Anti-endometrial antibodies in women measured by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure anti-endometrial antibody concentrations in the serum of women with endometriosis. Pooled cytosolic protein extracts from the endometrial gland cells of 10 women were used as an antigen source. Serum samples were obtained from women with endometriosis before (n = 51) and after 6 months treatment with danazol or nafarelin (n = 30). Control sera came from women with a normal pelvis at laparoscopy, performed for sterilization (n = 23) or the investigation of pain and/or infertility (n = 22), 13 women with Rokitansky syndrome, and 10 umbilical cord bloods and adult males. There were no significant differences in serum anti-endometrial antibody concentrations before and after treatment, or between women with endometriosis and without endometriosis. Concentrations were lower in male and cord blood serum than in female's serum (P < 0.0001). We conclude that the ELISA is not a useful diagnostic tool for endometriosis unless more specific antigens can be isolated.

Prostaglandin production by human peripheral blood monocytes changes with in vitro differentiation.

To investigate the influence of in vitro culture on prostaglandin (PG) production, human monocyte enriched peripheral blood mononuclear cells were isolated and incubated on gelatin-coated plates. On days zero, five and eleven of culture, the cells were examined microscopically and the production of PGF1 alpha, PGE2, PGD2, F metabolite (PGFM) and E metabolite (PGEM) were measured by radioimmunoassay. Differences in PG output were analyzed using the Wilcoxon and Friedman tests. Freshly isolated human peripheral blood monocytes produced mainly PGE2. In vitro, however, PGE2 production decreased from 196 (48-288) fmol/10(6) cells per 3h on day zero of culture to 28 (6-51) on day eleven (p = 0.04); median (range), n = 7. Prostaglandin D2 and PGEM output decreased similarly, but these differences failed to reach significance. Prostaglandin F2 alpha and PGFM output, on the other hand, increased from 32 and 19 fmol/10(6) cells per 3h, respectively, on day zero of culture to 127 (p < 0.05) and 58 (p = 0.01) on day eleven. Changes in PG output were associated with in vitro differentiation as evidenced by changes in cellular morphology. These result suggest that differentiation of human peripheral blood monocytes in vitro is accompanied by a shift in PG output from PGE2 and PGD2, towards PGF2 alpha.

Expression of granulocyte-colony stimulating factor and its receptor is regulated during the development of the human placenta.

The development of the placenta is dependent upon the regulated proliferation, invasion and differentiation of trophoblast. Expression of cytokines at the feto-maternal interface suggests that these molecules may participate in placentation. The expression of granulocyte-colony stimulating factor (G-CSF) and G-CSF receptor (G-CSFR) during the development of the human placenta was studied by immunohistochemistry using an anti-G-CSF monoclonal antibody (mAb) and two novel anti-G-CSFR mAbs. G-CSF was present in the stroma of fetal chorionic villi and maternal decidual tissues throughout pregnancy. G-CSFR was detected at high levels in fetal first and third, but not second trimester placental tissues. Staining for G-CSFR was undetectable in maternal decidual tissue from all gestational stages. In first trimester tissues, staining for placental G-CSFR was strongest in differentiated syncytiotrophoblast and invasive, extravillous cytotrophoblast, and weak staining was evident in undifferentiated cytotrophoblast. Immunohistochemical data suggesting temporal regulation of G-CSFR were corroborated by Western blotting and amplification by reverse transcription and PCR of G-CSFR mRNA. These data suggested that expression of G-CSFR in the human placenta is regulated both temporally and spatially, and that placental G-CSF is involved in paracrine regulation, and indicate a role for G-CSF and G-CSFR in trophoblast growth or function during placentation.

Bone marrow-derived cell populations in uterine and ectopic endometrium.

Uterine endometrium contains numerous bone marrow-derived cells. The spectrum of cell types is different from that of any other tissue, and the differences in endometrium from women with endometriosis may reflect a different endometrial phenotype in these women. The cell types of bone marrow origin found in ectopic endometrium may indicate the degree of differentiation of the tissue. It was found that, in normal endometrium, the CD45+ cell population comprised T cells, macrophages, CD56+ large granular lymphocytes, some CD16+ cells and a few B cells. Changes in these cell populations during the menstrual cycle were similar in endometrium from both controls and patients with endometriosis, and resembled that reported previously by others. In ectopic endometrium, the frequency of CD45+ cells remained within the same range as that of uterine endometrium but without any obvious pattern of change during the menstrual cycle. CD56+ large granular lymphocytes, an immune cell type characteristic of uterine endometrium, were also found in ectopic endometrium. Our results indicate that ectopic endometrium, as well as comprising both glandular and stromal cells, contains bone marrow-derived cell populations similar to those of uterine endometrium. This suggests that the same processes of cell migration and/or differentiation occur in ectopic and uterine endometrium.

Plasminogen activators in ectopic and uterine endometrium.

OBJECTIVE: To investigate the expression of the plasminogen activator (PA)-plasmin system components in ectopic endometrium and in uterine endometrium from women with and without endometriosis. DESIGN: Plasminogen, PAs (urokinase and tissue plasminogen activator), and PA inhibitors (1 and 2) were detected by immunohistochemistry using a alkaline phosphatase staining method. RESULTS: No differences in staining were found between uterine endometrium of women with endometriosis and women without endometriosis with any of the antibodies used. However, we did find differences between uterine and ectopic endometrium. Although the expression of the components of the PA-plasmin system reflected the cyclic changes in the hormonal levels in uterine endometrium, ectopic endometrium maintained a very high level of plasminogen and urokinase in every sample. We were unable to detect the presence of PA inhibitors in either uterine or ectopic endometrium. CONCLUSIONS: There is no evidence that uterine endometrium from women with endometriosis is originally more able to implant than that of women without the disease because of an increase in their PA expression. The high levels of urokinase and plasminogen in ectopic endometrium may reflect a more invasive nature of the endometriotic implants in the peritoneal cavity.

Interleukin-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia.

OBJECTIVES: Generalised maternal endothelial cell dysfunction appears to be an underlying problem in pre-eclampsia presumed to be caused, directly or indirectly, by one or more circulating factors derived from the placenta. Recently it has been suggested that tumour necrosis factor (TNF) may play an important role in pre-eclampsia and contribute to endothelial activation. This study was designed to investigate this proposal. DESIGN: Plasma TNF-alpha, IL-6 and both forms of soluble TNF receptors (p55 and p75 TNF-R) have been measured by ELISA in 31 pre-eclamptic patients and 31 pregnant controls matched for age, parity and gestational age. RESULTS: Levels of IL-6, TNF-alpha and soluble TNF-R (p55 and p75) were significantly higher in pre-eclamptic patients, compared with age and gestation matched controls with a wide variation in levels between pre-eclamptic individuals. There was a correlation between levels of IL-6 and TNF or TNF-R and between TNF and TNF-R levels. However, when the pre-eclamptic patients were subdivided on the basis of the severity of their disease, the median values of plasma concentrations of IL-6, TNF-alpha and TNF-R were all higher in the group with lower platelet counts. CONCLUSIONS: These new findings are consistent with the concept that the maternal syndrome of pre-eclampsia is associated with endothelial dysfunction and provide evidence that at least part of this dysfunction could arise from excessive release of TNF-alpha into the circulation.

Comment: effect of cytokines on prolactin production by human decidual stromal cells in culture: studies using cells freed of bone marrow-derived contaminants.

Human decidua contains resident decidual cells alongside a population of bone marrow-derived cells, among which macrophages and large granular lymphocytes are most abundant. We hypothesized that soluble effectors produced by bone marrow-derived cells may modulate the function of the decidual cells. To investigate this, a cell purification protocol was devised that involved digestion of first-trimester decidua with collagenase and hyaluronidase to produce a mixed stromal cell suspension from which the bone marrow-derived cells were removed using immunomagnetic beads coated with anti-CD45. The resulting stromal cells were maintained in culture in the presence of progesterone and were found to produce PRL. The effect of a panel of cytokines on PRL production was examined. Tumor necrosis factors-alpha and -beta had a dose-dependent inhibitory effect, and tumor necrosis factor receptors were identified on the cells. Interleukin 1 alpha and 1 beta, platelet-derived growth factor, and transforming growth factor-beta 1 were also found to inhibit PRL production, and platelet-derived growth factor and transforming growth factor-beta 1 stimulated cell proliferation. These findings suggest an interaction between the immune and endocrine systems in regulating the maternal environment of early pregnancy.

Expression of tumour necrosis factor alpha and its receptors in carcinoma of the breast.

The expression of tumour necrosis factor alpha (TNF-alpha) and its two distinct receptors, TNF-R p55 and TNF-R p75, was assessed by immunocytochemistry in 28 primary breast cancer and three reduction mammoplasty specimens ('normal' breast tissue). Expression of TNF-alpha or TNF-R p75 was not detectable in normal breast tissue or in non-malignant breast tissue adjacent to the tumours. By contrast, TNF-R p55 was expressed by occasional stromal cells in normal tissue. TNF-alpha was expressed focally in 50% of the tumours studied, being largely localised to macrophage-like cells in the stroma. TNF-R p55 was expressed by a population of stromal cells in all the tumours examined, and a varying proportion of neoplastic cells in 75% of these tissues. TNF-R p75 was detected in about 70% of the tumours, immunoreactivity being confined mainly to cells in the stroma. In this preliminary study there was no association between the above cytokine parameters and such measures of tumour biology as lymph node status, tumour grade, proliferative activity or degree of angiogenesis. However, there was a correlation between the expression of TNF-R p55 by blood vessels and the number of leucocytes present.

The effect of placental syncytiotrophoblast microvillous membranes from normal and pre-eclamptic women on the growth of endothelial cells in vitro.

OBJECTIVES: To determine if placental syncytiotrophoblast microvillous (STBM) membranes contain factors which could cause the maternal endothelial cell disturbance thought to be central to the pathophysiology of the maternal syndrome of pre-eclampsia. DESIGN: STMB membranes isolated from pre-eclamptic or normal placentae were added to cultures of endothelial cells and their effect on the proliferation (measured by 3H-thymidine incorporation), viability (measured by 51Cr release) and growth as a monolayer of these cells was determined. Membranes prepared from red blood cells, and non-endothelial adherent and nonadherent cell lines were used as specificity controls. SUBJECTS: STBM membranes were isolated from the placentae of primigravid women, 10 having caesarean sections for breech presentations and 10 for pre-eclampsia. RESULTS: STBM membranes from the placentae of normal and pre-eclamptic women suppressed endothelial cell proliferation to a similar extent and disrupted the cell monolayer to form a honeycomb-like pattern. This change in morphology was seen before significant endothelial cell death occurred. Red blood cell membranes had no effect on either endothelial cell proliferation, viability or monolayer integrity. Endothelial cells from human umbilical arteries and bovine adrenal capillaries were similarly suppressed, but comparable concentrations of STBM membranes had no effect on non-endothelial cell lines. CONCLUSIONS: Syncytiotrophoblast microvillous membranes specifically interfered with endothelial cell growth in vitro. Our results demonstrate that there are trophoblast products which could cause the maternal syndrome of pre-eclampsia through endothelial cell damage.

Antigenic heterogeneity of human cytotrophoblast and evidence for the transient expression of MHC class I antigens distinct from HLA-G.

Expression of MHC class I antigens on trophoblast populations in first trimester human chorionic villous tissue was assessed by immunohistology. Antibodies used were W6/32 which recognizes a non-polymorphic framework determinant of HLA- A, -B, -C, MHM5 specific for HLA-B, C and 4E and B23.1 which are specific for HLA-B. Syncytiotrophoblast and villous cytotrophoblast were negative with all the anti (HLA class I) antibodies tested. Interstitial trophoblast cells within the maternal decidua were identified with a new antibody, NDOG5, which is specific for extravillous cytotrophoblast. Double labelling showed that they bind W6/32 but not 4E, MHM5 or B23.1; consistent with the expression of the monomorphic HLA-G. In contrast the cytotrophoblast cells of the cell islands and cytotrophoblast shell, which also express the NDOG5 antigen, were positive with W6/32, 4E, MHM5 and B23.1. Cell column cytotrophoblast cells were negative with all four MHC class I antibodies. These results suggest that differentiation of cytotrophoblast from noninvasive to invasive forms is associated with transient expression of class I antigens other than HLA-G on cytotrophoblast shell and cell island cytotrophoblast.

Anti-endometrial and anti-endothelial auto-antibodies in women with endometriosis.

The present study investigates the specificity of anti-endometrial antibodies present in serum of women with endometriosis. A two colour indirect immuno-histochemical method was used, so that the antigenic reactivity of endogenous immunoglobulins was blocked and specific anti-endometrial antibodies were readily distinguishable. Serum was collected from women with endometriosis, before and after 6 months of treatment, and from women without the disease. The reactivity of serum with uterine and ectopic endometrium from women with and without the disease was studied. The frequency of anti-endometrial antibodies in the serum of women with endometriosis was higher (P < 0.001) than in control sera. Most antibodies specifically reacted with glands in ectopic and uterine endometrium. Antibody reactivity was strongest with endometrium from control women, compared with uterine and ectopic endometrium from women with endometriosis (P < 0.01). A proportion of sera containing anti-endometrial antibodies also reacted with vascular endothelium. Binding was strongest to vessels in uterine and ectopic endometrium from women with endometriosis compared to endometrium from women without the disease (P < 0.01). The presence of anti-endometrial antibodies was associated with infertility.

Prostaglandin D2 production by term human decidua: cellular origins defined using flow cytometry.

OBJECTIVE: To measure prostaglandin (PG) D2 output by human decidua at term and to identify the cell population(s) responsible for its production. METHODS: Decidual cell suspensions were prepared enzymatically and the cells were labeled with either of two monoclonal antibodies: F10/89/4, which recognizes the leukocyte common antigen (CD45), or L243, which labels HLA-DR and is specific for macrophages in this tissue. Cells were sorted on a Coulter EPICS 541 flow cytometer. Prostaglandin levels were measured by radioimmunoassay. RESULTS: Human decidua is an important intrauterine source of PGD2 at term. Twenty-nine percent (median) of decidual cells were CD45-positive and 12% were HLA-DR-positive; sorted cell populations were 95% pure. Prostaglandin D2 output (fmol/10(6) cells per 3 hours) by bone marrow-derived (CD45-positive) cells was significantly higher than that by non-bone marrow-derived cells: median 63 (range 35-67) versus 20 (13-21), respectively; HLA-DR-positive cells (macrophages) had the highest PGD2 production rate (186, range 97-288 fmol/10(6) cells per 3 hours). Under basal conditions, PGD2 production by unsorted term decidual cells was not influenced by labor. CONCLUSION: Bone marrow-derived cells (macrophages) are the major source of decidual PGD2 at term. Further studies are required to investigate the possible role of PG production by human decidual macrophages in the mechanism of term and/or preterm labor.

Tumor necrosis factor-alpha selectively stimulates prostaglandin F2 alpha production by macrophages in human term decidua.

OBJECTIVE: Our objective was to investigate the effect of tumor necrosis factor-alpha on prostaglandin production by human term decidual cell subtypes in vitro. STUDY DESIGN: We measured the effect of tumor necrosis factor-alpha on prostaglandins F2 alpha, E2, D2, F metabolite, and E metabolite production by decidual cells (n = 4) with radioimmunoassay. We used flow cytometry after labeling with an antibody to histocompatibility antigen DR, L243, which is specific for macrophages in this tissue, to prepare pure populations of decidual macrophages (n = 3). Differences in prostaglandin output were analyzed by Wilcoxon and Kruskal-Wallis tests. RESULTS: Tumor necrosis factor-alpha stimulated prostaglandin F2 alpha output by unfractionated decidual cells, without altering the output of any other prostaglandins. Tumor necrosis factor-alpha (10 nmol/L) increased prostaglandin F2 alpha production from decidual macrophages (HLA-DR-positive cells) by about threefold: from a median of 727 (range 423 to 1226) to a median of 1974 (range of 1550 to 2201), fmol/10(6) cells per 18 hours, as compared with a 1.4-fold increase from nonmacrophages from a median of 247 fmol/10(6) cells per 18 hours (range 125 to 611) to a median of 340 fmol/10(6) cells per 18 hours (range 201 to 505). CONCLUSION: Stimulation of decidual prostaglandin F2 alpha production by tumor necrosis factor-alpha may be important in the etiology of spontaneous labor at term or preterm labor associated with infection.

Production of granulocyte colony-stimulating factor at the materno-foetal interface in human pregnancy.

A bioassay specific for human granulocyte colony-stimulating factor (G-CSF) was developed and used to measure G-CSF production in human pregnancy tissues. G-CSF was secreted by both foetal chorionic villous and maternal decidual tissues taken in the first trimester and at term. The level of G-CSF production by placental tissue was 6750 (1250-10,000) units of bioactivity per g of tissue in 48 hr in the first trimester and 104 (83-190) U/g at term. Bioactive G-CSF was also secreted by decidual tissue, more in the first trimester than at term. ELISA immunoassays measured 75 (10-820) ng/g/48 hr of G-CSF antigen from first trimester placenta, 15 (10-50) ng/g from first trimester decidua and less than 2 ng/g from term placenta. RNA isolated from decidual and chorionic villous tissue or from cells purified by flow cytometry, contained G-CSF mRNA in both tissues. In decidua, mRNA for G-CSF was confined to the macrophages, and cytotrophoblast from term amniochorion contained no detectable G-CSF mRNA. No G-CSF, measured as bioactivity or as mRNA, was detectable in choriocarcinoma cell lines.

Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres.

Isolation of pure preparations of the different cell populations of human endometrium is a prerequisite for studies of in-vitro function. Sieving of dispersed endometrial cells, followed by adsorption onto immunomagnetic microspheres coated with antibody to Thy-1 was used to separate glandular and stromal cells. The purity of these cell populations was checked with antibodies to cytokeratin and Thy-1. The stromal cells were 98% pure and 90% viable, gland cells were 82% pure with 76% viability. The purified cells were able to proliferate in vitro as shown by thymidine incorporation.

Isolation and characterisation of a subpopulation of human chorionic cytotrophoblast using a monoclonal anti-trophoblast antibody (NDOG2) in flow cytometry.

Human cytotrophoblast cells, isolated from term amniochorion by enzymic digestion and Percoll gradient centrifugation, were characterised by flow cytometry. A panel of 12 anti-trophoblast monoclonal antibodies was screened for labelling of these cells in flow cytometry and the results compared with immunoperoxidase labelling of cytospin preparations and tissue sections. All 12 antibodies were positive for trophoblast on tissue sections, 11/12 were positive on cytospins but only two (NDOG2 and GB25) gave consistent results in flow cytometry. Two-colour labelling with NDOG2 and W6/32, an antibody to HLA-A, -B, -C, demonstrated that 88% of the NDOG2-positive cells also express Class I major histocompatibility complex (MHC) antigens. The NDOG2-positive cytotrophoblast subpopulation was isolated by flow cytometry in sufficient purity (greater than 95%) and yield (3.1 x 10(6)) for use in functional studies in vitro.

Identification by flow cytometry of the prostaglandin-producing cell populations of term human decidua.

Human term decidua produces prostaglandins (PGs) which have been implicated in the initiation of human parturition. Using flow cytometry to isolate pure cell populations, we have investigated the cell types responsible for decidual PG production. Cell dispersions were prepared enzymatically from decidua vera isolated from term placentae, and were incubated in Dulbecco's Modified Eagle's Medium containing 0.25% bovine serum albumin at 37 degrees C. PGF2 alpha and PGE2 output were measured by radioimmunoassay of the conditioned medium. Production of PGF2 alpha (fmol/10(6) cells per 3 h) exceeded that of PGE2 at 273 (108-322) versus 97 (38-127) respectively (median (range]. The decidual cell dispersions were then incubated with monoclonal antibodies (anti-CD45 which labels the leukocyte common antigen or anti-human leukocyte antigen class II (HLA-DR) which is specific for macrophages in this tissue) and sorted by flow cytometry. The resultant antibody-positive and -negative cell populations were incubated and PG production was measured. Controls showed that antibody labelling and sorting did not alter PG production. PGF2 alpha and PGE2 output by bone marrow-derived (CD45-positive) cell populations exceeded that of non-bone marrow-derived (CD45-negative) cells. Furthermore, we were able to demonstrate that the HLA-DR-positive macrophage population had the highest PGF2 alpha and PGE2 production rates in human term decidua in vitro.

Expression of intermediate filament in endometrial glands changes with the onset of pregnancy and in endometriosis.

Appropriate endometrial differentiation is believed to be a prerequisite for pregnancy success. This study investigates the expression of two intermediate filament proteins, cytokeratin and vimentin, in human endometrium and first trimester decidua and in ectopic endometrium from women with endometriosis. Stromal elements, including vascular endothelial cells, were consistently vimentin-positive and cytokeratin-negative. Surface and glandular epithelial cells of human endometrium co-expressed vimentin and cytokeratin during all stages of the menstrual cycle, but failed to express vimentin after the onset of pregnancy. This suggests that intermediate filaments, and especially vimentin, may have a role to play in the proliferation and/or differentiation of the endometrial glands during decidualization. Ectopic endometrium showed a staining pattern similar to normal endometrium.

Expression on cells of early human pregnancy decidua, of the p75, IL-2 and p145, IL-4 receptor proteins.

Immunohistological studies of human first trimester pregnancy decidua demonstrated the presence of the p75 interleukin-2 receptor (IL-2R) and the p145 interleukin-4 receptor protein (IL-4R) on cells in the decidual stroma; there was no expression of CD25, the p55 IL-2R. The IL-4R was also expressed on the basal face of the glandular epithelial cells. Flow cytometric analysis of antibody-labelled decidual cell dispersions confirmed these results. Double antibody labelling demonstrated that p75 was expressed exclusively on the CD56-positive decidual large granular lymphocytes (LGL), whereas the IL4-R was expressed on some decidual LGL, and most decidual macrophages and T cells. In vitro incubation of decidual cells with IL-2 failed to induce expression of p55 or to increase the expression of either p75 or the p145 IL-4R. Purified decidual LGL proliferated in vitro in response to IL-2, and IL-4 inhibited this IL-2-induced proliferation.

Variation during the menstrual cycle of immune cell populations in human endometrium.

Morphometric analysis and immunohistology of tissue sections have been used to assess variation, during the normal menstrual cycle, of the bone marrow-derived cell populations in human endometrium. Levels of T cells and macrophages were found to be relatively constant throughout the cycle. In contrast, numbers of large granular lymphocytes, identified as being CD56-positive, were generally low between days 10 and 19, but increased sharply in the latter part of the luteal phase, decreasing again after menstruation. This LGL population is known to be abundant in first trimester pregnancy decidua, and is presumed to play a role in early pregnancy success.