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

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1 Human Reproduction Vol.20, No.11 pp , 2005 Advance Access publication July 21, doi: /humrep/dei181 The effect of metriosis, cycle, lymphocyte suppression and pregnancy on CA-125 levels in peritoneal fluid and serum in baboons H.Falconer 1,2, C.S.Bambra 2, D.Chai 2, F.J.Cornillie 3, J.A.Hill 4 and T.M.D Hooghe 2,5,6 1 Department of Obstetrics and Gynaecology, Karolinska Hospital, Stockholm, Sweden, 2 Institute of Primate Research, Nairobi, Kenya, 3 Centocor B.V. Heverlee, Belgium, 4 New England Fertility Center, Reading, Massaschusetts, USA and 5 Leuven University Fertility Center, Department of Obstetrics and Gynecology, UZ Gasthuisberg, KU Leuven, Leuven, Belgium 6 To whom correspondence should be addressed at: Leuven University Fertility Center, UZ Gasthuisberg, Herestraat 49, B Leuven, Belgium. Thomas.dhooghe@uz.kuleuven.ac.be 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 metriosis 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 metriosis. METHODS: CA-125 levels were measured in 264 serum samples that were serially obtained during one menstrual cycle from 10 animals with and without metriosis. 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 metriosis. 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 metriosis. Serum CA-125 levels were unaffected by induction, lymphocyte suppression or pregnancy. Induction of metriosis 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 metrium whereas the main source of PF CA-125 seems to be the peritoneum or ectopic metrium. The baboon appears to be a valid model to further study the relationship between metriosis and CA-125. Key words: baboon/ca-125/metriosis Introduction CA-125 is a cell surface antigen which is expressed in tissues derived from embryonic coelomic epithelium such as metrium, cervix, Fallopian tubes, peritoneum, pleura and pericardium (Kabawat et al., 1983). It is present in low concentrations in serum from healthy women. Elevated concentrations are found in women with epithelial ovarian carcinoma (Bast et al., 1983). Various studies have demonstrated elevated serum CA-125 concentrations in serum of patients with metriosis (Barbieri et al., 1986; Patton et al., 1986; Pittaway and Fayez, 1986; Pittaway and Douglas, 1989). Similarly, increased serum CA-125 concentrations have been reported in rhesus monkeys with spontaneous metriosis (Pittaway et al., 1986). The use of serial CA-125 concentrations in serum has been recommended to monitor metriosis in infertile women (Pittaway, 1990). A valid method to measure CA-125 concentrations in peritoneal fluid (PF) has been reported by Kruitwagen et al. (1991a,b). These studies showed that the mean PF CA-125 concentration and total amount were significantly lower during the luteal phase when compared with other phases of the menstrual cycle. No correlation was found with the presence or absence of metriosis and adhesions. Due to ethical restrictions for performing serial laparoscopies in women, it is difficult to correlate the spontaneous evolution of metriosis with serum and PF CA-125 concentrations. Such studies can only be done in non-human primates with spontaneous metriosis. Additionally, the effect of lymphocyte suppression and of induction of metriosis on CA-125 levels can be studied in this animal model. A high prevalence of spontaneous metriosis (25%) has been reported in baboons of proven fertility (D Hooghe et al., 1991) and the disease seems to be progressive (D Hooghe et al., 1992, 1996a). The current study was undertaken to document the concentration of CA-125 in serum and PF during the menstrual cycle in baboons with metriosis and controls, and to assess the effect of the induction of metriosis (intrapelvic injection of menstrual metrium) (D Hooghe et al., 1995a), lymphocyte suppression (D Hooghe et al., 1995b) and pregnancy The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved For Permissions, please journals.permissions@oupjournals.org

2 H.Falconer et al. (D Hooghe and Debrock, 2002) on serum and PF CA-125 values. The following hypotheses were tested: (i) PF and serum CA-125 levels are increased in baboons during the menstrual phase when compared to the other phases of the cycle, as is known in women; (ii) serum and PF CA-125 levels are increased after induction of metriosis due to CA-125 production originating from ectopic metrium and/or other peritoneal tissues (D Hooghe et al.,1995a,2001); (iii) lymphocyte suppression increases serum and PF CA-125 levels in baboons with metriosis, since the progression of spontaneous metriosis appears to be faster in immunosuppressed baboons than in controls (D Hooghe et al.,1995b); (iv) pregnancy reduces serum and PF CA-125 levels in baboons with or without metriosis, since it is generally believed that pregnancy reduces the extent and activity of metriotic lesions (D Hooghe and Debrock,2002). Materials and methods Study design Part 1 This was a longitudinal study evaluating changes in serum CA-125 during the menstrual cycle in baboons with and without metriosis. Serial serum samples were collected throughout the menstrual cycle from 10 baboons with a normal pelvis (n = 6) or with metriosis (n = 4) (Table Ia). Samples were collected daily during the first 10 days and subsequently every second day from day 11 until the first day of the next menstruation. Part 2 This was a cross-sectional study of CA-125 in serum and PF samples obtained during different s of the menstrual cycle, to evaluate the impact of the induction of metriosis, lymphocyte suppression and pregnancy. These samples were collected from a total of 32 baboons during previously published studies (D Hooghe et al., 1991, 1992, 1995a, 1996a,b). This part of the study was subdivided into five parts (Table Ib): (i) determination of serum CA-125 values during different s of the menstrual cycle; (ii) measurement of CA-125 values in PF during the menstrual cycle; (iii) studying the impact of induction of metriosis on CA-125 in serum and PF; (iv) studying the impact of lymphocyte suppression by methylprednisolone and azathioprine on CA-125 in serum and PF; (v) studying the impact of pregnancy on CA-125 in serum and PF. Baboons The animals were all trapped in the wild and had been in captivity at the Institute of Primate Research between 9 and 47 months. The study protocol was reviewed and accepted by ISREC (Institutional and Table Ia. Description of the number of animals and samples for part 1 of the study 3034 Controls No. of animals 6 a 4 b No. of serum samples Menstrual phase Follicular phase Luteal phase a One animal excluded due to undetectable CA-125. b Two animals excluded due to undetectable CA-125. Spontaneous metriosis Scientific Ethical Committee). The animals were kept in single cages for 3 months prior to onset of the studies. Furthermore, all animals went through a diagnostic laparoscopy to screen the pelvic area for the presence of metriosis or other abnormalities. Monitoring of the menstrual cycle The menstrual cycle was monitored through daily observations of the perineal skin (sex skin) (D Hooghe et al., 1991; Bambra, 1993). No hormonal assays were performed in this study. The menstrual cycle in the baboon is divided into eight different s, with 7 being menstrual phase, 1 5 follicular phase and 6, 0 luteal phase. The sex skin is monitored every day by trained animal technicians. The progressive inflation of the perineum throughout the cycle correlates with the follicular growth and the subsequent deflation ( 6 and 0) to the luteal phase. Ovulation occurs during 5. An additional, 8, is similar to 0 (however more reddish) and becomes apparent during pregnancy (Hendrickx, 1971). The animals inflated and deflated normally during the studies and their cycle length was comparable to the cycle length they had established in the colony since captivity. Sample collection In the longitudinal part of the study, 264 peripheral blood (PB) samples were collected from the right or left forearm vein of the baboon. The PB was immediately centrifuged and sera were snap-frozen in liquid nitrogen and stored at 80 C before being analysed. In the cross-sectional part of the study, a total of 112 PF and 92 serum samples were analysed. These samples had all been obtained and archived from other studies performed at IPR. Serum samples were handled as described above. The PF was aspirated during laparoscopy before the animal was placed in the Trendelenburg position and stored at 80 C before being analysed, as described previously (D Hooghe et al.,1991). Both serum and PF samples were freeze-thawed only once during the studies. During the study period of 2 years, five animals with an initially normal pelvis developed metriosis at a later and were therefore used both as controls and as primates with spontaneous metriosis. Laparoscopies and induction of metriosis Laparoscopies and anaesthesia were carried out as previously described (D Hooghe et al., 1991). The pelvic area was thoroughly screened for the presence of metriosis. Spontaneous metriosis was classified according to the rafs (ASRM) classification system (D Hooghe et al.,1991). Lesions were characterized either as typical blue-black, red or white. Endometriosis was induced as previously described (D Hooghe et al., 1995a). Briefly, metrium obtained through transcervical curettage was seeded onto the pelvic area under laparoscopic vision. Lymphocyte suppression A daily injection of 0.8 mg/kg i.m. methylprednisolone and 2 mg/kg azathioprine was performed for 3 months in 11 baboons (three animals with induced metriosis, five baboons with spontaneous metriosis and three controls with a normal pelvis, Table I), as described previously (D Hooghe et al., 1995b). Pregnancy Serum and peritoneal fluid samples were analysed before pregnancy (Table I) and during the second trimester. Pregnancy was verified with ultrasound.

3 Cyclic changes of CA-125 in the baboon Table Ib. Description of the number of animals and samples for part 2 of the study (a) (b) (c) (d) Control Sp Ind Control Sp Ind Control Sp Ind Post LS Serum CA-125 No. of animals 21 a 14 b 8 c No. of serum samples M FP LP P PF CA-125 No. of animals No. of PF samples M FP LP P a Eight animals excluded due to undetectable CA-125. b Three animals excluded due to undetectable CA-125. c One animal excluded due to undetectable CA-125. PF = peritoneal fluid; Sp = spontaneous metriosis; Ind = induced metriosis; Post LS = post lymphocyte suppression; M = menstrual phase; FP = folllicular phase; LP = luteal phase; P = pregnancy. CA-125 assay The CA-125 assays were performed using kits from Centocor Inc. (Malvern, Pennsylvania, USA). For serum samples the one-step IRMA (immunoradiometric assay) was performed and the two-step for PF samples. All assays were performed in duplicates in one run. Intra- and inter-assay coefficients of variation were 3.8 and 5.7% respectively. The rationale for using two different assays is that early experiments with these kits revealed an artefact when analysing PF samples with a one-step IRMA (Williams et al., 1988; Hunter et al., 1990). Serially diluted serum samples showed excellent parallelism with the one-step assay but when repeated in PF, a marked hook-effect (limited parallelism to the calibration standard) was observed. However, with the two-step IRMA, the parallelism for diluted PF samples was equal to the parallelism observed in serum using the one-step IRMA. The two-step assay involves incubation of a 100 µl sample with capture-mab (monoclonal antibody) at room temperature, followed by 3 h of incubation with signal-mab for quantifying CA-125 in PF. Kruitwagen et al. (1991b) has recommended using the two-step IRMA for PF samples whereas the one-step is sufficient for serum samples. Statistical analysis Statistical analysis was done using linear regression, Kruskal Wallis, analysis of variance and Mann Whitney tests where appropriate and a value of P < 0.05 was considered to be statistically significant. Results Part 1 CA-125 was detectable in serum from 7/10 (70%) animals (two with spontaneous metriosis and five controls). The control animals (n = 5) had a mean cycle length of 31 ± 4 days and all had a similar length of follicular phase, luteal phase and menstrual phase (details not shown). The two baboons with spontaneous metriosis had a mean cycle length of 27 days. In both groups, the concentration of CA-125 in serum was highest during menstruation, with a significant (P < , linear regression) decline throughout the menstrual cycle (Figure 1). Part 2 In the serum samples from 12 baboons (28%; eight controls, three with spontaneous metriosis and one animal with induced metriosis) CA-125 was not detectable and these samples were excluded for further statistical analysis. No significant changes were observed in CA-125 concentration in serum during different s of the menstrual cycle in baboons with a normal pelvis, spontaneous or induced metriosis (Table II). In contrast to the serum samples, CA-125 was detected in all PF samples. In PF from baboons with a normal pelvis, the mean concentration of CA-125 in PF was significantly higher during the follicular phase than during the menstrual or luteal phase (Table II). However, no significant cycle changes were observed in CA-125 levels in PF from baboons with (spontaneous or induced) metriosis. During both the menstrual and luteal phase, the mean concentration of CA-125 in PF was significantly higher in baboons with (spontaneous or induced) metriosis than in primates with a normal pelvis (Table II). The concentration of CA-125 in serum was not affected by lymphocyte suppression, pregnancy or induction of metriosis. The concentration of PF CA-125 was significantly increased after induction of metriosis, but lymphocyte suppression and pregnancy had no effect (Table III). 3035

4 H.Falconer et al. (a) (b) (IU/ml) (IU/ml) 10 5 Controls (n=5) Menstrual cycle (days) Spontaneous metriosis (n=2) Menstrual cycle (days) means means Figure 1. Serial serum CA-125 levels during the menstrual cycle were detectable in five out of six baboons with a normal pelvis (a) and two out of four baboons with spontaneous metriosis (b). Linear regression analysis showed that the highest levels of serum CA-125 were observed during menstruation and that these levels declined progressively and significantly during follicular and luteal phase (P < ). Discussion Serum markers have gained much attention the past years in the development of non-invasive diagnostic tools for metriosis. Several alternatives, such as ultrasonography and magnetic resonance imaging, have failed to detect peritoneal metriosis (Mais et al., 1993; Takahashi et al., 1996; Guerriero et al., 1998). However, new non-invasive tests are under development, as it has been recently demonstrated that serum IL-6 and PF tumour necrosis factor (TNF)-α could be used to discriminate between patients with and without metriosis (Bedaiwy et al., 2002). CA-125 is the most extensively studied serum marker, with high specificity but, unfortunately, low sensitivity. However, some recent studies indicate that CA-125 could be used as an adjuvant together with other markers or methods. In a recent meta-analysis, it was suggested that routine use of CA-125 in subfertile patients could be justified to identify a subgroup likely to benefit from laparoscopy (Mol et al., 1998). Other authors have reported that a screening method combining CA- 125 with the level of a leukocyte subpopulation increases the positive predictive value in detecting metriosis (Gagne et al., 2003). In part 1 of the study, a serial analysis of serum CA-125 levels during the menstrual cycle revealed that the highest CA-125 levels were observed during menstruation with subsequent progressive decline during follicular phase and luteal phase, as has also been demonstrated in humans and in rhesus monkeys (Pittaway et al., 1986; Lanzone et al., 1991; Pittaway and Fayez, 1987; Fedele et al., 1988; Hornstein et al., 1992). These cycle-dependent changes of the CA-125 levels in serum indicate that the CA-125 originates mainly from the metrium and that the metrial breakdown is probably associated with direct secretion of CA-125 into the peripheral blood. This idea is shared by several authors (Jäger et al., 1988; Zeimet et al., 1993; Hompes et al., 1996) and supported by the detection of CA-125 in cultured metrium (Bischof et al., 1986) and positive indirect immunoperoxidase staining for CA-125 in metrial biopsies (Fedele et al., 1989). The purpose of the cross-sectional part of the study was to enable comparisons of serum CA-125 between groups (metriosis versus controls), since the number of animals was insufficient in the first part for these statistical analyses. Furthermore, the cross-sectional part allowed analyses of changes in PF CA-125 during the cycle. From a methodological point of view, a longitudinal study with serial samples of these changes is superior to the cross-sectional with only one sample/animal/cycle. However, this would require daily laparoscopies, introducing the bias of repeated surgery on PF content (inflammatory response to surgery) and CA-125 levels (D Hooghe et al., 1999). Table II. CA-125 (IU/ml) in peritoneal fluid and serum according to the cycle phase (means ± SD) in controls and in baboons with metriosis Peritoneal fluid CA-125 (IU/ml) Serum CA-125 (IU/ml) Controls (n = 15) Spontaneous metriosis (n = 15) Induced metriosis (n = 5) Controls (n = 13) Spontaneous metriosis (n = 11) Induced metriosis (n = 7) Menstrual 1406 ± 954 a,b 2788 ± 1136 b ND 20.5 ± ± ± 8.1 Follicular 3936 ± 3068 a 3586 ± ± 2190 c 24.7 ± ± ± 8.1 Luteal 1170 ± 1241 a,b 3179 ± 2057 b 1829 ± 511 c 21.1 ± ± The numbers of animals and samples are shown in Table I (part 2). In controls, the PF CA-125 concentration was significantly higher (P < 0.05) during the follicular phase than during the menstrual or luteal phases. In baboons with spontaneous metriosis, PF CA-124 levels were significantly higher than in controls during the menstrual (P < 0.05) and luteal (P < 0.05) phases. Analyses of CA-125 in serum showed no significant differences between groups. a,b P < c P < ND = no data. 3036

5 Cyclic changes of CA-125 in the baboon Table III. Effects of induction of metriosis (part 3a), lymphocyte suppression (part 3b) and pregnancy (part 3c) on the concentration of CA-125 (mean ± SD) in serum and peritoneal fluid (PF) Serum CA-125 (IU/ml) CA-125 in PF (IU/ml) Pre Post Pre Post Induction (A) 11.0 ± ± ± 456 a 2216 ± 127 a Induction (B) 14.0 ± ± ± 1171 b 2523 ± 836 b Lymphocyte 15.8 ± ± ± ± 2911 suppression (A) Lymphocyte 2088 ± ± 2260 suppression (B) Pregnancy 13.3 ± ± ± ± 313 The numbers of animals and samples are detailed in Table I (Part 3a, b, c). Separate analysis was performed on samples obtained from animals that were in the same cycle (analysis A) or in a different cycle (analysis B) before (pre) and after (post) induction of metriosis or lymphocyte suppression respectively. a P < b P < It is not surprising that the significant cycle-dependent changes of CA-125 seen in part 1 (longitudinal study) were not confirmed in part 2 (cross-sectional study), since there were important methodological differences between these two parts. In part 1, a longitudinal design (analysis of serial blood samples from the same baboon during one menstrual cycle) was applied and more samples (n = 264) with detectable serum CA-125 were available. Such longitudinal design is less influenced by interindividual variability than the cross-sectional method used in part 2, where only one sample per baboon was analysed and the lower number of samples (n = 58) was much lower. The origin of peritoneal fluid CA-125 is a puzzling question. Our results are consistent with studies in women reporting a poor correlation between the concentration of CA-125 in PF and serum (Ismail et al., 1994). Several authors have suggested that non-metrial sources of PF CA-125 are important, including peritoneum and metriotic lesions (Fedele et al., 1988; Kruitwagen et al., 1991a; Ismail et al., 1994). Indeed, Barbieri et al. (1986) demonstrated the presence of CA-125 on the cell surface of ovarian metrioma from three patients, whereas other investigators have failed to reproduce these results (Fedele et al., 1988, 1989). In our study, the induction of metriosis resulted in a significantly increased concentration of CA-125 in PF (Table III). These results are in line with previous observations in baboons showing an increase in inflammation markers, i.e. leukocytes, macrophages and inflammatory cytokines in PF after induction of metriosis (D Hooghe et al., 2001). Furthermore, a rise in the concentration of CA-125 in PF has also been demonstrated after abdominal surgery in men (Duk et al., 1986). Taken together, these data suggest that both damaged peritoneum and ectopic metrium are important sources of CA-125 in PF. In baboons with a normal pelvis (controls), a significantly higher concentration of CA-125 in PF was observed during the follicular phase when compared to menstrual or luteal phase. We hypothesize that it takes a few days before the inflammatory effects of retrograde menstruation in baboons (D Hooghe et al., 2001) result in peritoneal damage leading to increased PF CA-125 concentration during the follicular phase rather than during the menstrual phase. A similar but non-significant trend was observed in PF from baboons with spontaneous metriosis, probably since the CA-125 levels in PF were significantly higher during menstrual and luteal phase in baboons with spontaneous metriosis than in those with a normal pelvis (Table II), resulting in the loss of cyclical variation in PF CA-125 concentrations, as has been suggested before in women (Kruitwagen et al., 1991a). Although we reported that the progression of spontaneous metriosis appears to be faster in immunosuppressed baboons than in controls (D Hooghe et al., 1995a), our hypothesis that lymphocyte suppression increases serum CA-125 levels and PF CA-125 levels in baboons with metriosis was not confirmed in the current study. This can be explained by the small number of baboons tested, the heterogeneity in terms of presence of metriosis and cycle, and possibly a limited biological effect of general lymphocyte suppression with methylprednisolone and azathioprin on the CA-125 production by metrium, metriosis or peritoneum. In spite of these inconclusive results, modulation of the immune system and the inflammatory system are interesting fields of future research. Recently, blocking of the pro-inflammatory cytokine TNF-α has been demonstrated to reduce the extent of metriosis in baboons (Barrier et al., 2004). Several other cytokines and immunomodulators, such as interleukin-8 and pentoxifylline, could be interesting targets in the development of new drugs against metriosis. Although it is generally believed that pregnancy reduces the extent and activity of metriotic lesions in women, this was not clinically confirmed in baboons (D Hooghe et al., 1997). Therefore, it is not surprising that we could not confirm the hypothesis that pregnancy reduces serum CA-125 and PF CA-125 levels in baboons with or without metriosis. This is a relevant observation, since no paired comparisons between CA-125 levels before and during pregnancy are available in women with or without metriosis. In women, serum CA-125 levels show wide fluctuations during the first trimester with values above normal (Spitzer et al., 1998; Bon et al., 2001). The cause of undetectable serum CA-125 in 30% of the baboons is unclear. PF concentrations are generally 10-fold higher, which is the likely reason why CA-125 was detected in all PF samples as opposed to serum samples. The serum CA- 125 concentrations in baboons seem to be lower than serum CA-125 concentrations observed in women, and we speculate that the sensitivity of the human CA-125 kits might have been too low to detect very low values in serum from baboons. This may be related to the speculation by some authors that the molecular structure in PF CA-125 differs from that in serum (Kruitwagen et al., 1991a). Finally, inter-species differences between humans and baboons in CA-125 turnover and structure could be additional sources for undetectable values. In conclusion, serum CA-125 levels were increased during menstruation in all baboons whereas PF CA-125 levels were increased during the follicular phase in baboons with a normal pelvis when compared to other phases of the cycle. Higher PF CA-125 levels were also observed after induction of metriosis. Finally, PF CA-125 levels were higher in baboons with spontaneous metriosis when compared to baboons with a 3037

6 H.Falconer et al. normal pelvis during the menstrual and luteal phase. Collectively, these suggest that serum CA-125 originates mainly from bleeding eutopic metrium whereas CA-125 production in PF seems to come from ectopic metrium and/or peritoneal damage or inflammation, possibly in response to and following retrograde menstruation The baboon seems to be a valid model for further studies evaluating cause effect relationships between metriosis and the production of CA-125 in serum and peritoneal fluid. References Bambra C (1993) Veterinary Management and Research Techniques for Reproductive Studies in the Baboon: A Practical Approach. Institute of Primate Research, Nairobi. Barbieri RL, Niloff JM, Bast RC Jr, Scaetzl E, Kistner RW and Knapp RC (1986) Elevated serum concentrations of CA-125 in patients with advanced metriosis. 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