Bcl-2 and Fas expression in eutopic and ectopic human endometrium during the menstrual cycle in relation to endometrial cell apoptosis.

OBJECTIVE: Our purpose was to investigate Bcl-2 and Fas expression in human eutopic and ectopic endometrium during the menstrual cycle in relation to endometrial cell apoptosis. STUDY DESIGN: Eutopic and ectopic endometrial samples were obtained from 29 patients with endometriosis, and eutopic endometrial tissue samples were obtained from 9 patients with uterine myoma. Bcl-2 and Fas expression were examined by immunohistochemical staining with specific monoclonal antibodies; Bcl-2 expression in eutopic endometrium was also examined by Western blotting and apoptotic cells were detected by the labeling of deoxyribonucleic acid fragments. RESULTS: In eutopic endometrium Bcl-2 was strongly expressed during the proliferative phase. Endometrial glandular cells showed evidence of cyclic changes in Bcl-2 expression, but cyclic changes were not apparent in peritoneal and ovarian endometriotic tissue. Fas expression was observed on glandular cells but not on stromal cells, and no cyclic changes in expression occurred in either ectopic or eutopic endometrium. Apoptotic cells were observed primarily in the glandular cells of the basal layer in eutopic endometrium during the late secretory and menstrual phases. CONCLUSION: The current study indicated that there was no apparent correlation between Bcl-2 or Fas expression with endometrial cell apoptosis. The absence of cyclic changes in Bcl-2 expression in ectopic endometrium implied a difference in the mechanisms of proliferation or differentiation between eutopic and ectopic endometrium.

Progesterone stimulates the induction of human endometrial CD56+ lymphocytes in an in vitro culture system.

An unusual population of large granular lymphocytes (LGL) with the surface phenotype CD56(bright+)/CD16-/CD3- increases in the human endometrium during the late secretory phase and early pregnancy. To identify the factor(s) that induces CD56+ LGL in the human endometrium, we isolated endometrial leukocytes from nonpregnant human endometrium and investigated changes in CD56+ cells during culture with various factors. Isolated endometrial leukocyte-rich fraction and peripheral blood mononuclear cells were cultured for 6 days in the presence of progesterone, estradiol, PRL, or hCG, then nonadherent cells were collected and examined immunocytochemically. Endometrial leukocyte-rich fractions were composed of leukocytes and endometrial stromal cells, and 53.2 +/- 5.8% of them expressed CD45 antigen before culture. Therefore, leukocytes and endometrial stromal cells were cocultured in these endometrial leukocyte-rich fraction cultures. The percentage of CD56+ cells in endometrial leukocyte-rich fractions cultured with progesterone was significantly higher than that in fractions without progesterone. On the other hand, estradiol, PRL, and hCG did not significantly induce CD56+ cells in endometrial leukocyte-rich fractions. There was no significant difference in the percentage of CD56+ cells between peripheral blood mononuclear cell cultures with and without progesterone. These findings suggest that progesterone is an important factor for the in situ proliferation or differentiation of CD56+ LGL in human endometrium.

Endometrial stromal cell decidualization inhibits human chorionic gonadotrophin and human placental lactogen secretion by co-cultured trophoblasts.

We have developed an in-vitro co-culture system to examine the interaction between purified first trimester cytotrophoblasts and purified non-pregnant human endometrial stromal cells (ESC). ESC decidualization is an important step in endometrial maturation and may modulate embryo implantation. In order to investigate the effects of ESC decidualization on trophoblast function, we examined human chorionic gonadotrophin (HCG), human placental lactogen (HPL), progesterone and estrogen secretion by trophoblasts co-cultured in contact with ESC, either with or without decidualization induced by progesterone. Decidualized ESC inhibited basal HCG and HPL secretion for 3 days during the culture for HCG, and for 5 days during the culture for HPL (P < 0.01 and P < 0.03 respectively). After 5 days of co-culture, decidual transformation of ESC as indicated by prolactin production occurred in the control cultures due to progesterone and oestradiol secretion by the co-cultured trophoblasts, but no significant differences in HCG or HPL secretion were observed between the two groups. Although the type of trophoblast used in the present study is far from implantation, our results clearly demonstrated that HCG and HPL secretion by trophoblasts was inhibited by the presence of co-cultured decidualized ESC, and suggested that ESC decidualization may regulate trophoblast function at the human fetal-maternal interface.

Expression of leukaemia inhibitory factor (LIF) receptor in human placenta: a possible role for LIF in the growth and differentiation of trophoblasts.

Leukaemia inhibitory factor (LIF) is a cytokine that displays multiple activities in various tissues and is essential for blastocyst implantation in mice. In the human uterus, LIF is expressed in endometrial tissue and the decidua. To elucidate the role it plays, the mRNA levels for two LIF receptor (R) subunits, LIF-R and gp130, were examined in human endometrium, placenta and decidua by Northern blot hybridization. The expression of LIF-R gene was detected in the chorionic villus during the first trimester, in term placenta, and at lower levels in the decidua. The expression of LIF-R gene was not detectable in non-pregnant endometrium. The expression of the gp130 gene was detected in all tissues examined. During pregnancy, there was no significant change in the mRNA concentration of LIF-R in the placenta, while that of gp130 increased after the second trimester. The human choriocarcinoma cell line, BeWo, was found to express LIF-R and gp130. LIF inhibited forskolin-induced human chorionic gonadotrophin (HCG)-beta production by BeWo in a dose-dependent manner, and it ameliorated forskolin-induced growth suppression. These findings suggest that LIF plays a regulatory role in trophoblast growth and differentiation during pregnancy in human placenta.

Monoclonal antiphosphatidylserine antibody reactivity against human first-trimester placental trophoblasts.

OBJECTIVE: To investigate the binding of antibodies against negatively charged phospholipids (antiphospholipid antibodies) to human placenta, we tested the reactivity of three mouse monoclonal antiphospholipid antibodies against first-trimester human placenta. STUDY DESIGN: Formalin-fixed and frozen sections of first-trimester placentas were stained by immunoperoxidase with three mouse monoclonal antibodies. Each monoclonal antibody reacted differently with cardiolipin and phosphatidylserine, 3SB9b reacted with phosphatidylserine, D11A4 reacted with cardiolipin, and BA3B5C4 reacted with both. RESULTS: 3SB9b reacted strongly with the syncytiotrophoblastic layer of both formalin-fixed and frozen placental tissue. Sporadic reactivity was observed against the cytotrophoblastic layer. BA3B5C4 reacted strongly and specifically with cytotrophoblastic cells. D11A4 reacted minimally or, more commonly, not at all. CONCLUSION: The trophoblastic layer directly in contact with the maternal circulation is most reactive with antiphospholipid antibodies that react with phosphatidylserine rather than cardiolipin, suggesting that the trophoblasts may potentially be directly damaged by antiphospholipid antibodies through mechanisms unrelated to thrombosis. In addition, the differential reactivity of 3SB9b and BA3B5C4 suggests that the antigenic conformation involving phosphatidylserine on the cytotrophoblast is altered concurrent with fusion into the syncytium.

Expression of messenger ribonucleic acid for gonadal steroid receptors in the human pelvic peritoneum.

OBJECTIVE: To investigate the expression of messenger RNA (mRNA) for gonadal steroid hormone receptors in the human pelvic peritoneum. DESIGN: Analysis of estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR) mRNA expressions in the pelvic peritoneum was carried out using the quantitative reverse transcription-polymerase chain reaction (PCR) method. SETTING: Department of Gynecology and Obstetrics, Kyoto University Hospital, Kyoto, Japan. PATIENTS: Pelvic peritoneal tissues from patients with (n = 10) and without (n = 10) endometriosis who had undergone gynecological surgery were studied. RESULTS: Estrogen receptor, PR, and AR mRNAs were detected in all pelvic peritoneal samples analyzed. In the pelvic peritoneum of patients without endometriosis, ER mRNA levels were significantly lower in the luteal phase than in the follicular phase. This cyclic profile of ER mRNA expression was not observed in the pelvic peritoneum of patients with endometriosis. During the follicular phase, ER mRNA levels in the pelvic peritoneum of patients with endometriosis were significantly lower than those of patients with endometriosis. Neither PR nor AR mRNA levels in the pelvic peritoneum of either patient group showed significant cyclic variations throughout the menstrual cycle. A comparison of PR and AR mRNA levels in the pelvic peritoneum of the endometriosis and the nonendometriosis groups revealed no significant differences. CONCLUSIONS: These data indicate a decrease in ER gene expression in the pelvic peritoneum of patients with endometriosis during the follicular phase. This suggests that the possible responsiveness of peritoneal cells to estrogen may be related to the occurrence and/or development of endometriosis.

Hormonal regulation in the production of macrophage colony-stimulating factor and transforming growth factor-beta by human endometrial stromal cells in culture.

The effects of gonadal steroids on the secretion and gene expression of macrophage colony-stimulating factor (M-CSF) and on the secretion of transforming growth factor (TGF)-beta 1 and TGF-beta 2 by human endometrial stromal cells (ESCs) were examined by an in vitro system of ESC differentiation (decidualization). M-CSF production by ESCs was dose-dependently enhanced by the addition of progesterone or testosterone, while estradiol treatment had no effect. TGF-beta 2 secretion by ESCs was inhibited by progesterone, estradiol and testosterone treatment, and on the contrary, slight enhancement by estradiol was observed in TGF-beta 1 secretion. These findings indicate that human ESCs produce cytokines of M-CSF and TGF-beta s, which are important for the growth and differentiation of the peri-implantation embryo as well as local immune cells under direct control of gonadal steroidal actions, and suggest a novel network between endocrine and immune systems in the human endometrium.

Tumour necrosis factor alpha inhibits in-vitro decidualization of human endometrial stromal cells.

Interleukin-1 (IL-1) has been reported previously to inhibit the in-vitro decidualization of human endometrial stromal cells as assessed by progesterone-induced prolactin production and morphological transformation. In this study we examined whether other cytokines, such as tumour necrosis factor-alpha (TNF alpha), interferon-beta (IFN beta), IFN gamma or granulocyte-macrophage colony-stimulating factor (GM-CSF), could affect the decidualization of human endometrial stromal cells in vitro. Of these cytokines, TNF alpha significantly suppressed prolactin production in a dose-dependent manner, with no apparent effect on cell number. The morphological transformation of endometrial stromal cells was also inhibited by TNF alpha. TNF alpha and IL-1 significantly suppressed cAMP-stimulated prolactin production by endometrial stromal cells. Neither the progesterone concentration in the supernatant of the endometrial stromal cell culture system nor intracellular calcium concentration of the endometrial stromal cells were affected by the addition of TNF alpha or IL-1. These results indicated that TNF alpha and IL-1 suppress both progesterone-induced and cAMP-mediated prolactin production in endometrial stromal cells, and that this inhibition was not attributable to direct effects on progesterone metabolism or related to Ca(2+)-mediated signal transduction. These experiments suggested that a local increase of TNF alpha and IL-1 under certain pathological conditions in vivo may disturb blastocyst implantation and/or the maintenance of pregnancy by inhibiting the decidualization of endometrial stromal cells.

Progesterone-dependent secretion of macrophage colony-stimulating factor by human endometrial stromal cells of nonpregnant uterus in culture.

Recent studies have suggested that macrophages colony-stimulating factor (M-CSF), a hematopoietic glycoprotein essential to the proliferation and differentiation of mononuclear phagocytes and their progenitor cells, is also involved in the reproductive process in mice and humans. In this study, we examined, by enzyme-linked immunosorbent assay, the supernatants of stromal cell-enriched fraction (SF) of human nonpregnant endometrium for the presence of M-CSF during culture with progesterone (P) or estrogen. The bioactivity of M-CSF was assessed in a colony-forming assay of murine bone marrow cells. In addition, the M-CSF level in the culture supernatant of SF further purified by subculture, of epithelial cell-enriched fraction purified from human endometrium, and of peripheral blood lymphocytes, including about 10% monocytes, was examined with or without P, because SF is contaminated by epithelial cells and macrophages, both of which are suggested to secrete M-CSF. During 2-week culture, the level of M-CSF in the supernatants of SF cultured with P was markedly higher than that of control culture and estrogen-treated culture on any day tested, except for the first 2 days. P had a dose-dependent effect on M-CSF production by SF. Estrogen also enhanced M-CSF production by SF, but did not show dose dependency. The SF culture supernatants showed a colony-forming activity that was completely blocked by neutralizing anti-M-CSF antibody. SF subcultured three times, which was confirmed to be of more than 99% purity, secreted M-CSF in a P-dependent manner. M-CSF was also detected in the culture supernatants of epithelial cell-enriched fraction and peripheral blood lymphocytes, but P-dependent M-CSF production was not shown in these cultures. These results suggest that human endometrial stromal cells themselves can secrete bioactive M-CSF in a P-dependent manner in vitro, indicating that the M-CSF reported to be present in human endometrium is secreted in part by stromal cells and may play a role in the regulation of endometrial function under P control.

Bestatin, a potent aminopeptidase-N inhibitor, inhibits in vitro decidualization of human endometrial stromal cells.

We have reported that human endometrial stromal cells (ESC) express a cluster of differentiation-13 antigen/aminopeptidase-N, and the expression of this peptidase antigen was shown to increase with the decidualization of ESC. To clarify the role of this peptidase in human endometrium, the effect of bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-(S)-leucine), an inhibitor of aminopeptidase-N, on the decidualization of ESC in vitro was examined. Purified human ESC were cultured for 12 days in the presence of 10(-6) mol/L progesterone with or without bestatin. Decidualization was assessed by PRL production and morphological transformation. The effects of a stereoisomer of bestatin and of pepstatin were similarly examined using the same culture system. Bestatin inhibited progesterone-induced PRL production in a dose-dependent manner, with no effect on cell number or viability, whereas neither its stereoisomer nor pepstatin inhibited aminopeptidase activity or PRL production. The morphological transformation of ESC was also inhibited by bestatin, but not by its stereoisomer or pepstatin. These findings demonstrate that the inhibition of aminopeptidase-N activity blocks the in vitro decidualization of ESC and suggest an important role for this peptidase in the functional differentiation of human ESC.

Androgens induce prolactin production by human endometrial stromal cells in vitro.

Although there is a significant quantity of androgens in the endometrium, the function of these hormones has not been clarified, except for being estrogen precursors. Human endometrial stromal cells (ESC) were cultured in the presence of testosterone (T) and 5 alpha-dihydrotestosterone. Following culture, prolactin (PRL), a biochemical marker of stromal cell differentiation (decidualization) which is produced by ESC, was examined. T induced PRL production in a time- and dose-dependent manner, as reported previously for progesterone (P) stimulation. In addition, 5 alpha-dihydrotestosterone, which cannot be converted to estrogens, similarly induced PRL production. T in combination with P enhanced PRL production in cultured ESC significantly more than either P or T stimulation alone. A specific androgen receptor blocker, flutamide, when added to cultures containing T, inhibited PRL production in a dose-dependent manner, but did not affect the production of PRL induced by P. These results indicate that in vitro PRL production by human ESC is induced not only by P, but also by androgens through specific receptors and further suggest that androgens play an important role in human endometrial differentiation.