University of Wisconsin, Harlow Center for Biological Psychology, 22 N. Charter St, Madison, WI 53715, USA
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Abstract |
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Key words: endometriosis/intestinal microflora/Lactobacillus/rhesus monkey
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Introduction |
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Monkeys experience many of the same reproductive disorders as humans and, thus, have been used to investigate reproductive physiology and to model many gynaecological procedures, such as human IVF and ICSI (Sutovsky et al., 1996). Moreover, like humans, rhesus monkeys develop spontaneous endometriosis (Bertens et al., 1982
) characterized by ectopic implantation of endometrial glands and stroma (Hadfield et al., 1997
; Giudice et al., 1998
). In many human patients with more severe forms of the disease, the endometrial implants grow along the serosal surface of the gastrointestinal tract (Prystowsky et al., 1988
), where they become a source of significant pain and excretory problems. Female monkeys with this gynaecological condition show a particular predilection for having implant sites on the serosa of the lower intestine and colon (Coe et al., 1998
). Because of the impact endometriosis can have on the gastrointestinal tract, we sought to compare gut microflora of monkeys with endometriosis to age-matched, healthy females with a particular focus on one genus of bacteria, Lactobacillus. This species of bacteria is known to be beneficial, and limits enteric infection by producing bacteriocin-like, antibacterial substances (Bernet-Camard et al., 1997
) and by competing with pathogens for binding sites on the epithelium of the intestine (Bernet et al., 1994
).
There are several factors that predispose monkeys to endometriosis, such as prolonged periods of menstruation without intervening pregnancies (D'Hooghe et al., 1996), estrogenic treatments (Dizerega et al., 1980
; Hadfield et al., 1997
), and exposure to certain environmental toxicants (Rier et al., 1993
). Endometriosis may emerge years later, when the ectopic implants can be found aggressively invading normal tissue throughout the peritoneal cavity (Giudice et al., 1998
). Both the epithelial cells of the damaged tissues, and the leukocytes responding to the implants and inflamed tissues, secrete growth factors and proinflammatory cytokines, which in turn may affect gastrointestinal secretions and motility (Taché and Saperas, 1992
; Collins, 1996
). Because Lactobacilli gain an ecological advantage over other bacterial species by being resistant to the low gastric pH, events that suppress acid secretion, such as intestinal inflammation, could create an internal environment less conducive to Lactobacilli proliferation. Therefore, we hypothesized that monkeys with endometriosis would have an altered profile of intestinal microflora, particularly altered Lactobacilli. In addition, a second retrospective study was conducted on the necropsy reports of >400 female rhesus monkeys to confirm that endometriosis is associated with intestinal inflammation in this species.
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Materials and methods |
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Three methods of estimating the integrity of the gastrointestinal microflora were utilized. Total non-fastidious aerobic and facultatively anaerobic bacteria were enumerated by pour plating with brainheart infusion agar (Difco Laboratories, Detroit, MI, USA). This agar is rich in nutrients and allows growth of non-fastidious aerobic and facultatively anaerobic bacteria, such as members of the genera Streptococcus, Staphylococcus, Klebsiella, Proteus, and the family Enterobacteriaceae. The differential agar eosinmethylene blue (EMB; Difco Laboratories) was utilized to quantify Gram-negative aerobic and facultatively anaerobic bacteria. Although all members of the family Enterobacteriaceae will grow on EMB agar, most of the colonies are Escherichia coli or members of the genus Enterobacter (unpublished observations). Aerobic members of the genus Lactobacillus, including L. acidophilus, L. caseii and L. amylovorous, were enumerated by spread plate analysis on selective LBS agar (Becton Dickinson & Co., Cockeysville, MD, USA). After incubating the agar plates for 30 h at 37°C in a humidified atmosphere of air + 5% CO2, colonies were counted and the number of colony-forming units (CFU) per gram of faecal matter was determined.
Statistical analysis: Ageing research is typically conducted on monkeys aged >20 years, which is equivalent to >50 years in humans. Because age could impact the intestinal microflora (Hopkins et al., 2001), animals were assigned to the following groups prior to culturing based on their age and health: 1019 years (n = 6 healthy middle-aged controls, n = 4 with endometriosis), and 2031 years (n = 4 healthy aged controls, n = 4 with endometriosis). Although this resulted in small sample sizes, a power analysis indicated that an n = 4 was sufficient to detect differences in microflora considered to be biologically relevant. To determine whether the presence of ectopic endometrial growths influenced the intestinal microflora, a two-factor analysis of variance was performed, with the age of the animal (1019 versus 2031 years) and health status (control versus endometriosis) evaluated as the independent variables. A log10 transformation was performed on all bacterial concentrations prior to statistical analysis and the data are expressed as medians and the quartile range.
Study II
Subjects: To evaluate the association between endometriosis and gastrointestinal inflammation, a survey of necropsy records from a large cohort of deceased female rhesus monkeys was conducted. Necropsies have been performed for over 3 decades on all monkeys who have died from: (i) natural ageing, (ii) humane euthanasia for an incurable disease, or (iii) killed as part of a terminal research protocol. The necropsies are performed immediately after euthanasia, or within 8 h of death from natural ageing. The necropsies used for this study were performed between 1981 and 1998 and reflect the normal mortality at the WRPRC during this 18-year period. Initially, all necropsy reports were screened for the study; however, reports were excluded if monkeys were aged <10 years at the time of death, or, if it was not possible to determine the age at the time of death, resulting in 413 reports. The reports included 100 monkeys diagnosed with endometriosis by microscopic analyses of ectopic endometrial glands and stroma, as well as 313 control females without detectable endometriosis.
Descriptive statistics: Age at the time of death, presence of endometriosis, and evidence of intestinal inflammation were recorded for all females. Monkeys were categorized as having endometriosis if any ectopic endometrial growths were identified during necropsy and confirmed histologically. Endometriosis was rated as mild, moderate or severe depending on the number and distribution of implants. This schema has been used previously with monkeys (Coe et al., 1998) and is based on the rAFS classification system for characterizing endometriosis in humans (American Fertility Society, 1985
). Mild disease was defined as few implants and limited fibrous adhesions; moderate endometriosis was assigned when there were more fibrous adhesions; severe endometriosis was characterized by multiple loci with extensive fibrous adhesions. In addition to endometriosis, the monkeys were recorded as having nonspecific intestinal inflammation if infiltration of leukocytes into intestinal tissue, intestinal lesions, watery intestinal contents, or evidence of chronic diarrhoea were noted in the necropsy report.
Statistical analysis: The distribution of intestinal inflammation in monkeys with and without endometriosis was examined initially by 2 analysis. To determine if the age at the time of death was associated with the prevalence of intestinal inflammation, the distribution of intestinal inflammation in monkeys with and without endometriosis was examined in adult monkeys (1019 years old) as well as in aged monkeys (
20 years old) by
2 analysis. Autolysis of intestinal tissue can occur rapidly after death and may result in misinterpretation of intestinal inflammation. Therefore, the prevalence of intestinal inflammation was determined in separate analyses for animals that were killed as part of a terminal research protocol or for humane purposes (with necropsy immediately following) and compared with those that died from natural causes (with necropsy occurring up to 8 h later).
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Results |
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Discussion |
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It was not possible to determine whether the magnitude of the microflora alterations was associated with the stage of endometriosis (i.e. mild to severe). However, microflora alterations are more likely to occur with severe disease, because monkeys with endometriosis typically do not show overt symptoms until several peritoneal organs are impacted (McClure et al., 1971; La Regina and Vogler, 1984
). Moreover, the five animals that were confirmed as having endometriosis at abdominal surgery and post-mortem necropsy had at least three of their organs affected by endometriosis. This profile is consistent with severe endometriosis according to the rAFS classification system (American Fertility Society, 1985
).
Although extrauterine endometrial cells most often invade the ovaries and exterior of the reproductive tract in women with milder forms of endometriosis (Giudice et al., 1998), with increasing severity, the gastrointestinal tract, and occasionally the urinary tract, can become inhabited by the ectopic implants (Prystowsky et al., 1988
). Like uterine endometrium, the ectopic endometrial cells proliferate and mature in response to cyclic changes in steroid hormone concentrations, which becomes a significant source of abdominal pain. The cyclic proliferation can also contribute to widespread inflammation in the peritoneal cavity of women with endometriosis (Giudice et al., 1998
). In our study, monkeys with endometriosis were significantly more likely to have inflamed intestines at the time of death compared with healthy animals. This finding gives support to a hypothesized mechanism through which endometriosis could affect the intestinal microflora. Endometrial cells and leukocytes, particularly macrophages and monocytes that are recruited to inflamed tissues, secrete large quantities of proinflammatory cytokines, such as interleukin (IL)-1, IL-6, and tumour necrosis factor (Rier and Yeaman, 1997
). In addition to their immunomodulatory role, cytokines can have widespread effects on the gastrointestinal tract by suppressing gastric acid secretion (Taché and Saperas, 1992
) as well as gut motility (Plaza et al., 1997
). Although our study did not establish the causal mechanism for the change in the microflora, it is likely that pro-inflammatory mediators affected the gut, which in turn altered the profile of intestinal microflora, a hypothesis warranting further investigation in humans.
Disruption of the intestinal microflora can have adverse health consequences. In addition to aiding with proper digestion and absorption of macromolecules, the intestinal microflora act as a barrier to gut pathogens by blocking attachment to gut binding sites (Bernet et al., 1994), which is the first step of pathogenicity. For example, feeding germ-free immunodeficient rodents with Lactobacilli reduces colonization of Candida albicans after oral infection (Wagner et al., 1997
). In addition, several members of the microflora, including Lactobacilli, produce antibacterial substances that can have direct inhibitory effects on pathogenic organisms (Bernet-Camard et al., 1997
). Disturbance of these non-specific defences ultimately increases susceptibility to enteric infection, and could account for the increased occurrence of food allergies and opportunistic C. albicans infections in women with endometriosis (Lamb and Nichols, 1986
; Nichols et al., 1987
). Our study clearly demonstrates that endometriosis is associated with profoundly altered intestinal microflora, particularly Gram-negative members and members of the genus Lactobacillus. Although the specific mechanism through which endometriosis influences the intestinal microflora needs to be ascertained, it is likely that secretion of inflammatory mediators contributes to altering the make-up of the intestinal microflora. Our data thus offer a unique view of the pathology of endometriosis.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 26, 2001; resubmitted on January 14, 2002; accepted on March 7, 2002.