Jouko Halme

Increased activation of pelvic macrophages in infertile women with mild endometriosis

Pelvic fluid was collected from 66 women undergoing laparoscopic sterilization or diagnostic laparoscopy for evaluation of infertility. Cells consisting mainly of macrophages were separated, counted, and subjected to histochemical staining for acid phosphatase and myeloperoxidase as markers of cell irritation. Pelvic fluid was analyzed for acid phosphatase, neutral protease, and extractable prostaglandin E2 and F2 alpha. A higher proportion (46% versus 15%) of the macrophages in the group with mild endometriosis exhibited positive staining for acid phosphatase as compared with the fertile group. Pelvic fluid from patients with mild endometriosis had higher acid phosphatase and neutral protease activity than that from fertile patients (p less than 0.05, p less than 0.01). The content of either prostaglandin was not significantly higher in the endometriosis group as compared with the fertile group. The results suggest that mild endometriosis is associated with activation of macrophages and release of active substances into peritoneal fluid that may be responsible for the associated infertility.

Altered maturation and function of peritoneal macrophages: possible role in pathogenesis of endometriosis

Human peritoneal macrophages from healthy women and patients with endometriosis were analyzed with flow cytometry for size distribution, cell membrane antigen expression, and membrane function. Endometriosis was associated with a significantly increased number of peritoneal macrophages and a higher proportion of large macrophages with increased expression of three antigen markers. Peritoneal macrophages from normal patients exhibited diminished cell membrane capping function as compared with that of endometriosis-related macrophages or blood monocytes. On the basis of these findings, a hypothesis is formulated suggesting that endometriosis is associated with an increased influx of macrophages that are allowed to undergo further maturation-activation. The resultant population of large macrophages may contribute to the maintenance of the disease or associated infertility.

Accentuated cyclic activation of peritoneal macrophages in patients with endometriosis

Peritoneal fluid was collected from 107 women undergoing laparoscopic sterilization or diagnostic laparoscopy for evaluation of infertility. Cells consisting mainly of macrophages were separated and subjected to sophisticated flow fluorocytometric analysis. In this way more detailed information was obtained about activational characteristics of the pelvic macrophage population during the menstrual cycle. In normal women the macrophages, as compared to peripheral monocytes, showed evidence of elevated baseline activation, and a gradual increase in several markers occurred during the menstrual cycle. Cells increased in size, lost their ability to stain for myeloperoxidase, and increased in activity of both endoenzymes and ectoenzymes. These results suggest that female peritoneal macrophages are continuously responding to stimuli. The macrophage irritation was much more pronounced in women with mild endometriosis. This accentuated cyclic activation may represent an inflammatory response to bleeding from ectopic implants or retrograde menstruation or may be a consequence of some defect in the cell-mediated immune response in endometriosis.

Pelvic macrophages in normal and infertile women: The role of patent tubes

The volume of peritoneal fluid and its macrophage content were examined in 80 women undergoing laparoscopy. The amount of pelvic fluid was not dependent on the patency of the fallopian tubes, and no statistical difference in the fluid volume was observed between the infertile groups, including those with endometriosis, as compared to the fertile group. The number of pelvic macrophages in fertile women was extremely high during the menstrual period, and during the rest of the cycle, it remained at a basal level. The number of pelvic macrophages in women with occluded tubes was low. Four of 21 infertile patients with mild endometriosis had very high numbers of pelvic macrophages, even in the luteal phase. These results suggest that the passage of endometrial irritants through the tubes elicits a macrophage response in the peritoneal cavities of both normal women and patients with endometriosis. Although most patients with endometriosis had normal numbers of peritoneal macrophages, it is possible that these macrophages differ qualitatively from normal, and this could explain the associated infertility.

Effect of peritoneal fluid from endometriosis patients on endometrial stromal cell proliferation in vitro.

Late proliferative phase endometrial stromal cells grown in short-term culture were used as a model for the stromal component of endometriotic implants. Cells were grown in medium alone and in medium supplemented by 5, 10, and 20% concentrations of the cell-free fractions of peritoneal fluid obtained from patients with and without endometriosis. Nine fluid-sample pairs were matched based on the presence or absence of endometriosis at laparoscopy and the similarity of peritoneal fluid estradiol concentrations. Stromal cell proliferation as reflected by 3H-thymidine incorporation during sequential cell harvests over 72 hours was greater for cells exposed to endometriosis peritoneal fluid than for those exposed to non-endometriosis peritoneal fluid. This reached statistical significance with exposure to peritoneal fluid concentrations of 10 and 20% (P less than .05). A linear dose-response relationship between 3H-thymidine incorporation and peritoneal fluid concentration could be derived only for stromal cells exposed to fluid samples obtained from endometriosis patients (r = 0.51; P less than .001). In addition, proliferation over 72 hours was significantly greater for cells grown in 20% endometriosis peritoneal fluid than for those grown in nutrient medium alone (P less than .001). These data imply that factor(s) secreted into the peritoneal fluid of endometriosis patients may play a role in the proliferation or maintenance of disease implants.

Semen parameters and fertilization of human oocytes in vitro: a multivariable analysis

Semen parameters in 195 couples undergoing in vitro fertilization and embryo transfer were studied using multivariable analysis. Semen parameters that correlated most closely with reduced ability to fertilize apparently mature oocytes were a slow rate of foreward progression of sperm and the presence of excess numbers of white cells in semen. In men with semen parameters within the normal range, the hamster egg penetration assay (HEPA) test did not add additional predictive power. In men with suspected semen abnormalities, however, a low attachment rating added some, but minimal, predictive value. None of the predictive methods reported thus far in this or other studies offers sufficient accuracy to reliably identify the men who will prove infertile for in vitro fertilization treatment.

The effect of growth factors on the proliferation of human endometrial stromal cells in culture

OBJECTIVE Development of ectopic implants of endometriosis is associated with both an inflammatory response by macrophages and endometrial stromal cell proliferation. Macrophages are capable of releasing a variety of inflammatory mediators, including growth factors. To assess the impact of such factors on endometrial tissue, we have studied the effects of recombinant growth factors, fibroblast growth factor, epidermal growth factor, transforming growth factor-alpha, and inflammation mediators transforming growth factor-beta, and tumor necrosis factor-alpha on human endometrial stromal cell proliferation. STUDY DESIGN Increasing concentrations of these compounds were added to cultures of primary, secondary, and long-term stromal cells and the cells were harvested at 24, 48, and 72 hours. RESULTS Epidermal growth factor, transforming growth factor-alpha, transforming growth factor-beta, and fibroblast growth factor induced a statistically significant, dose-dependent increase in stromal cell thymidine uptake of 1.5- to fivefold. The cytokine tumor necrosis factor had no effect alone, but the combination of fibroblast growth factor and tumor necrosis factor had a synergistic effect, increasing cell proliferation 25% to 84% over fibroblast growth factor alone. CONCLUSION The stromal cell response to a wide range of cell growth effectors and the potential of mediators like tumor necrosis factor-alpha to synergize suggest that such macrophage-secretory products may contribute to proliferation of endometrial implants in vivo.

Peritoneal fluid environment and infertility.

The PF environment is one that hosts the processes of ovulation, gamete transportation, fertilization, and early embryonic development. The cellular and acellular constituents of this dynamic fluid are in a constant interactive state, being influenced by the physiologic events of the menstrual cycle and pelvic disease processes; these constituents probably influence disease manifestation and reproduction. The importance of understanding this zone of early reproductive life has been now recognized. We hope that future investigations will define the exact role(s) of known components and some yet-to-be defined substances of PF in disease processes that affect reproductive function. With better understanding of normal and abnormal events in this pelvic microenvironment, we can develop rationales for novel treatment modalities.

Role of Peritoneal Inflammation in Endometriosis-associated Infertility

This paper has discussed the evidence for the presence of infertility in patients with endometriosis and more critically reviewed some of the studies that have addressed the impact of various potential local peritoneal mechanisms that may lead to subfertility. Substantial evidence supports the notion that patients with endometriosis have reduced fecundability. Although several mechanisms, including, e.g., anatomic factors and ovulatory dysfunction, are possible, recent studies have pointed towards local inflammatory cells and their secretory products as being important mediators of subfertility. Ample evidence exists for the presence of an altered peritoneal inflammatory environment in patients with endometriosis. In addition, in vitro studies have identified peritoneal macrophages and their secretory products, specifically TNF-alpha as the most likely contributors to the reduced fecundability through effects on sperm function.

Effect of pelvic fluid from endometriosis patients on human sperm penetration of zona-free hamster ova.

Peritoneal fluid (PF) samples from fertile women, infertile women without signs of endometriosis, and infertile women with endometriosis were examined for effects on motility of normal spermatozoa and for effects on their fertilizing capacity as measured by the interspecies penetration test (ISPT). All unheated peritoneal fluid samples caused rapid destructive changes in the female hamster gametes, presumably due to the presence of unidentified serum components, and caused clumping of spermatozoa but no change in sperm motility. Heat-treated fluid samples were therefore used in subsequent experiments to assess their effect on the ISPT. The mean penetration rate for fertile women was 58%; for infertile women without endometriosis, 55%; and 54% for infertile women with endometriosis. The differences between the penetration rates in these patient groups were not significant. No adverse thus found on the fertilizing capacity of fertile spermatozoa.