly had minimal endometriosis. Similarly, minimal to mild endometriosis has been reported in >80% of women of proven fertility with endometriosis who are undergoing laparoscopic sterilization (28-34). This population of asymptomatic, parous women may be comparable to the baboons of proven fertility in the wild that were evaluated in our study. Initially, all the baboons with endometriosis had minimal disease (39). During a follow-up period of 10 to 12 months, the number of lesions increased, but no progression beyond minimal endometriosis was observed (87). However, follow-up studies over 20 months (86) showed progression to mild or moderate disease in four of six baboons. Moderate and severe endometriosis also could be induced experimentally by the intrapelvic injection of menstrual endometrium (26). It may, be argued that experimentally induced endometriosis is different from spontaneous disease and should not be used as a model to study endometriosis in women. Indeed, in all previous reports, autotransplantation of endometrium obtained by hysterotomy during the follicular or luteal phase (18, 19) was used, and this artificial way of inducing endometriosis causes iatrogenic adhesions. However, the baboon study was designed to test the hypothesis that endometriosis could be induced experimentally by imitating an event of massive retrograde menstruation, based on the original hypothesis of Sampson (44) that endometriosis may be caused by increased retrograde menstruation with subsequent pelvic implantation of endometrial cells. In baboons with an accessible uterus, menstrual endometrium was obtained by transcervical biopsy and was injected into the pelvis under laparoscopic control (26). This method of induction resulted in typical and subtle lesions that were macroscopically similar to the spontaneous endometriosis observed both in women and baboons. The proportion of subtle red lesions was higher in baboons with experimental endometriosis than in those with spontaneous disease, supporting the concept that red lesions represent younger and more active endometriosis (6). Experimental induction of endometriosis offers the opportunity to make serial observations in the same animal before and after induction, enabling investigators to identify factors in PB and peritoneal fluid that are the consequence of endometriosis. Further, baboons with experimental endometriosis often had advanced stages of disease, offering potential models for the study of medical and/or surgical treatment of this disease. It may be argued that the revised AFS classification for endometriosis (85) cannot be applied to baboons with endometriosis, because they did not have ovarian disease. The revised AFS classification is a Vol. 68, No. 4, October 1997

descriptive instrument used to determine the extent of endometriosis in women, but it has poor intraobserver and interobserver reproducibility and is not based on correlations with pain or infertility (96). For our study, the AFS scoring system was adapted for the five times smaller size of the female baboons when compared with women. Our diagnosis of moderate and severe endometriosis was determined by the extent of peritoneal disease, posterior cul-desac obliteration, and the presence of adnexal adhesions. Therefore, we propose that the baboon can be used as an interesting nonhuman primate model to study the effects of peritoneal endometriosis without ovarian involvement. The baboon data provide new insights into the pathogenesis of endometriosis. The Sampson hypothesis (44) that endometriosis is caused by retrograde menstruation is supported by the increased incidence of retrograde menstruation in baboons with spontaneous endometriosis (47), the observation that cervical occlusion could cause retrograde menstruation and endometriosis (501, and the finding that intrapelvic injection of menstrual endometrium caused experimental endometriosis similar to the spontaneous disease (26). Research is needed to identify specific growth factors, cytokines, and integrins that may explain why endometriosis could be induced more successfully with menstrual endometrium than with luteal endometrium. Further studies also are needed to quantify the amount and implantation potential of endometrial cells in PF during the menstrual cycle in women with and without endometriosis. The role of the immune system in the pathogenesis of endometriosis needs to be addressed further in studies with well-defined study and control groups. Although endometriosis in baboons was associated with changes in WBC subpopulations (741, normal NK cell activity was observed and the clinical relevance of decreased antiendometrial cytotoxicity was questioned (72,79,97X General immunosuppression with methylprednisolone and azathioprine for 3 months did not result in a dramatic progression of spontaneous endometriosis and did not cause endometriosis in baboons with a previously normal pelvis (80). It is possible that immunosuppression for 3 months is too short a period to have a significant effect on the development of endometriosis. Baboons with minimal endometriosis and those with a normal pelvis were similar in several ways. The incidence of luteinized unruptured follicle syndrome was comparable in baboons with a normal pelvis and in those with minimal endometriosis (66). Fertility was not decreased in baboons with minimal endometriosis when compared with those with a normal pelvis in two independent studies (94, 95). PeD’Hooghe

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ripheral blood immune cell populations also were not different between primates with minimal endometriosis and those with a normal pelvis (74). However, the presence of minimal endometriosis was a risk factor for the development of more advanced disease, as supported by our observation (86) that spontaneous endometriosis was moderately progressive in most baboons over a 2-year period. It cannot be excluded that repetitive laparoscopies in the same baboons may cause peritoneal trauma and facilitate the development of endometriosis (98). However, this possibility was not supported by the observation that scarification of the posterior uterine surface at the time of induction did not favor the implantation of autologous endometrium (26, 54). Remodeling of endometriotic lesions (transition between subtle and typical lesions, and between subtle red and subtle white implants) was observed for the first time in baboons with spontaneous endometriosis (87,861, and also was confirmed later in primates with experimental disease (26) and in women with endometriosis (90). The decreased cycle fecundity and PR in baboons with mild, moderate, and, to a lesser extent, severe endometriosis when compared with animals with a normal pelvis or minimal disease offered evidence in a prospective study that endometriosis-associated subfertility is correlated negatively with the stage of disease (95). Recurrent luteinized unruptured follicle syndrome seems to be a possible mechanism explaining the subfertility associated with mild endometriosis in baboons (66). The results of these fertility studies in baboons (94, 95) support the rationale for treating minimal endometriosis to prevent progression and the infertility associated with more advanced stages of this disease. The association between pelvic pain and endometriosis was not evaluated in this project. Hypothetically, future research could assess the effect of experimental induction of endometriosis on general behavior during menses and nonmenstrual phases of the cycle in baboons. However, the association between dysmenorrhea, dyspareunia, and endometriosis will be virtually impossible to determine in baboons, regardless of the skill of the observer and the cooperation of the test subjects. Despite some limitations (e.g., a low prevalence of ovarian endometriosis, mostly minimal endometriosis), the baboon offers unique opportunities to study important aspects of human endometriosis, including pathogenesis, prevalence, incidence, spontaneous evolution, and endometriosis-associated subfertility. In women, it is impossible to perform the serial and controlled observational studies that are required for understanding completely this enigmatic disease. Further, pelvic injection of menstrual 622

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endometrium offers an in vivo culture model for the study of interactions between the endometrium, peritoneum, and PF. This induction method can cause advanced stages of experimental endometriosis and may lead to an attractive baboon model to assess the effects of medical and surgical treatment. Because of intrinsic advantages with respect to its strength, size, reproductive anatomy, and physiology, the baboon also may offer a suitable model for the study of other aspects of reproductive biology. In conclusion, our study demonstrated that spontaneous endometriosis in the baboon provides a primate model for the study of human endometriosis and that the baboon offers the potential to be developed as a model for severe disease and its sequelae, which may facilitate the diagnostic, therapeutic, and preventative strategies needed to combat this enigmatic and problematic disease.

Acknowledgments. The authors thank the mentors of this project: Charanjit S. Bambra, Ph.D., Nairobi, Kenya; Philippe R. Koninckx, M.D., Ph.D., Leuven, Belgium; and Joseph A. Hill, Harvard Medical School, Boston, MA. We are grateful for critical comments from the collaborating European centers: Maurice Bruhat, M.D., Clermont-Ferrand, France; Joaquin Calaf, M.D., Barcelona, Spain; Hans Evers, M.D., Ph.D., Maastricht, The Netherlands; Jef Raus, M.D., Ph.D., Diepenbeek, Belgium; and Marc Dhont, M.D., Ph.D., Gent, Belgium. The help of advisers, Arthur F. Haney, M.D., Duke University, Durham, NC; Daniel C. Martin, Memphis, TN; Robert S. Schenken, M.D., San Antonio, TX, and Egbert R. te Velde, M.D., Ph.D., University of Utrecht, The Netherlands, is greatly appreciated. The laparoscopy equipment was kindly donated by Mrs. Storz-Rehling (Storz Company, Tuttlingen, Germany). The authors also thank Mr. Zakariah Maheli Karanja, Mr. Samuel Kago, and Mr. Sammy Kisara for their clinical assistance and the WOB (Flemish Organization for Development Programs Abroad, Belgium) for logistic and administrative support. REFERENCES 1. Haney AF. Endometriosis: pathogenesis and pathophysiology. In: Wilson EA, editor. Endometriosis. New York: AR Liss, 1987:23-51. 2. Hill JA. Immunological factors in endometriosis and endometriosis-associated reproductive failure. Infertility and Reproductive Medicine Clinics of North America 1992;3:583-96. 3. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis in the rat. Fertil Steril 1985;44:684-94. 4. Jansen RPS, Russell P. Nonpigmented endometriosis: clinical, laparoscopic and pathologic definition. Am J Obstet Gynecol 1986;155:1160-3. 5. Martin DC, Hubert GD, Vander Zwaag R, El-Zeky FA. Laparoscopic appearances of peritoneal endometriosis. Fertil Steril 1989;51:63-7. 6. Nisolle M, Casanas-Roux F, Anaf V, Mine J, Donnez J. Morphometric study of the stromal vascularization in peritoneal endometriosis. Fertil Steril 1993;59:681-4. 7. McCann TO, Myers RE. Endometriosis in rhesus monkeys. Am J Obstet Gynecol 1970; 106:516-23. 8. Digiacomo RF. Gynecologic pathology in the rhesus monkey (Macaca mulatta). Vet Path01 1977; 14:539-46. Fertility and Sterility@

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over 2 years shows a normal monthly fertility rate (MFR) in baboons with stage I endometriosis and a decreased MFR in primates with stage II and stage III-IV disease. Fertil Steril 1996;66:809-13. 96. Hornstein MD, Gleason RE, Orav J, Haas ST, Friedman AJ, Rein MS, Hill JA, Barbieri RL. The reproducibility of the revised American Fertility Society classification of endometriosis. Fertil Steril 1993;59:1015-21. 97. D’Hooghe TM, Hill JA. Natural killer cell activity and endometriosis [letter]. Fertil Steril 1995;64:226-8. 98. van der Linden PJQ, de Goeij AFPM, Dunselman GAJ, Erkens HWH, Evers JLH. Endometrial cell adhesion in an vitro model using intact amniotic membranes. Fertil Steril 1996;65:76-80.

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