Expression of nuclear factor kappa B components in human endometrium.

Nuclear factor kappa B (NFkappaB) is a family of transcription factors involved in signalling between IL1 and TNFalpha receptors and cytokines and adhesion molecules in a number of cell types, including those of the human endometrium. In this study, we used immunocytochemistry to investigate the in vivo expression of the p50, IkappaBalpha and IkappaBbeta NFkappaB components in endometrium obtained from normal fertile women throughout the menstrual cycle. All three components were expressed by both the stromal and epithelial cells of the endometrium and staining was predominately seen in the cytoplasm of the cells. Staining for p50 was more intense in the epithelial compartment than the stromal compartment. Staining in the stromal compartment was low to moderate throughout the cycle but, in the epithelial compartment, staining was cycle dependent and increased slightly during the mid-secretory phase. The staining patterns for IkappaBalpha and IkappaBbeta were similar. As for p50, staining for both proteins was greater in the epithelial compartment compared to the stromal compartment and stromal cell staining was low to moderate throughout the cycle. However, in contrast to p50, staining for the IkappaB proteins in epithelial cells decreased during the mid-secretory phase of the cycle. Although the immunocytochemistry technique used is only semi-quantitative, the results suggest an increased expression of the active and a decreased expression of the inhibitory NFkappaB components by the endometrium at the time of implantation. If confirmed, it would suggest that NFkappaB is involved in the control of factors important in the implantation process.

Expression of interleukin-11 receptor alpha and interleukin-11 protein in the endometrium of normal fertile women and women with recurrent miscarriage.

Interleukin-11 (IL-11) is a member of the IL-6 family of cytokines. Previous studies have suggested that IL-11 may play a role in human endometrial function. In this study, we have used immunocytochemistry to compare endometrial IL-11Ralpha and IL-11 expression in precisely timed peri-implantation biopsies from 9 normal fertile women and 16 recurrent miscarriage (RM) women. Immunocytochemistry was semi-quantified by obtaining an H-score value, which showed increased expression of both IL-11 and IL-11Ralpha in epithelial cells compared to stromal cells in all biopsies. There was a significant (P<0.01) reduction in epithelial cell IL-11, but not stromal cell IL-11, expression in endometrium from RM women compared to normal fertile women. There were no significant differences in expression of IL-11Ralpha protein in both stromal and epithelial cells in endometrium from the two groups of women. This work shows the presence of IL-11 and IL-11Ralpha within the endometrium of RM women during the peri-implantation period. The decreased expression of IL-11 in epithelial endometrium in RM women suggests that this cytokine may play a role in preventing miscarriage.

Do androgens have a direct effect on endometrial function? An in vitro study.

OBJECTIVE: To test the hypothesis that androgens have a direct effect on the function of endometrial epithelial cells. DESIGN: In vitro study. SETTING: Academic research center. PATIENT(S): Endometrial epithelial cells were prepared from biopsy samples obtained from normal fertile women. INTERVENTIONS: Cells were incubated with androstenedione, testosterone, dihydrotestosterone, and DHEA. MAIN OUTCOME MEASURE(S): Secretion of glycodelin A into the culture fluid was used to assess secretory activity. Uptake of (3)H-thymidine and immunostaining for Ki67 was used to assess cell growth. The specific action of the androgens was confirmed by incubation with an antiandrogen, cyproterone acetate. RESULT(S): Androstenedione (10(-6) M and 10(-7) M) caused a dose-dependent decrease in glycodelin A secretion, uptake of (3)H-thymidine, and percentage of positive Ki67 cells in cultured human endometrial epithelial cells. Testosterone, dihydrotestosterone, and DHEA had no effect on glycodelin A secretion or (3)H-thymidine uptake. The direct effect of androgens on endometrial function were confirmed by demonstrating the presence of androgen receptors in cultured endometrial epithelial cells and showing that the direct effects of the androgens were not observed when cyproterone acetate was added to the cultures. CONCLUSION(S): The results suggest that androstenedione can inhibit human endometrial cell growth and secretory activity. Infertility and miscarriage associated with high androgen levels (e.g., that caused by the polycystic ovary syndrome) may be due to an adverse effect of high androgen levels on the endometrium.

Serum androgen levels in women who have recurrent miscarriages and their correlation with markers of endometrial function.

OBJECTIVE: To compare plasma androgen concentrations in women who have recurrent miscarriages and in fertile women, and to correlate the results with concentrations of the endometrial protein PP14 in uterine flushings and plasma from women who have recurrent miscarriages. DESIGN: Retrospective study. SETTING: Hospital research unit. PATIENT(S): Women attending a recurrent miscarriage clinic and normal fertile volunteers. Ten of the women with recurrent miscarriages had polycystic ovary disease (PCOD) as assessed by ultrasonography or increased follicular LH levels. INTERVENTION(S): Plasma samples were obtained from the women on days LH-7, LH-4, LH+0, and LH+7 or LH+10 of a cycle. An endometrial flushing sample and a biopsy specimen were taken from women with recurrent miscarriages on day LH+7 or LH+10. MAIN OUTCOME MEASURE(S): Androstenedione, testosterone, and sex hormone-binding globulin (SHBG) were measured in the plasma samples. The endometrial protein PP14 was measured in the uterine flushings and in the LH+7 or LH+10 plasma samples from the women with recurrent miscarriages. RESULT(S): Testosterone concentrations were higher in the women with recurrent miscarriages both with and without PCOD on days LH-7 and LH-4 of the cycle. Concentrations of androstenedione also were higher in the women with recurrent miscarriages, but without PCOD on day LH-7. Testosterone SHBG ratios were higher in the women with recurrent miscarriages, without PCOD compared with the controls on days LH-7, LH+0, and LH+7. Mean follicular testosterone concentrations were correlated negatively with both uterine (r = -0.47) and plasma (r = -0.49) PP14 levels on day LH+10. Mean luteal phase testosterone SHBG ratios were correlated negatively with uterine PP14 concentrations on day LH+7 of the cycle (r = -0.674). CONCLUSION(S): Androgen levels are higher in women who have recurrent miscarriages than in normal fertile controls. These high levels of androgens may have a detrimental effect on endometrial function.

Impact of high body mass index on endometrial morphology and function in the peri-implantation period in women with recurrent miscarriage.

There is evidence that women with a high body mass index may have a higher risk of miscarriage. It is not known if this is due to an endometrial or embryo defect. The aim of this retrospective study was to examine markers of endometrial function in overweight and obese women with recurrent unexplained miscarriage. A total of 136 women were included in the study and classified according to their body mass index (BMI) into two groups, normal BMI (< 25 kg/m(2), n = 70) and high BMI (> or = 25 kg/m(2), n = 66). Endometrial morphology was examined in all patients. A subgroup of 28 patients was examined for endometrial oestrogen and progesterone receptors in different components of the endometrium, and in a further subgroup of 28 patients, endometrial glandular leukaemia inhibitory factor and leukocyte populations were examined. A modest increase in the BMI (30.4 +/- 0.71 kg/m(2)) does not have a significant impact on endometrial steroid receptors, leukocyte populations or endometrial morphology. However, there was a significant negative correlation between endometrial glandular leukaemia inhibitory factor concentrations and the BMI (r = -0.4, P = 0.02), warranting further investigation in prospective studies that include patients with higher BMI levels.

Cytokine expression in the endometrium of women with implantation failure and recurrent miscarriage.

One potential cause of reproductive failure such as infertility and recurrent miscarriage may be an endometrial defect. Numerous studies in mice have suggested the importance of various different cytokines in successful pregnancy outcome. This article reviews the literature available on the role of T helper cytokines and IL-1, IL-11, LIF, IL-12 and IL-18 in infertility and recurrent miscarriage, with particular emphasis on the role that endometrial cytokines may play. Although there are numerous studies on cytokines in recurrent miscarriage, much less has been reported on their role in infertility with or without failure after IVF. There is also considerable variation in the results obtained from various different studies, which may be due to different populations studied, the different timing of the sample collection, and whether the cytokines were measured in whole tissue or a specific cell population. The presence of complicated networks of cytokines and their overlapping biological activities means that alteration of one cytokine is likely to affect others and this also makes the study of their role in implantation failure very difficult. There is an urgent need to re-examine the role played by various cytokines in reproductive failure through carefully planned and vigorously designed studies and to compare the different types of reproductive failure.

Endometrial factors in recurrent miscarriage.

Recurrent pregnancy loss may be a consequence of an abnormal embryonic karyotype, or maternal factors affecting the endometrium resulting in defective implantation. In order to study the endometrial factors responsible for recurrent pregnancy loss, endometrial biopsy samples should be precisely timed according to the LH surge, and the investigation should be carried out in a non-conception cycle, prior to the next pregnancy. The various methods of studying the endometrium including morphological studies, morphometry, immunohistrochemistry, measurement of endometrial protein in plasma and uterine flushings, cytokine expression in endometrial cells, leukocyte populations in the endometrium and ultrasonographic and hysteroscopic studies, were reviewed. The clinical relevance of the observed abnormality depends on whether or not the abnormality is persistent in subsequent cycles, and if the observed abnormality is of significant prognostic value. Very little is known about the treatment of endometrial defect associated with recurrent pregnancy loss, but preliminary data suggest that the use of HMG may be of benefit.

Expression of interleukin (IL)-11 receptor by the human endometrium in vivo and effects of IL-11, IL-6 and LIF on the production of MMP and cytokines by human endometrial cells in vitro.

Interleukin (IL)-6, leukaemia inhibitory factor (LIF) and IL-11 belong to the same family of cytokines whose receptors utilize gp130 as the signalling molecule. We have investigated the expression of the IL-11 receptor, IL-11Ralpha, protein in the human endometrium in vivo and the effects of IL-6, LIF and IL-11 on the production of metalloproteinases (MMPs) and cytokines by cultured endometrial epithelial and stromal cells. Immunostaining showed that IL-11Ralpha was expressed in both epithelial and stromal cells, with epithelial staining being more intense than stromal staining and little variation in staining in either compartment throughout the cycle. Incubation of both stromal and epithelial cells with IL-6, LIF and IL-11 had no effect on MMP-2, -7, -9, transforming growth factor (TGF)beta or IL-1beta production or cell growth. IL-6 and LIF also had no effect on tumour necrosis factor (TNF)alpha production, but IL-11 caused a dose-dependent decrease in TNFalpha production by epithelial cells. IL-6 receptor, LIF receptor and gp130 were all expressed by cultured stromal and epithelial cells, showing that the lack of effect is not due to lack of expression of the receptor components. The results show that although IL-6, LIF and IL-11 signal through the same molecule, they may have different effects in endometrial cells, suggesting the activation of different signalling pathways, which may ultimately be important in the control of endometrial function.

Expression of nuclear factor kappa B in human endometrium; role in the control of interleukin 6 and leukaemia inhibitory factor production.

Expression of the rel-A component of nuclear factor kappa B (NFkappaB) by human endometrial cells was investigated by immunocytochemical analysis of cryostat sections cut from endometrial biopsy material and of cultured endometrial epithelial cells. In-vivo expression of rel-A was low in epithelial cells in endometrium obtained during the proliferative phase of the cycle, but increased in these cells during the secretory phase and was maximal at the time of implantation. In-vivo expression of rel-A by stromal cells did not vary greatly throughout the cycle, but showed a slight peak at the time of ovulation. In contrast similar expression of rel-A was seen in short-term cultures of epithelial cells prepared from both proliferative and secretory endometrium. Addition of the NFkappaB inhibitor SN50 (5 microg/ml) to confluent cultures of endometrial epithelial cells inhibited interleukin (IL)-1alpha (10 ng/ml) and tumour necrosis factor alpha (TNFalpha) (10 ng/ml) stimulated IL-6 (P < 0.001 and P < 0.01 respectively) and LIF (P < 0.01 and P < 0.05 respectively) production. The proteasome inhibitor MG132 (0.3 and 3 micromol/l) also caused a dose-dependent decrease in IL-1alpha and TNFalpha-stimulated IL-6 (P < 0.001 and P < 0.001 respectively) and leukaemia inhibitory factor (LIF) (P < 0. 001 and P < 0.001 respectively) production by endometrial epithelial cells. The results support the hypothesis that NFkappaB mediates signalling between IL-1 and TNFalpha receptors and the expression of LIF and IL-6 in endometrial epithelial cells.

The production of tumour necrosis factor alpha (TNF-alpha) by human endometrial cells in culture.

The production of tumour necrosis factor alpha (TNF-alpha) by cultured human endometrial epithelial and stromal cells prepared from endometrium obtained at different stages in the menstrual cycle has been investigated. TNF-alpha was not detectable in the supernatants of stromal cell cultures prepared from endometrial tissue obtained at any time in the menstrual cycle. TNF-alpha production by endometrial epithelial cells in culture varied depending on the time in the cycle at which the endometrial tissue was taken. Cells prepared from tissue obtained during the late proliferative phase of the cycle produced more TNF-alpha than those prepared from tissue obtained at other times in the cycle. In addition, a small increase in TNF-alpha production was seen by cells prepared from tissue obtained during the mid-secretory phase of the cycle. Interleukin 1 (IL-1) (1.4-140 pmol/l) caused a dose-dependent increase in TNF-alpha production by cells prepared from both proliferative and secretory endometrium. Maximum IL-1-stimulated increases in TNF-alpha production were similar in cells from both proliferative and secretory endometrium and typically reached from four to 10 times basal values. High doses of progesterone, either alone or in the presence of oestradiol, also affected TNF-alpha production by epithelial cells. TNF-alpha production by cells prepared from proliferative endometrium was increased by progesterone. In contrast, TNF-alpha production by cells prepared from secretory endometrium was decreased in the presence of progesterone. The effects of steroids on TNF-alpha production were less marked than that of IL-1, with values increasing or decreasing to a maximum of three times the basal value. Placental protein 14 (PP14) (0.18 and 1.8 nmol/l) also increased TNF-alpha production by cells prepared from proliferative tissue, but had no effect on its production by cells prepared from secretory endometrium. PP14-stimulated TNF-alpha levels typically only reached a maximum of two times basal values.

A review of immune cells and molecules in women with recurrent miscarriage.

Immunological rejection of the fetus due to recognition of paternal antigens by the maternal immune system, resulting in abnormal immune cells and cytokine production, is postulated to be one cause of unexplained pregnancy loss. Although there is evidence for this in rodents, there is less evidence in humans. This article focuses on studies in humans, and reviews the recent literature on the differences in immune cells and molecules in normal fertile women and women with recurrent miscarriage (RM). Although much of the evidence is contradictory, these studies do suggest differences in the expression of some immune cells and molecules in women with RM. Differences in the CD56+ population of cells are seen, and there is some evidence for an alteration in the ratio of Th1 and Th2 cytokines produced by peripheral blood monocytes (PBMCs) and clones of decidual CD4+ cells. There is also some evidence for differences in endometrial cytokine production, and in particular decreased production of pro-inflammatory cytokines such as interleukin-6. Possible reasons for the variations in data are discussed, and the importance of compartment (peripheral blood, endometrium or decidua) in which the cells and molecules are measured and the timing of the sampling, both with respect to the menstrual cycle and pregnancy (at the time or just after miscarriage) is emphasized.

The production of placental protein 14 by human uterine tubal epithelial cells in culture.

Cells prepared from the mucosal layer obtained from the fimbrial, proximal ampullary and distal ampullary regions of the human uterine (Fallopian) tube have been grown in monolayer culture. Immunocytochemistry with anti-cytokeratin, anti-vimentin or anti-CD 45 antibodies indicated that the overwhelming number of cells were epithelial in nature and were free of fibroblasts and leukocytes. Basal and steroid-stimulated placental protein 14 (PP14) production was investigated in tissue obtained from nine patients undergoing hysterectomy, by addition of oestradiol and/or progesterone to confluent cultures. Basal PP14 production varied considerably between experiments, probably due to differences between individuals from whom the tissue had been obtained. However, there was no difference in basal PP14 production by cells prepared from the fimbrial, proximal ampullary and distal ampullary parts of the tube obtained from the same patient. When total PP14 production by cells obtained from an individual uterine tube was pooled both progesterone and oestradiol significantly (P < 0.05) stimulated the production of PP14 but the effect of progesterone either alone or in the presence of oestradiol was numerically greater than that of oestradiol alone. Considering PP14 production by cells prepared from the different regions of the tube showed that cells from the fimbrial region were more responsive to steroid stimulation than cells prepared from either the proximal or the distal ampullary regions. All combinations of hormonal supplementation stimulated PP14 production by cells from the fimbrial region on all days measured (P < 0.05 - P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The production of leukaemia inhibitory factor by human endometrium: presence in uterine flushings and production by cells in culture.

The concentration of leukaemia inhibitory factor (LIF) was measured in uterine flushings obtained from normal fertile women, from women with unexplained infertility and from women who suffered recurrent miscarriage. In normal fertile women, LIF was not detected in flushings obtained on days luteinizing hormone (LH)+0 to LH+6 of the cycle, but concentrations gradually increased from day LH+7 to a maximum at day LH+12. The amount of LIF in flushings obtained from women with unexplained infertility was significantly lower than in those from normal fertile women on day LH+10 (P < 0.05). The production of LIF by cultured human epithelial and stromal cells was also investigated. LIF was not detectable in the supernatants of cultured stromal cells. Basal LIF production by epithelial cells varied according to the stage in the cycle at which the biopsy was taken. Significantly more LIF was produced by epithelial cells from late proliferative and early secretory endometrium compared with amounts produced by cells from early proliferative (P < 0.001) and late secretory (P < 0.01) endometrium. High doses of progesterone and oestradiol caused a small decrease in epithelial cell LIF production: the combined effect of progesterone and oestradiol (P < 0.01) was greater than the effect of either steroid alone (P < 0.05). The results show, for the first time, the capability of human endometrium to produce LIF in vivo. The fact that maximum LIF concentrations are present at implantation and that decreased concentrations occur in women with unexplained infertility suggest the importance of this cytokine in embryo implantation.

Expression of leptin receptor isoforms in vitro: lack of effects of leptin on endometrial cytokine and MMP production.

PROBLEM: Leptin has a key role to play in human female reproduction. Its receptor is expressed highly throughout the reproductive tract. Cytokines have an important role in preparing the endometrium for implantation and leptin is known to modulate cytokine production in other tissues. We, therefore, investigated the possible role of leptin in endometrial growth and function. METHOD OF STUDY: Reverse transcriptase polymerase chain reaction and immunocytochemistry were used to determine the pattern of expression of leptin receptor isoforms in primary human endometrial epithelial and stromal cells in culture. The effect of leptin on cell growth and on the production of cytokines [Leukaemia Inhibitory Factor (LIF), interleukin 6 and tumour necrosis factor-alpha] and matrix metalloproteinases (MMP) (MMP2 and MMP-9) was also investigated. RESULTS: Expression of the long form of the leptin was restricted to the cultured endometrial, epithelial cells. Both cultured endometrial stromal and epithelial cells expressed the short and variant isoforms of the receptor. Incubation of epithelial and stromal cell cultures with varying concentrations of leptin (0-1000 ng/mL) had no significant effect on cell growth or levels of MMP-2 or MMP-9 production. Leptin also had no significant effect on cytokine production by epithelial cells. CONCLUSIONS: This study shows for the first time, the presence of leptin receptor isoforms on endometrial, epithelial and stromal cells in culture. Leptin had no effect on cytokine and MMP production by these cells. However, it is possible that leptin affects other factors within the endometrium not investigated here.