Embryotoxicity of peritoneal fluid in women with endometriosis. Its relation with cytokines and lymphocyte populations

María-José Gómez-Torres1, Pedro Acién1,3,4, Adolfo Campos2 and Irene Velasco2

1 Departments of 1Gynaecology and 2 Immunology, School of Medicine, Miguel Hernández University, and 3 Service of Obstetrics and Gynaecology, San Juan University Hospital, Campus San Juan, 03550 Alicante, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The goals of the present work were to study the embryotoxic effects of peritoneal fluid (PF) in women with or without endometriosis, and to relate any embryotoxicity to the severity of endometriosis, infertility or achievement of pregnancy, cytokine concentrations and lymphocyte populations. METHODS: Sixty-six consecutive women of reproductive age, 54 with endometriosis (21 infertile) and 12 infertile without endometriosis, and another 12 fertile women as control group, were included in this study. They all underwent laparoscopy or laparotomy in the second half of the cycle, and PF was collected from the pouch of Douglas. The embryotoxicity of the PF was assessed by means of a mouse embryo assay, and expressed as the number of embryos that did not reach blastocyst stage. Cytokines and lymphocyte populations present in PF were also studied and correlated with embryotoxicity. RESULTS: PF embryotoxicity was increased in women with endometriosis, but there was little correlation with the severity of the disease. However, although a clear relationship to the presence of infertility was not found, embryotoxicity appeared to be lower in those infertile patients with endometriosis who later became pregnant. We found a significant increase in embryotoxicity in the presence of high cytokine concentrations, especially with interleukin-6, and less so with interleukin-8 (P < 0.05). No good correlation was observed with lymphocyte populations, but CD56 (NK) cells were significantly increased in the PF of women with endometriosis. In general, the correlations for embryotoxicity were better when PF was diluted at 20% (91.4 ± 17 versus 68.1 ± 31, P < 0.01). CONCLUSIONS: These results suggest that alteration in the production of cytokines in the PF, especially IL-6, besides contributing to the endometriosis and its evolution, probably increases embryoxicity. However, no correlation was found between the latter and associated infertility.

Key words: embryotoxicity/endometriosis/immunology/peritoneal fluid


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Many studies (Haney et al., 1981Go; Syrop and Halme, 1987Go; Halme and Surrey, 1990Go) have reported that macrophages, cytokines and other products present in the peritoneal fluid (PF) from patients with endometriosis could be responsible for a change in the peritoneal environment that generates embryotoxic activity, and could thus be related to the frequently-associated infertility.

The presence of endometriosis produces an i.p. inflammatory response. Patients with endometriosis have elevated levels of proinflammatory cytokines, such as interleukin-6 (IL-6) and tumour necrosis factor {alpha} (TNF{alpha}) (Ho et al., 1996Go; Overton et al., 1996Go; Calhaz-Jorge et al., 2000Go). IL-6 has been reported to play a role in the regulation of ovarian steroid production and early implantation events. Therefore, this cytokine can be an important factor in infertility in women with endometriosis (Punnonen et al., 1996Go). In addition, it has been reported that endometriotic cells spontaneously release IL-6 in culture. This secretion is increased by the presence of IL-1ß and TNF{alpha} (Akoum et al., 1996Go), proinflammatory cytokines produced mainly by activated macrophages and also elevated in PF of these patients.

On the other hand, deficient T cell function and decreased peritoneal NK cell activity have been proposed as important factors in the pathogenesis of endometriosis. The suppression of these activities is supported by the increase in IL-6, IL-10 and IL-12 and the decrease in IFN-{gamma} production described in PF of patients with endometriosis (Ho et al., 1996Go; Ho et al., 1997Go). The establishment and maintenance of immunological tolerance to endometriosis may depend on the balance between TH1 and TH2 cytokine profiles (Braun et al., 1998Go). Impairment of macrophage function supports the theory that an inappropriate immunological response of the peritoneal environment to misplaced endometrium may act in the initial phases of endometriotic implants (Calhaz-Jorge et al., 2000Go). Similarly, secretory leukocyte protease inhibitor (SLPI), a potent inhibitor of human leukocyte elastase found in the PF of endometriosis patients, may contribute to the pathogenesis of endometriosis (Shimoya et al., 2000Go).

A third of the patients with endometriosis are infertile and evidence for the last few years suggest that embryotoxic factors could be responsible for this reproductive failure. Several studies suggest the presence of soluble factors in sera from women with endometriosis (INF-{gamma}) that have a deleterious effect on embryo development (Damewood et al., 1990Go; Cameo et al., 1999Go). In this study, we have analysed the embryotoxicity of PF present in the pouch of Douglas of women with or without endometriosis and with or without infertility. This is also a prospective study of the association between cytokines, lymphocyte populations and embryotoxicity and future pregnancy outcome in infertile endometriosis patients.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
We have studied PF collected from the pouch of Douglas of 66 patients of reproductive age, who underwent laparoscopy (in some cases, laparotomy) due to suspicion of endometriosis. Many of these were infertile women. Endometriosis was confirmed in 54, and was not observed in 12. Another 12 fertile women who underwent laparoscopy for tubal sterilization were included as a control group. Therefore, 54 women with endometriosis (21 with infertility and 33 fertile) and 24 patients without endometriosis (12 with infertility and 12 control women) were studied. Among the 33 non-infertile endometriosis patients, there were 14 fertile women and 19 women with unproven fertility (single or not wishing pregnancy). According to the revised American Fertility Society classification of endometriosis and its posterior modification in 1996 (reported in Fertility and Sterility, 1997), 12 cases had minimal/mild, 16 moderate, and 26 severe endometriosis. Among the infertile women, eight out of 21 patients with endometriosis became pregnant spontaneously without specific treatment in the following year (38.1%), but only one out of 12 infertile women without endometriosis did (8.3%). Most of these latter cases presented male factor for infertility (oligoasthenospermia) and laparoscopy was performed to eliminate minimal/mild endometriosis and as part of the research to fulfil an infertility study.

Infertile patients with endometriosis had infertility of 3.7 ± 2.4 years duration (1–10 years) and infertile patients without endometriosis 5.1 ± 2.7 years duration (1.5–10 years). The mean age of women with endometriosis was 30.6 ± 4.4 years old, and that of the infertile women without endometriosis was 32.5 ± 2.9 years old.

Peritoneal fluid collection
All patients underwent laparoscopy or laparotomy in the second half of the cycle. The blood-free PF from the pouch of Douglas was collected into heparinized tubes and was immediately transported to the experimental reproduction laboratory. This PF was centrifuged for 10 min at 300 g, 4°C, to remove cells. The supernatant was aliquotted and immediately frozen and stored at –80°C. The pellet was rinsed three times and used for cytometric analysis of lymphocyte populations. Samples of supernatants were thawed for embryotoxicity analysis and cytokine concentrations.

Mouse embryo assay
CB6F1 female mice, 8–10 weeks of age, were injected with 10 IU of human menopausal gonadotrophin (HMG; Lepori®; Farma-Lepori SA, Barcelona, Spain) on day 1 of stimulation cycle, followed by 15 IU of human chorionic gonadotrophin (HCG; Lepori®), 48 h after HMG. After administration of HCG, each female was placed in a cage with a male and checked the next morning for a copulation plug. Twenty-four hours later, the females were killed by cervical dislocation, the oviduct–uterus segments were excised and the oviducts were flushed with M2 medium using a blunt 30-gauge needle. The embryos thereby retrieved were rinsed and placed in a Falcon organ culture dish containing M16 medium supplemented with a 10, 20 or 50% solution of peritoneal fluid. Control embryos in M16 medium alone were cultured simultaneously. The total volume in each well was 1 ml, and 5–10 embryos were placed in each well. The culture conditions were 37°C and 5% of CO2. Embryos were checked at 72 h post-incubation to evaluate the ability of PF to promote the growth of murine embryos. The embryotoxicity was expressed as the percentage of embryos that did not reach the blastocyst stage at each PF concentration.

Local institutional approval was obtained and the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals were followed in all experiments.

Cytokine assays
The interleukins (IL) IL-2, IL-6, IL-8, and interferon (INF) INF-{gamma} levels were determined in the PF using commercially available ELISA kits from Quantikine R&D Systems (Minneapolis, USA). The samples were diluted and assayed according to kit instructions. The assays were linear for 4–1024 pg/ml (IL-2, INF-{gamma}), 1–512 pg/ml (IL-8) and 5–1800 pg/ml (IL-6). Sensitivity for detection of the cytokines assessed in this study ranged from <1 pg/ml (INF-{gamma}) to 10 pg/ml (IL-8). When cytokine concentrations were higher than the top range, samples were half-diluted and results were increased twice. We considered a sample as elevated when its value was above the mean + 2 SD of controls.

Cytometric analysis
The lymphocyte populations present in peritoneal fluid were quantified by cytometric analysis using the following monoclonal antibodies: CD3-FITC, CD4-FITC, CD29-FITC, CD45-PE, CD8-PE, CD16-PE, CD56-PE, CD11b-FITC (Pharmigen BD, Heidelberg, Germany). Then, 1x106 mononuclear cells of peritoneal fluid were incubated for 10 min with 10 ml of each mononuclear antibody. Analysis was carried out using the FACS Vantage flow cytometer (Becton & Dickinson, Aalst, Belgium) equipped with an INNOVA 621 II Enterprise Ion Laser, and filter settings for FITC (530 nm) and PE (585 nm) were used. Live lymphocytes were gated for cell size and granularity. Ten thousand events were acquired and analysed. Data samples were analysed using Cell Quest software (Becton & Dickinson).

Statistical analysis
All statistical calculations were performed using the SPSS Windows Release 8.0 software package. Following application of the Kolmogorov–Smirnov test, data were analysed by the paired Student's t-test for normal distributions, or the Mann–Whitney U-test for non-normal distributions. Differences were considered significant if P < 0.05. Correlation coefficients between lymphocyte populations and embryotoxicity were also analysed.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The embryotoxicity of the PF in concentrations at 10 and 20% was significantly higher in those women with endometriosis than in the control group (Figure 1AGo). This higher embryotoxicity of the PF was seen in all endometriosis patients, fertile, infertile and with unproven fertility. However, to our surprise, embryotoxicity was also significantly higher in fertile women than in infertile ones, with the concentration of PF at 20% (91.4 ± 17 versus 68.1 ± 31, P < 0.01). Though embryotoxicity also seemed slightly increased in relation to the severity of the endometriosis, no significant changes were observed in this sense (data not shown). Similar observations were recorded with cytokine concentrations, especially for IL-6, though the results were not very homogeneous. A significant increase was observed only for the IL-8 in endometriosis patients with unproven fertility (Figure 1BGo). Although the cytokine increase was higher in patients with moderate and severe endometriosis, the differences were not significant either. In the severe endometriosis and the fertile patients, INF-{gamma} values were similar to those of the control group. IL-2 was not produced at detectable concentrations in the PF analysed.



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Figure 1. Percentages of embryotoxicity according to the proportion of peritoneal fluid (A), values of cytokines (IL-6, IL-8 and INF-{gamma}) (B), and percentages of lymphocyte populations (CD4, CD8, CD29 and CD56) (C) in the peritoneal fluid of control women (n = 12) and endometriosis patients (n = 54) separating the latter in fertile (n = 14), with unproven fertility (n = 19) and infertile women (n = 21). Mean values and SEs are shown.*P < 0.05; **P < 0.01; ***P < 0.001 comparing endometriosis and control women; ++P < 0.01 comparing fertile and infertile endometriosis patients.

 
The lymphocyte populations in the PF were not significantly modified in the patients with endometriosis except CD56 (NK) cells, which showed a significant increase in the endometriosis fertile and unproven fertility patients (Figure 1CGo), without relation to the severity. CD29+ cells also seemed to increase in the PF of endometriosis patients with the severity of the disease, although the differences were not significant. No changes were observed in CD3, CD4, CD8, CD16 and CD11b populations.

Therefore, the relationship between infertility and embryotoxicity is not clear, because the embryotoxicity of the PF was higher in the endometriosis fertile patients; and concerning the causes of infertility, when there were other causes apart from the endometriosis, there was also higher embryotoxicity, although the differences were not significant either (data not shown). Neither were there significant differences in infertile women without endometriosis.

Figure 2Go shows the relationship between normal or increased cytokine concentrations and embryotoxicity of the PF at 20% concentration. In the presence of normal values of cytokines, embryotoxicity was higher in women with endometriosis than in those without it, though the differences were not significant. Embryotoxicity increased in the presence of high concentrations of cytokines in endometriosis patients, especially for IL-6 (P < 0.05).



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Figure 2. Comparison between cytokine concentrations and the percentages of embryotoxicity of peritoneal fluid at proportion of 20% in women with or without endometriosis. High values were considered more than mean + 2SD, the rest were considered normal values. *P < 0.05 comparing normal and high values.

 
Significant negative correlations were observed between the number of NK+ cells (CD16) and the embryotoxicity of PF at 50% in all endometriosis patients (r = –0.318, P < 0.05), and between CD4+ and CD4CD29+ cells and the embryotoxicity of PF at 20% in infertile patients with endometriosis (r = –0.583, P < 0.05 and r = –0.553, P < 0.05). The correlation was also significant and negative between CD4+ cells and embryotoxicity of PF at 10% in endometriosis-infertile (r = –0.566, P < 0.05) and in all endometriosis patients (r = –0.433, P < 0.01).

Finally, we analysed the relationship between embryotoxicity, cytokines and lymphocyte populations and the subsequent achievement of pregnancy by infertile women. There were no significant differences, but higher levels of embryotoxicity and cytokines were observed in those patients who did not achieve pregnancy. Likewise, the percentage of CD56+ and CD29+ cells was higher, and that of CD4+ and CD8+ cells lower in this same group of patients, though the differences were not significant either. There were significant differences only in the embryotoxicity at 50% in the PF, very high in those endometriosis infertile patients without subsequent achivement of pregnancy (Figure 3Go).



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Figure 3. Percentages of embryotoxicity of peritoneal fluid in endometriosis infertile patients according to subsequent achievement of pregnancy (n = 8) or not (n = 13). Mean values and SE are shown. *P < 0.05 comparing subsequent and no pregnancy.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Some authors (Ramey and Archer, 1993Go; Ho et al., 1996Go; Koninckx et al., 1998Go; Calhaz-Jorge et al., 2000Go; Shimoya et al., 2000Go) have suggested that alterations in PF are involved in the development of endometriotic implants. Patients with endometriosis have high levels of proinflammatory cytokines (IL-6; TNF{alpha}), and different growth factors and adhesion molecules seem to have an important role for paracrine activity in the peritoneal cavity in the establishment and maintenance of endometriosis (Kupker et al., 1998Go). Furthermore, factors present in PF have been related to the mechanisms that cause associated infertility (Gleicher et al., 1987Go; Sueldo et al., 1987Go), such as an increase in sperm phagocytosis and the presence of cytotoxic factors derived from activated macrophages (Halme and Surrey, 1990Go). In addition, abnormal levels in cellular populations and cytokines (interleukins, tumour necrosis factors, interferons) could exert a negative influence on the fertility of women with endometriosis. However, as the blastocyst stage is reached within the Fallopian tube, the negative influence on fertility is more likely to be exerted by factors present in the serum (Damewood et al., 1990Go; Simon et al., 1992Go; Cameo et al., 1999Go) than by the embryotoxic activity of the PF. Moreover, some authors (D'Hooghe et al., 1999Go) have suggested that the laparoscopic diagnosis could cause peritoneal inflammation and be responsible for changes in the cytokine and lymphocyte populations of the PF, although it is also true that PF is usually withdrawn for study at the beginning of laparoscopy. Therefore there is not sufficient time for these changes in composition to happen.

PF embryotoxicity has been studied in women with and without endometriosis, with controversial results (Morcos et al., 1985Go; Sherif et al., 1987Go; Acién et al., 1999Go; Illera et al., 2000Go). Illera et al. have observed that the PF of women with endometriosis has a detrimental effect on the embryonic implantation in the mouse model (Illera et al., 2000Go), perhaps because it adversely affects uterine receptivity. In our study, we also observed significant differences in PF embryotoxicity between patients with or without endometriosis (control cases), but not in relation to infertility. However, embryotoxicity was significantly decreased in those infertile patients with endometriosis who later got pregnant. Other authors (Sherif et al., 1987Go), however, have not found any influence of PF from women with endometriosis on the development of two-cell embryos up to the blastocyst stage. Morcos et al. observed that such PF is more toxic for embryonic development than similar concentrations of fluid from patients without endometriosis (Morcos et al., 1985Go). Yet, they also observed that high concentrations of PF are toxic in both groups of patients, a fact that suggests that the toxic factor/s are not unique to patients with endometriosis. Our results are in good agreement with those of the above authors, since a better correlation of the values of embryotoxicity with the presence of endometriosis, cytokines and lymphocyte populations was observed at the 20% concentration of PF.

Some studies (Harada et al., 1997Go) have shown that the PF from women with endometriosis contains a high number of activated macrophages, which secrete great amounts of local products such as growth factors and cytokines, and that endometriotic implants can also produce them (Schroder et al., 1996Go; Gazvani et al., 1998Go). Likewise, several authors (Badawy et al., 1989Go; Dmowski et al., 1994Go; Ho et al., 1995Go) have pointed out that activated T cells are decreased both in the blood and in the PF from endometriosis patients, a fact that could play an important role in the functional defect of NK cells. This decrease in NK cytotoxicity seems to be correlated with the severity of the disease. However, among the data from our patients, the percentage of NK cells was increased in the PF of endometriosis patients, though not in proportion to its severity. Yet, there are no studies correlating PF embryotoxicity to cytokine concentrations, NK cells and T cells present in it. In our material, although all the cytokines studied were increased in patients with endometriosis, especially IL-6 and IL-8, we only observed significant correlation between IL-6 and PF embryotoxicity at 20%. Although there was no correlation between cytokine concentrations and associated infertility, patients with endometriosis who did not get pregnant seem to have higher cytokine concentrations than those who became pregnant later. However, the differences were not significant and therefore many questions about the role of cytokines in endometriosis and infertility still remain unsolved.

Finally, the correlation between embryotoxicity and lymphocyte populations was also weak, though it was significantly negative for CD4+ and CD29+ cells in endometriosis-infertile patients. A significant increase in CD56 (NK) cells was also observed in patients with endometriosis, but not so large as in endometriosis infertile patients.

In summary: (i) the most significant results in the analysis of embryotoxicity of PF were obtained with a 20% concentration; (ii) embryotoxicity was increased in women with endometriosis, but was not correlated with the severity of the disease; (iii) embryotoxicity was decreased in those women with endometriosis and infertility who later got pregnant; (iv) cytokine concentrations, especially IL-6, showed a good correlation with the embryotoxic activity of the PF; and (v) among the lymphocyte populations, the increase of CD56 (NK cells) was only significant in endometriosis patients, but did not appear to be related to the severity of the condition or to associated sterility.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We wish to thank the physicians at the Service of Obstetrics and Gynaecology of San Juan University Hospital and nurses at the Gynaecology operating theatre who collaborated with the protocol and the extractions of blood and peritoneal fluid. We also wish to thank Dr J.J.Prieto from the Histology Department, School of Medicine, `Miguel Hernandez' University, Campus de San Juan, Alicante, Spain, for reviewing the English. This study was supported by the `Fondo de Investigaciones Sanitarias, FIS 94/0768'. Ministry of Health, Madrid, Spain.


    Notes
 
4 To whom correspondence should be addressed at: Departamento/División de Ginecología, Facultad de Medicina, Universidad Miguel Hernández, Campus de San Juan, Apartado de Correos 18, 03550 San Juan de ALICANTE, Spain. E-mail: acien{at}umh.es Back

Submitted on February 20, 2001; resubmitted on August 20, 2001


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 Discussion
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 References
 
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accepted on October 29, 2001.





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