The effect of endometriosis, cycle stage, lymphocyte suppression and pregnancy on CA-125 levels in peritoneal fluid and serum in baboons.

BACKGROUND: Serum CA-125 during the mid-follicular phase has been reported to be a clinically useful and reproducible marker in the diagnosis of advanced endometriosis in women. This study was undertaken to document the effect of the menstrual cycle, pregnancy and lymphocyte suppression on CA-125 levels in peritoneal fluid (PF) and serum in baboons with a normal pelvis and baboons with endometriosis. METHODS: CA-125 levels were measured in 264 serum samples that were serially obtained during one menstrual cycle from 10 animals with and without endometriosis. In addition, CA-125 levels were determined in 204 archived samples (serum, n = 112 and PF, n = 92) obtained from 32 female baboons with or without endometriosis. The CA-125 assays were performed by radioimmunoassay using kits from Centocor (Malvern, PA, USA). RESULTS: Serum CA-125 levels were at their highest during menstruation and decreased progressively during the follicular and luteal phase. PF CA-125 levels were increased during the follicular phase in baboons with a normal pelvis, but no cyclic changes were observed in animals with endometriosis. Serum CA-125 levels were unaffected by induction, lymphocyte suppression or pregnancy. Induction of endometriosis resulted in increased PF CA-125 levels, whereas lymphocyte suppression or pregnancy had no effect. CONCLUSION: In baboons, serum CA-125 originates mainly from eutopic endometrium whereas the main source of PF CA-125 seems to be the peritoneum or ectopic endometrium. The baboon appears to be a valid model to further study the relationship between endometriosis and CA-125.

Effect of menstruation and intrapelvic injection of endometrium on inflammatory parameters of peritoneal fluid in the baboon (Papio anubis and Papio cynocephalus).

OBJECTIVE: Our purpose was to test the hypothesis that menstruation and intrapelvic injection of endometrium for the induction of endometriosis affect inflammatory parameters in peritoneal fluid from baboons. STUDY DESIGN: In the first part of this study, 107 laparoscopies were performed in 62 female baboons with a normal pelvis during menstruation, the follicular phase, and the luteal phase. In the second part of this study, 21 baboons were studied during paired laparoscopies in the follicular phase and the luteal phase of the cycle. In the third part of this study, 11 baboons were studied by paired laparoscopies during menses and during the nonmenstrual phase of the cycle. In the fourth part of this study, paired laparoscopies were performed in 7 baboons before and after intrapelvic injection of endometrium. Peritoneal fluid was aspirated and measured in all laparoscopies and assessed for leukocyte concentration. In the third and fourth parts of the study, peritoneal fluid was analyzed for the concentrations of inflammatory cytokines and for the proportions of cells with immunohistochemical staining positive for these cytokines. RESULTS: During menstruation, in comparison with nonmenstrual phases of the cycle, the leukocyte concentration of the peritoneal fluid was increased significantly, as were the proportions of peritoneal fluid cells with positive staining for tumor necrosis factor alpha, transforming growth factor beta(1), and intercellular adhesion molecule 1 and the peritoneal fluid concentrations of transforming growth factor beta(1) and interleukin 6. After intrapelvic injection of endometrium, the peritoneal fluid leukocyte concentration and the proportions of peritoneal fluid cells with positive staining for tumor necrosis factor alpha, transforming growth factor beta(1), CD3, and human leukocyte antigen (DR locus) significantly increased. CONCLUSION: Subclinical peritoneal inflammation occurs in baboons during menstruation and after intrapelvic injection of endometrium.

Pelvic inflammation induced by diagnostic laparoscopy in baboons.

OBJECTIVE: To test the hypothesis that diagnostic laparoscopy can cause pelviperitoneal inflammation. DESIGN: Retrospective analysis of data collected during a prospective controlled study in baboons. SETTING: An academic research environment. ANIMAL(S): Samples were collected during laparoscopies in female baboons at the Institute of Primate Research, Kenya. INTERVENTION(S): In the first part of the study, 44 laparoscopies were performed in 16 baboons (5 with a normal pelvis, 11 with endometriosis) during the luteal phase, with a time interval of 1 month. In the second part of the study, 53 laparoscopies were performed in 15 baboons (6 with a normal pelvis, 9 with endometriosis) during the late follicular and luteal phases of one cycle, with a median time of 3-4 days between each laparoscopy. MAIN OUTCOME MEASURE(S): Peritoneal fluid (PF) was measured and analyzed for white blood cell (WBC) concentrations and, in the second part of the study, for the distribution of lymphocyte subsets (CD3, CD4, CD8, and CD20) and for the presence of cytokines transforming growth factor-beta1, interleukin (IL)-6, and IL-10. RESULT(S): In the first part of the study, PF volumes and WBC concentrations were comparable at the baseline and follow-up laparoscopies. In the second part of the study, PF obtained at the second laparoscopy showed a 10-fold increase in volume, a 3-fold increase in WBC concentration, a 10-fold increase in IL-6 concentration, and a 2-fold increase in transforming growth factor-beta1 concentration when compared with PF obtained at the first laparoscopy. The PF subset of granulocytes and CD3-positive cells was higher and the PF subset of macrophages was lower at follow-up laparoscopies than at the baseline laparoscopy. CONCLUSION(S): Diagnostic laparoscopy can cause peritoneal inflammation in baboons.

Current status of reproductive behaviour in Africa.

The current annual population growth rate of 3.2% in Africa will double the population by the year 2025. The majority of this population is below 15 years of age, and Africa concurrently also has the highest incidence of infertility in the world. Sexual behaviour, which has been poorly studied in Africa, has a direct impact on reproductive health [including fertility, infertility and sexually transmitted diseases (STDs)]. The multiple cultures and religions which characterize the African continent also affect reproductive health. Factors that have a significant effect on reproductive health in Africa include greater prevalence of extramarital/commercial sexual activity, polygamy, lower prevalence of contraceptives, reliance on traditional practices, high incidence of STDs and teenage pregnancies. High risk reproductive behaviours are predominantly displayed by adolescents, and the prevalence of STDs, including HIV (human immunodeficiency virus), is very high in this group. Pregnancy-related complications are the major cause of health-related problems in 15-19 year old girls. Maternal mortality rates in most countries remain high. Literacy rates affect these behaviours. It is apparent that changing the sexual behaviour of adolescents is one way of reversing the adverse trends, such as STD transmission, unwanted pregnancy and poor general reproductive health.

DNA typing of primate major histocompatibility complex (Mhc)-DQA1 locus by PCR and dot blot hybridization.

Non-human primates (NHPs) are increasingly utilized as models to investigate different aspects of immune responses against self (autoimmunity) and foreign antigens. These animals provide valuable models for testing the efficacy of candidate vaccines against pathogens such as human immunodeficiency virus (HIV) and also fertility regulating agents (immunocontraceptives). In order to fully understand the effects of vaccination, it may be necessary to elucidate the immunogenetic background of these animals. The major histocompatibility complex (Mhc) molecules play an important role in the generation of effective immune responses. Serological techniques have been used in the identification of human leukocyte antigens (HLA) necessary for cross-matching organs and tissues for transplantation. However, the application of this technique for typing monkey Mhc alleles has been hampered by unavailability of well characterized immunological reagents. Polymerase chain reaction (PCR)-based techniques such as restriction fragment length polymorphism (RFLP) and sequence-specific oligonucleotide probe hybridization (SSOP) have been extensively used for typing HLA-DP, DQ and DR alleles. A commercially available Kit (AmpliTypeR) designed for amplification and typing of HLA DQalpha alleles is routinely used in typing DNA samples for forensic casework. In the present study, we have evaluated this kit for possible application in routine typing of primate DQA1 alleles. Genomic DNA from ten African primate species (23 individuals) was isolated from peripheral blood lymphocytes and polymorphic second exon of DQA1 locus amplified using GH26 and GH27 PCR primers. The PCR products were hybridized on a nylon membrane containing immobilized sequence-specific oligonucleotide probes. Our results show seven of the nine probes hybridizing with primate DQA1 alleles, indicating that typing of equivalent primate alleles can be accomplished at lower stringency conditions. However, it may be necessary to design additional oligonucleotides probes (based on available primate DQA1 sequences) to improve the discriminating power of this kit for use in routine typing of Old World monkey DQA1 alleles.

Colinear synthesis of an antigen-specific B-cell epitope with a 'promiscuous' tetanus toxin T-cell epitope: a synthetic peptide immunocontraceptive.

Carrier conjugation is commonly used to provide T-cell help for small, linear peptides containing antigen-specific B-cell epitopes. However, carrier conjugation is expensive, variable and often results in adverse side effects if the conjugate is administered repeatedly. To eliminate the need for carrier conjugation, we examined two synthetic peptides for their ability to elicit sustained antibody titres in female rabbits and baboons. One peptide (hC1-20) was based on the sequence of the sperm-specific isozyme of human lactate dehydrogenase (LDH-C4). This peptide stimulates helper T-cell responses. The other peptide (bC5-19:TT) was a chimera between an LDH-C4 B-cell epitope and a 'promiscuous' T-cell epitope from tetanus toxin which has been shown to bind to and stimulate many different major histocompatibility complex alleles. Both peptides were immunogenic in rabbits and baboons. The chimera elicited consistently high antibody titres and was immunogenic in 19/19 wild-caught female baboons. When 14 bC5-19:TT immunized baboons were mated, their fertility was reduced by 62% compared with controls (P < 0.02). This carrier-free construct can be incorporated into biodegradable microspheres which may provide long-term protection from pregnancy with a single dose.

Analysis of primate major histocompatibility complex (MHC)-DQA1 locus by PCR-single strand conformation polymorphism (SSCP).

Major histocompatibility complex (Mhc) gene products play an important role in the immune responses against pathogens and autoimmunity, disease resistance and transplantation. Non-human primates (NHPs) are increasingly being utilized as models to test the safety and efficacy of candidate vaccines. Mhc typing of NHPs is an important component of the vaccine trial studies and in the investigations of any associations between Mhc alleles and disease. Routine typing of primate Mhc alleles has been hampered by unavailability of well characterised immunological reagents. In this study, we have used PCR amplification and SSCP for screening polymorphisms in the primate DQA1 locus. Using this technique, 9 African primate species (36 individuals) were analyzed. Ten individuals showed three or four electrophoretic band patterns and the rest two-band patterns indicating this technique can be used to discriminate homozygous and heterozygous individuals prior to DNA sequencing. This method may also be used to screen primates for Mhc-DQA1 allelic polymorphism. However, practical application of this technique for routine typing of primate Mhc-DQA1 alleles depends on availability of adequate nucleotide sequence information.

Flow cytometric analysis on reactivity of human T lymphocyte-specific and cytokine-receptor-specific antibodies with peripheral blood mononuclear cells of chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and squirrel monkey (Saimiri sciureus)

There are relatively few monoclonal antibodies (mAb) that have been characterized for their applicability in studies on the immune system of various nonhuman primates. In the present study, we identified a large number of mAb that can be used in future immunological studies in three different nonhuman primates, i.e., chimpanzees, rhesus macaques, and squirrel monkeys. The reactivity of 161 anti-human mAb to T-cell antigens and cytokine receptors were tested on peripheral blood mononuclear cells (PBMC) from the three primate species by flow cytometric analysis. A total of 105 (65%), 73 (45%), and 68 (42%) antibodies reacted with PBMC from chimpanzees, rhesus macaques, and squirrel monkeys, respectively. Out of the 161 mAb, 38 reacted with all three species and 112 reacted with one or two of the species. No specific reaction was observed with mAb to receptors to GM-CSF, 4-1BB, FLT3, FLX2, common beta-chain, IL-1 (type I receptor), and IL-8.

The effect of pregnancy on endometriosis in baboons (Papio anubis, Papio cynocephalus).

BACKGROUND: This study was performed to test the hypothesis that endometriosis undergoes regression during pregnancy. METHODS: This study was performed on 11 baboons with histologically proven endometriosis, housed at the Institute of Primate Research, Nairobi, Kenya. In each individual baboon paired laparoscopies were performed prior to and during pregnancy (6 during first and 5 during second trimester of gestation) with an interval of 5 +/- 3 months. During each laparoscopy the number, size and type of endometriosis implants were noted in detail on a pelvic map; the endometriosis score and stage were calculated according to the revised American Fertility Society (AFS) classification. In each baboon the observations prior to and during pregnancy were compared and analysed by Wilcoxon signed rank test (two-tailed). RESULTS: No significant change was observed in the AFS score or stage of endometriosis, or in the number, size and type of endometriotic lesions in baboons during gestation when compared to the nonpregnant state. CONCLUSION: In baboons pregnancy had no significant effect on endometriosis during the first or second trimester of gestation.

The cycle pregnancy rate is normal in baboons with stage I endometriosis but decreased in primates with stage II and stage III-IV disease.

OBJECTIVES: To test the hypothesis that fertility is reduced in baboons with endometriosis. DESIGN: Prospective controlled follow-up study. SETTING: Institute of Primate Research, Nairobi Kenya. SUBJECTS: Seventy-one baboons, including 34 controls (normal pelvis) and 37 animals with histologically proven endometriosis (16 spontaneous and 21 induced). Baboons with endometriosis had minimal (n = 9), mild (n = 14), moderate (n = 7), or severe (n = 7) disease. INTERVENTIONS: Seventy-one baboons were mated during 286 cycles. MAIN OUTCOME MEASURES: Cycle fecundity rate, cycle pregnancy rate, cycle abortion rate. RESULTS: Cycle pregnancy rate (excluding spontaneous abortions) was 15% (44/286) and was comparable between controls (19%, 20/104) and baboons with endometriosis (13%, 24/182). The cycle pregnancy rates were 24%, 10%, 7.5%, and 12.5% in animals with minimal, mild, moderate, and severe endometriosis, respectively. The cycle pregnancy rate was significantly lower in baboons with stage III-IV endometriosis (9%, 6/64) and with stage II disease (10%, 7/73) than in animals with stage I endometriosis (24%, 11/45) or in controls with a normal pelvis (19%). CONCLUSION: The normal cycle pregnancy rate in baboons with stage I endometriosis and the decreased cycle pregnancy rate in baboons with stage II and stage III-IV disease suggest that fertility decreases with the stage of endometriosis in baboons.

Increased prevalence and recurrence of retrograde menstruation in baboons with spontaneous endometriosis.

This study was done to test the hypothesis that the incidence and recurrence of retrograde menstruation is higher in baboons with spontaneous endometriosis than in those without. A total of 399 laparoscopies was performed on 113 female baboons. Group 1 consisted of 84 animals with a normal pelvis (including 23 that later underwent induction of endometriosis and were assigned to group 4), group 2 comprised nine baboons with spontaneous endometriosis acquired during the last 2 years of the study, group 3 had 18 baboons with long-term spontaneous disease, and group 4 comprised 25 animals with induced endometriosis. Retrograde menstruation was defined by the presence of blood-stained peritoneal fluid (red or dark brown) during menses. Recurrence of retrograde menstruation was analysed during the first two laparoscopies in 13 baboons. Peritoneal fluid was 10 times more frequently blood-stained during menses (62%) than during non-menstrual phases (6%). Retrograde menstruation was observed more frequently in animals with spontaneous disease (groups 2 and 3, 83%) than in animals with a normal pelvis (group 1, 51%). Recurrence of retrograde menstruation was observed more frequently in baboons with spontaneous endometriosis (5/5) than in those without (3/8). The results of this study demonstrate that retrograde menstruation is common in baboons, with a higher prevalence and recurrence in animals with spontaneous endometriosis than in those without.

Development of spontaneous endometriosis in baboons.

OBJECTIVE: To determine the incidence of spontaneous endometriosis over a 32-month period in baboons with initially normal pelves. METHODS: In this observational longitudinal study, which was performed at the Institute of Primate Research, Nairobi (Kenya), 24 baboons with laparoscopically confirmed normal pelves underwent 67 serial laparoscopies (mean 2.8 +/- 1.9, median 3, range 1-6) after 1-3 months (n = 8), 4-6 months (n = 11), 7-9 months (n = 9), 10-12 months (n = 17), 13-15 months (n = 6), 16-18 months (n = 4), 19-21 months (n = 3), 22-24 months (n = 4), 25-27 months (n = 1), and 30-32 months (n = 4). 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 were tabulated according to the revised classification of the American Fertility Society. Taking into account the variable length of follow-up, we used life-table analysis to calculate the cumulative incidence of endometriosis. RESULTS: The cumulative incidence of minimal endometriosis (proven by histology) was 64% up to 32 months of follow-up. The eight baboons that developed proven endometriosis were followed over longer periods of time and had undergone more laparoscopies than the animals that did not develop the condition. CONCLUSION: There is a high incidence of minimal endometriosis in baboons, which increases with the time of follow-up and the number of repeat laparoscopies.

Effect of endometriosis on white blood cell subpopulations in peripheral blood and peritoneal fluid of baboons.

In women with endometriosis, changes in peripheral blood and peritoneal fluid white blood cell (WBC) populations have been reported, but it is known whether these alterations are casually related to or a consequence of endometriosis. The purpose of this study was to test the hypothesis that peripheral blood and peritoneal fluid WBC populations are altered in baboons with spontaneous and induced endometriosis compared to animals without disease. Peripheral blood and peritoneal fluid samples were obtained at laparoscopy from 60 baboons with a normal pelvis (n = 23), spontaneous endometriosis (n = 19) and induced disease (n = 12), luteal phase (n = 20), pregnancy or nursing (n = 11) and in non-cycling animals (n = 8). The WBC concentration was analysed with a Coulter counter and fluorescent antibody cell separation (FACS) analysis was used to measure cluster designation (CD)2, CD4, CD8, interleukin (IL)2R and leucine (Leu) M5 subsets. In peripheral blood, the percentage of CD4+ and IL2R+ cells was increased in baboons with stage II-IV spontaneous or induced endometriosis, suggesting that alterations in peripheral blood WBC populations may be an effect of endometriosis. In peritoneal fluid the WBC concentration and percentages of Leu M5+ macrophages and CD8+ lymphocytes were only increased in baboons with spontaneous endometriosis and not in animals with induced disease, suggesting that alterations in peritoneal fluid WBC populations may lead to the development of endometriosis. In summary, the results of this study suggest that peripheral blood and peritoneal fluid immune cell populations are affected in baboons with endometriosis.

Disappearance of the ovulation stigma in baboons (Papio anubis, Papio cynocephalus) as determined by serial laparoscopies during the luteal phase.

OBJECTIVE: To investigate how long an ovulation stigma remains visible as determined by serial laparoscopies performed during the luteal phase in baboons. SUBJECTS AND SETTING: Sixteen female baboons with a normal pelvis (n = 6) and with endometriosis (n = 10) housed at the Institute of Primate Research, Nairobi, Kenya. INTERVENTIONS: Fifty-six laparoscopies were carried out before ovulation (n = 7) and serially during the luteal phase (n = 49; 3 +/- 1 per baboon): 1 to 2 days (n = 2), 4 to 5 days (n = 15), 8 to 9 days (n = 11), 12 to 13 days (n = 12), and 16 to 17 days (n = 9) after ovulation. MAIN OUTCOME MEASURE: During each laparoscopy the ovaries were screened systematically for the presence and size of an ovulation stigma and/or corpus luteum (CL). RESULTS: When the laparoscopy was done within 5 days after ovulation, a fresh ovulation stigma was observed in all nine baboons with a normal pelvis or minimal endometriosis, but only in four of seven animals with mild to severe disease. If a fresh ovulation stigma had been observed within 5 days after ovulation (n = 13), it gradually became smaller but remained visible 8 to 9 days after ovulation in 91%, at 12 to 13 days after ovulation in 75%, and at 16 to 17 days after ovulation in 50% of the primates. CONCLUSION: If a fresh ovulation stigma was observed in baboons within 5 days after ovulation, it diminished in size but remained visible up to 8, 12, and 16 days after ovulation in 91%, 75%, and 50% of animals, respectively. Therefore, diagnostic laparoscopies for the detection of an ovulation stigma in baboons should be performed in the early luteal phase.

Increased incidence and recurrence of recent corpus luteum without ovulation stigma (luteinized unruptured follicle syndrome?) in baboons with endometriosis.

OBJECTIVE: To test the hypothesis that a recent corpus luteum (CL) without fresh ovulation stigma (OS) can be found with a higher incidence and recurrence in baboons with endometriosis and is associated with a reduced egg recovery rate after uterine flush. METHODS: One hundred thirty-eight laparoscopies were performed in the early luteal phase of 52 cycles in 32 female baboons with a normal pelvis (controls) and of 86 cycles in 21 animals with endometriosis. The incidence and recurrence of a recent CL without OS was studied during each laparoscopy and during the first three laparoscopies in 15 baboons (five controls, ten with endometriosis), respectively. The association between the presence of a recent CL without OS and the egg recovery rate was evaluated during 65 cycles in 21 baboons after uterine flush. RESULTS: When all 138 laparoscopies were analyzed, the incidence of a recent CL without OS was higher (P < .03) in animals with stage II-IV endometriosis (35%) than in those with stage I disease (19%) or controls (11%). Both the incidence (seven of ten) and recurrence (three of ten) of a recent CL without OS were higher in baboons with endometriosis than in controls (zero of five, P < .02) when studied during the first three laparoscopies. Laparoscopies in primates having a recent CL without OS were associated with a lower egg recovery rate (13%) than in animals with recent CL and OS (54%, P = .005). CONCLUSION: A recent CL without OS was associated with a reduced egg recovery rate and could be found with a higher incidence and recurrence in baboons with endometriosis than in controls, suggesting that luteinized unruptured follicle syndrome exists and could contribute to endometriosis-associated subfertility.

Serial laparoscopies over 30 months show that endometriosis in captive baboons (Papio anubis, Papio cynocephalus) is a progressive disease.

OBJECTIVE: To test the hypothesis that spontaneous endometriosis is a progressive disease in baboons. DESIGN AND SETTING: Prospective cohort study, Institute of Primate Research, Nairobi, Kenya. PARTICIPANTS: Twelve baboons with histologically proven spontaneous endometriosis. INTERVENTIONS: Fifty-four serial laparoscopies (4.2 +/- 1.9 per animal, range 1 to 7) were performed after 1 to 3 months (n = 2), 4 to 6 months (n = 1), 7 to 9 months (n = 6), 10 to 12 months (n = 17), 13 to 15 months (n = 7), 16 to 18 months (n = 1), 19 to 21 months (n = 8), 22 to 24 months (n = 8), 25 to 27 months (n = 2), and 30 to 32 months (n = 2). MAIN OUTCOME MEASURE: 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 both endometriosis score and stage were calculated. Remodeling was defined as a change in laparoscopic appearance (typical, subtle, suspicious) of an individual endometriotic lesion. Data were analyzed by a signed rank test. RESULTS: Endometriosis was progressive as shown by the significant increase in the number and surface of lesions and in endometriosis scores after 24 months. The total number of endometriotic lesions after 24 months consisted of 69% new (and mostly subtle) implants, 10% remodeled lesions, and 21% unchanged implants. CONCLUSIONS: This study demonstrated that endometriosis in captive baboons undergoing repeated laparoscopies is a dynamic and moderately progressive disease with periods of development and regression and active remodeling between different types of lesions.

The prevalence of spontaneous endometriosis in the baboon (Papio anubis, Papio cynocephalus) increases with the duration of captivity.

BACKGROUND: It is not known whether stress affects the prevalence of endometriosis in women. Baboons with spontaneous endometriosis may be interesting models to study the human disease. For baboons, living in captivity is a period of chronic stress without continuous exposure to pregnancy. This study was done to compare the prevalence of endometriosis between baboons recently captured in the wild and those living in captivity for several years. METHODS: A diagnostic laparoscopy was performed for screening endometriosis and obtaining biopsies in 104 female baboons including animals that had been captured in the wild less than 1 year ago (n=52, Group I), primates that had been living in captivity for one to two years (n=30, Group II) and animals that had been captured for more than two years (n=22, Group III). RESULTS: The prevalence of clinical and biopsy-proven endometriosis in all baboons was 17% and 12%, respectively. Clinical endometriosis was found more frequently in group III (32%) than in group II (17%) or group I (11%). The prevalence of biopsy-proven endometriosis was also significantly higher in group III (27%) than in groups I and II combined (8%, p=0.03). CONCLUSIONS: The results indicate that the prevalence of endometriosis increases with the time spent in captivity. This trend may be explained by more menstrual cycles uninterrupted by pregnancy in captive than in wild baboons, and possibly also by age-related factors or by captivity-associated stress.