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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: embryotoxicity/endometriosis/immunology/peritoneal fluid
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (TNF
) (Ho et al., 1996
; Overton et al., 1996
; Calhaz-Jorge et al., 2000
). 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., 1996
). 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
(Akoum et al., 1996
), 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- production described in PF of patients with endometriosis (Ho et al., 1996
; Ho et al., 1997
). The establishment and maintenance of immunological tolerance to endometriosis may depend on the balance between TH1 and TH2 cytokine profiles (Braun et al., 1998
). 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., 2000
). 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., 2000
).
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-) that have a deleterious effect on embryo development (Damewood et al., 1990
; Cameo et al., 1999
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Infertile patients with endometriosis had infertility of 3.7 ± 2.4 years duration (110 years) and infertile patients without endometriosis 5.1 ± 2.7 years duration (1.510 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, 810 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 oviductuterus 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 510 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- 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 41024 pg/ml (IL-2, INF-
), 1512 pg/ml (IL-8) and 51800 pg/ml (IL-6). Sensitivity for detection of the cytokines assessed in this study ranged from <1 pg/ml (INF-
) 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 KolmogorovSmirnov test, data were analysed by the paired Student's t-test for normal distributions, or the MannWhitney 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 2 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).
|
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 3).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PF embryotoxicity has been studied in women with and without endometriosis, with controversial results (Morcos et al., 1985; Sherif et al., 1987
; Acién et al., 1999
; Illera et al., 2000
). 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., 2000
), 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., 1987
), 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., 1985
). 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., 1997) 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., 1996
; Gazvani et al., 1998
). Likewise, several authors (Badawy et al., 1989
; Dmowski et al., 1994
; Ho et al., 1995
) 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
Submitted on February 20, 2001; resubmitted on August 20, 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akoum, A., Lemay, A., Paradis, I. et al. (1996) Secretion of interleukin-6 by human endometriotic cells and regulation by proinflammatory cytokines and sex steroids. Hum. Reprod.,11,22692275.[Abstract]
American Society for Reproductive Medicine (1997) Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil. Steril.,67,817821.[ISI][Medline]
Badawy, S., Cuenca, V., Kaufman, L. et al. (1989) The regulation of inmunoglobulin production by B cells in patients with endometriosis. Fertil. Steril.,51,770773.[ISI][Medline]
Braun, C.M., Huang, S.K., Kagey-Sobotka, A. et al. (1998) Co-regulation of antigen-specific T lymphocyte responses by type I and type II cyclic AMP-dependent protein kinases (cAK). Biochem. Pharmacol., 56, 871879.[ISI][Medline]
Calhaz-Jorge, C., Costa, A.P., Barata, M. et al. (2000) Tumour necrosis factor alpha concentrations in the peritoneal fluid of infertile women with minimal or mild endometriosis are lower in patients with red lesions only than in patients without red lesions. Hum. Reprod.,15,12561260
Cameo, M., Fontana, V., Cameo, P. et al. (1999) Similar embryotoxic effects of sera from infertile patients and exogenous interferon-gamma on long-term in-vitro development of mouse embryos. Hum. Reprod.,14,959963.
D'Hooghe, T.M., Bambra, C.S., Raeymaekers, B.M. et al. (1999) Pelvic inflammation induced by diagnostic laparoscopy in baboons. Fertil. Steril.,72,11341141.[ISI][Medline]
Damewood, M.D., Hesla, J.S., Schlaff, W.D. et al. (1990) Effect of serum from patients with minimal to mild endometriosis on mouse embryo development in vitro. Fertil. Steril.,54,917920.
Dmowski, W.P., Gebel, H.M. and Braun, D.P. (1994) The role of cell-mediated immunity in pathogenesis of endometriosis. Acta Obstet. Gynecol. Scand., 73, 714.[ISI]
Gazvani, M.R., Chistmas, S., Quenby, S. et al. (1998) Peritoneal fluid concentrations of interleukin-8 in women with endometriosis: relationship to stage of disease. Hum. Reprod.,13,19571961.[Abstract]
Gleicher, N., Roeiy, A., Confino, E. et al. (1987) Is endometriosis an autoimmune disease? Obstet. Gynecol.,70,115122.[Abstract]
Halme, J. and Surrey, E. (1990) Endometriosis and infertility: the mechanisms involved. Prog. Clin. Biol. Res., 323, 157178.[Medline]
Haney, A.F., Muscato, J.J. and Weinberg, J.B. (1981) Peritoneal fluid cell populations in infertility patients. Fertil. Steril.,35,696698.[ISI][Medline]
Harada, T., Yoshioka, H., Iwabe, T. et al. (1997) Increased interleukin-6 level in peritoneal fluid of infertile patients with active endometriosis. Am. J. Obstet. Gynecol.,176,593597.[ISI][Medline]
Ho, H.N., Chao, K.H., Chen, H.F. et al. (1995) Peritoneal natural killer cytotoxicity and CD25+ CD3+ lymphocyte subpopulation are decreased in women with stage IIIIV endometriosis. Hum. Reprod.,10,26712675.[Abstract]
Ho, H.N., Wu, M.Y., Chao, K.H. et al. (1996) Decrease in interferon gamma production and impairment of T-lymphocyte proliferation in peritoneal fluid of women with endometriosis. Am. J. Obstet. Gynecol.,175,12361241.[ISI][Medline]
Ho, H.N., Wu, M.Y., Chao, K.H. et al. (1997) Peritoneal interleukin-10 increases with decrease in activated CD4+ T lymphocytes in women with endometriosis. Hum. Reprod., 12,25282533.[Abstract]
Illera, M.J., Juan, J., Stewart, C.L. et al. (2000) Effect of peritoneal fluid from women with endometriosis on implantation in the mouse model. Fertil. Steril., 74,4148.[ISI][Medline]
Koninckx, P.R., Kennedy, S.H., Barlow, D.H. (1998) Endometriotic disease: the role of peritoneal fluid. Hum. Reprod. Update, 4, 741751.
Kupker, W., Schultze-Mosgau, A. and Diedrich, K. (1998) Paracrine changes in the peritoneal environment of women with endometriosis. Hum. Reprod. Update, 4, 719723.
Morcos, R.N., Gibbons, W.E. and Findley, W.E. (1985) Effect of peritoneal fluid on in vitro cleaveage of 2-cell mouse embryos: possible role in infertility associated with endometriosis. Fertil. Steril., 44,678683.[ISI][Medline]
Overton, C., Fernández-Shaw, S., Hicks, B. et al. (1996) Peritoneal fluid cytokines and the relationship with endometriosis and pain. Hum. Reprod., 11,380386.[Abstract]
Punnonen, J., Teisala, K., Ranta, H. et al. (1996) Increased levels of interleukin-6 and interleukin-10 in the peritoneal fluid of patients with endometriosis. Am. J. Obstet. Gynecol., 174,15221526.[ISI][Medline]
Ramey, J. and Archer, D. (1993) Peritoneal fluid: its relevance to the development of endometriosis. Fertil. Steril., 60,114.[ISI][Medline]
Schroder, W., Gaetje, R. and Baumann, R. (1996) Interleukin-6 and soluble interleukin-6 receptor in peritoneal fluid and serum of patients with endometriosis. Clin. Exp. Obstet. Gynecol., 23,1014.[Medline]
Sherif, G.A., Chad, I.F., Amin, U.H. et al. (1987) Local peritoneal factors: their role in infertility associated with endometriosis. Am. J. Obstet. Gynecol., 157,12071214.[ISI][Medline]
Shimoya, K., Moriyama, A., Ogata, I. et al. (2000) Increased concentrations of secretory leukocyte protease inhibitor in peritoneal fluid of women with endometriosis. Mol. Hum. Reprod., 6,829834.
Simon, C., Gomez, E., Mir, A. et al. (1992) Glucocorticoid treatment decreases sera embryotoxicity in endometriosis patients. Fertil. Steril., 58,284289.[ISI][Medline]
Sueldo, C.E., Lambert, H., Steinleitner, A. et al (1987) The effect of peritoneal fluid from patients with endometriosis on murine spermoocyte interaction. Fertil. Steril., 48,697699.[ISI][Medline]
Syrop, C.H. and Halme, J. (1987) Cyclic change of peritoneal fluid parameters in normal and infertile patients. Obstet. Gynecol., 69,416418.[Abstract]
accepted on October 29, 2001.