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BACKGROUND Several studies have suggested that endometrial interleukin 15 (IL-15) and the leukaemia inhibitory factor (LIF) may be important in embryo implantation. IL-15 is postulated to play a role in the control of uterine natural killer (uNK) cell proliferation and function, and uNK cells are also known to play a role in implantation. The aims of this study was to (1) compare endometrial levels of IL-15 and the LIF in women with recurrent implantation failure (RIF) after IVF with those in fertile women (controls) and (2) examine the relation of IL-15 and LIF levels to the uNK cell number. METHODS We investigated IL-15 and LIF in precisely timed endometrial biopsies (days LH + 7-LH + 9, where the day of the LH surge is LH + 0) obtained from control women (n = 15) and women with RIF (n = 45) by immunohistochemistry. A semi-quantitative analysis was performed by the H-score analysis of staining intensity in the stroma, glandular epithelium and luminal epithelium, separately. We also correlated expression of LIF and IL15 with uNK cell numbers (obtained in an earlier study of the same samples). RESULTS The quantity of the LIF protein in endometrial glandular epithelium in women with RIF [median and range; 179 (70–365)] was lower (P = 0.01) than in control women [median and range; 247 (120–287)]. In contrast, the level of the IL-15 protein in the stroma in women with RIF [median and range; 90 (0–175)] was higher (P = 0.009) than in control women [median and range; 60 (15–150)]. There was a significant correlation between the uNK cell number and stromal expression of IL-15 (r = 0.427, P = 0.001). No correlation between the LIF expression in any compartment and the uNK cell number was seen. CONCLUSIONS The results show an altered expression of LIF and IL-15 in the endometrium of women with RIF. Despite the limitation of not identifying uNK cells by phenotypic markers, the correlation between the uNK cell number and the stromal cell IL-15 suggests that IL-15 may play a role in the control of endometrial uNK cell function or proliferation. [ABSTRACT FROM AUTHOR]

BACKGROUND CD56+ cells in peripheral blood or the endometrium may be increased in women with reproductive failure. However, the relationship between numbers of peripheral blood CD56+ and endometrial CD56+ cells is uncertain. The aim of this study was (i) to compare the numbers of CD56+ cells in peripheral blood and endometrium in samples taken simultaneously and (ii) to compare measurements by flow cytometry and immunohistochemistry of CD56+ cells in the same endometrial biopsy. METHOD Endometrial biopsies and blood were obtained from women with recurrent miscarriage (n= 25) on days LH+7–LH+9 of the cycle. The total number of CD56+, CD56+ CD16− and CD56+CD16+ cells in blood was measured by flow cytometry; the number of CD56+ cells in the endometrium was determined by immunohistochemistry. Endometrial samples were also obtained from fertile women (n= 20) and used to measure CD56+ and CD45+ cells, by both flow cytometry and immunostaining. RESULTS There was no correlation between the numbers of total CD56+, CD56+CD16− or CD56+CD16+ in peripheral blood and the number of endometrial CD56+ cells in the same women. In endometrium from fertile women, a significant correlation was found between the numbers of CD56+ cells measured by flow cytometry and immunohistochemistry (correlation= 0.497, P= 0.026, when expressed as % total cells; correlation= 0.570, P= 0.009 when expressed as % CD45+ cells). CONCLUSIONS Measurements of CD56+ cells in peripheral blood do not correlate with endometrial CD56+ cell numbers and therefore should not be extrapolated to events in the endometrium. In contrast, measurements of endometrial CD56+ cells by flow cytometry and immunostaining correlate well. [ABSTRACT FROM AUTHOR]

BACKGROUND Although several studies have suggested that CXCL12 and its receptor, CXCR4, may play a role in embryo implantation, there are limited reports of expression of CXCR4 and CXCL12 in human endometrium. The aim of this study was to investigate CXCL12 and CXCR4 expression in human endometrium and to see if CXCL12 could affect matrix metalloproteinase (MMP) production by endometrial stromal and epithelial cells. METHODS Quantitative real-time RT–PCR (qRT–PCR) was used to detect the expression of CXCL12 and CXCR4 mRNA in endometrial biopsy samples obtained from fertile women (n = 30). Immunohistochemical analysis was carried out to determine where in the endometrium CXCL12 and CXCR4 were expressed. Primary cell culture followed by qRT–PCR and zymography was used to investigate whether CXCL12 affected MMP-2 and -9 production by endometrial stromal and epithelial cells. RESULTS Both CXCL12 and CXCR4 were detected in the endometrium. There was no difference in CXCL12 expression at different times in the cycle, but expression of CXCR4 mRNA was significantly higher in the early proliferative (P < 0.01) compared with late proliferative and secretory phases of the cycle. CXCL12 expression was strongest in the epithelial compartment, and weaker in blood vessel walls. CXCR4 immunostaining was strong in the epithelium and blood vessel walls and weaker in the stroma. CXCL12 (10 and 100 ng/ml) had no effect on mRNA expression or activity of MMP-2 or MMP-9 in either stromal or epithelial cells. CONCLUSIONS The results show that the expression of CXCL12 in human endometrium does not alter during the menstrual cycle, while the endometrial expression of its receptor, CXCR4, is highest in the early proliferative phase. In contrast to its effects in other cells, CXCL12 had no effect on MMP-2 or MMP-9 production by endometrial stromal or epithelial cells. [ABSTRACT FROM PUBLISHER]

BACKGROUND Abnormal human embryo implantation leads to poor foetal development and miscarriage, or pre-eclampsia. Ethical and practical considerations concerning implantation limit its investigation, and it is often difficult to extrapolate findings in laboratory animals when implantation processes show diverse species differences. Therefore, it is important to develop new in vitro models to study the earliest events of human implantation. The aim of this study was to derive trophoblast cell lines from human embryonic stem cells (hESCs) by a robust protocol and co-culture of these cells with an established endometrial cell culture system to validate a model of trophoblast invasion at implantation. METHODS Derivation of trophoblast cell lines from hESC lines was established by spontaneous and induced differentiation of embryoid bodies and by initial measurement of hCGβ secretion by enzyme-linked immunosorbent assay and their phenotype investigated using gene- and protein-expression markers. Vesicles formed from an aggregating trophoblast were co-cultured with decidualized human endometrial stromal cells in hypoxic (2% oxygen) and normoxic (20% oxygen) environments. RESULTS Derived villous cytotrophoblast cell (CTB) lines further differentiated to invasive, extra-villous CTBs. Eventually, cells lost their proliferative capacity, with some lines acquiring karyotypic changes, such as a gain in the X chromosome. Cell-invasion assays confirmed that the extra-villous CTBs were invasive and erosion of the endometrial stromal layer occurred, particularly under hypoxic conditions in vitro. CONCLUSIONS Trophoblast cell lines derived from hESCs differentiate and adapt in vitro and can be used as a model to study the mechanisms of early trophoblast invasion. [ABSTRACT FROM AUTHOR]

Abstract: 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-11Rα 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-11Rα 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-11Rα 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-11Rα 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. [Copyright &y& Elsevier]

Postmenopausal women taking oestradiol 17-β 2 mg daily were randomized to receive either 25 or 50 μg gestodene from day 17 to 28 of the cycle in a double-blind study. Placental protein P14 (PP14) and CA 125 concentrations in uterine flushing, endometrial morphology and irregular bleeding after 12 cycles of study were observed. Eleven and 12 women in the 25 and 50 μg groups respectively completed the study. There were no significant differences in pre-treatment biochemical and morphological indices between the groups. The median PP14 concentration increased from 332 to 5800 ng/ml (P < 0.001) and from 145 to 27 160 ng/ml (P < 0.001) in the 25 and 50 μg gestodene groups respectively. No between-group significant rise of PP14 was observed. Similarly, no significant change was seen between the initial and post-treatment concentrations of CA 125 for either group. All biopsies were atrophic at inception of the study, and both regimens produced secretory endometrial transformation in the majority of biopsies. No between-group difference was observed in the morphometric indices measured, or any significant correlation between the concentrations of PP14 or CA 125 and morphology. The mean number of days of withdrawal bleeding (3.8 and 4.2 days for 25 and 50 μg respectively) were similar. In conclusion, both regimens produced a significant rise in uterine flushing concentrations of PP14, but not CA 125. PP14 is a sensitive biochemical marker in the assessment of endometrial response to hormone replacement therapy. [ABSTRACT FROM PUBLISHER]

MUC1 is a cell-surface and secretory product of endometrial epithelium. Immunohistoehemical studies carried out using two different antibodies to the mucin-type tandem repeat region of MUC1 indicate a cell-surface location in proliferative phase glands, with intracellular deposits accumulating in the early secretory phase. Commencing 3–4 days after the luteinizing hormone (LH) peak and continuing into the late secretory phase, secretory MUC1 appears in gland lumens. Uterine flushings were collected as a function of time after the LH peak and were analysed using a two-site enzyme-linked immunosorbent assay for MUC1. Low but measurable concentrations were observed up to day 7, while on days 7–13 much higher values were obtained. In women suffering from recurrent spontaneous miscarriage, the concentration of MUC1 in flushings wassignificantly lower than in the controls on day LH+10. Lower values were also observed on days 7 and 13. Reduced epithelial secretory function and a resultant change uterine fluid composition are features of endometrium from recurrent miscarriage patients. [ABSTRACT FROM PUBLISHER]

The distribution of keratan sulphate (KS) in normal endometrium has been studied by immunohistochemistry using monoclonal antibody 5D4. KS is present in association with glandular epithelium throughout the normal cycle. Production is hormonally regulated, as indicated by the significant increase observed in the secretory phase of the cycle. Intracellular immunoreactivity increases to a maximum 3 days after the luteinizing hormone (LH) peak. This is followed by an increase in secreted KS from 4 days after the LH peak, and essentially all endometrial glands showed immunoreactive deposits by 5 days after the LH peak. Beginning on day 7 after the LH peak, a fraction of glands was observed to have lost the epitope. Nevertheless, KS remained in a significant proportion of the gland lumens 9 days after the LH peak. The data suggest that analysis of secretory KS in women gives an index of hormonally regulated epithelial differentiation in the peri-implantation phase. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate whether the endometrium of women with unexplained infertility differs in some immunological aspects from the endometrium of normal fertile women. Endometrial biopsies were obtained from 24 normal fertile women (group I) and 24 women suffering from unexplained infertility (group II) at 4, 7, 10 and 13 days following the luteinizing hormone (LH) surge. Endometrial granulated lymphocytes were assessed morphometrically in 2 microns resin sections. A panel of 11 monoclonal antibodies was employed to characterize the leukocyte subsets in frozen sections. Semi-quantification was performed with a Quantimet 970 image analyser. Data were analysed using one- and two-way analysis of variance. Compared with fertile controls, women with unexplained infertility had significantly lower numbers of CD8+ (T suppressor/cytotoxic) cells at each post-LH date. In contrast, the number of CD4+ (T helper/inducer) cells was significantly higher in group II. Throughout the luteal phase, infertile women had fewer CD56+ cells than normal fertile controls. The volume fraction of endometrium occupied by the nuclei of endometrial granulated lymphocytes did not alter with the cycle stage but the mean nuclear diameter and axial ratio decreased from LH+7 to LH+13. The differences observed in endometrial leukocytic subpopulations between fertile and infertile women may contribute to unexplained infertility probably by affecting the embryonic maternal dialogue during the implantation and early placentation period. [ABSTRACT FROM AUTHOR]

A retrospective study was designed to examine the relationship between luteinizing hormone (LH) concentrations in the follicular phase and endometrial development in the luteal phase of natural and artificial cycles. Two types of cycle were studied: natural cycles ( = 51) in subjects with unexplained infertility were divided into two subgroups, depending on whether LH measurements in the late follicular phase were based on urine ( = 24) or plasma ( = 27) samples; and artificial cycles ( = 17), produced by the administration of a standard hormone replacement therapy, in two subgroups of women, those with premature ovarian failure ( = 10) in whom plasma LH concentrations were high, and those with unexplained infertility ( = 7) who had their hypothalamic pituitary — ovarian axis down-regulated and in whom plasma LH concentrations were low. The correlation between plasma or urine concentrations of LH in the follicular phase and the results of endometrial biopsy obtained in the luteal phase was calculated. In natural cycles, LH concentrations were similar in those with normal or retarded endometrium, and there was no significant correlation between high LH concentration and retarded endometrial development. In artificial cycles, endometrial development was not different between those with low LH concentrations (down-regulated by Zoladex) and those with high LH concentrations (premature ovarian failure). Endometrial development in the peri-implantation period does not appear to be influenced by LH concentration in the follicular phase. The reported association between high LH concentration and poor reproductive performance cannot therefore be explained by abnormal implantation consequent upon retarded endometrial development. [ABSTRACT FROM PUBLISHER]

In the present study attempts have been made to express in quantitative terms the fine structure of the endometrial glandular epithelium in well-timed biopsies from fertile women during the early luteal phase. Biopsies from women with unexplained infertility have also been examined. Certain abnormalities of glandular maturation were found in these women. The nuclear channel system was not present at day luteinizing hormone (LH) + 4 in the infertile group while at LH + 6 there were more cells containing this feature than seen in the fertile group. There also seem to be abnormalities associated with intracellular deposits of glycogen-rich material and mitochondrial elaboration in the infertile women. Changes in the secretory apparatus and rough endoplasmic reticulum were observed. The evidence from the present study suggests that the endometria in the examined group of infertile women is responsive to steroidal stimulation. This response however, may be delayed or inadequate. [ABSTRACT FROM AUTHOR]

To examine the value of the measurement of oestradiol/progesterone ratios in the peri-implantation period, three different types of cycle were studied; natural cycles (n = 13), cycles treated with anti-oestrogens (n = 19), and artificial cycles (n = 41) produced by six different hormone replacement regimens. Normal endometrial development was found to occur across a wide range of oestradiol/progesterone ratios. There was no significant correlation between the oestradiol/progesterone ratios and results of endometrial dating. The results suggest that oestradiol/progesterone ratios are of limited value in predicting endometrial development at the time of implantation. [ABSTRACT FROM AUTHOR]

The complications of a consecutive series of 90 uterine washings performed among 52 women were studied. The amount of discomfort was significantly less than that of endometrial biopsy but greater than that of cervical smear or blood sampling. Incomplete recovery of fluid (< 8 ml from 10 ml flushing fluid) was reported in only 3% of cases. Vaginal bleeding, pelvic infection and delayed abdominal pain were not encountered. The techniques of uterine washing are simple and may have important implications in the study of endometrial function. [ABSTRACT FROM AUTHOR]

In the light and electron microscopical study reported here, we document the structure of the nucleus, nucleolus and nuclear channel system (NCS) in the uterine glandular epithelium in both fertile and infertile women during the early luteal phase. Nuclear volume was found to be larger in the infertile group at day 5 after the luteinizing hormone surge (LH + 5) compared to day-matched fertile subjects. A two-way analysis of variance performed on nucleolar volume data from fertile and infertile women biopsied on days LH + 4, +5 and +6 revealed a significant effect of condition but no effect of day or interaction. Nucleolar volume decreased from day LH + 2 to day LH + 6 in the fertile group, the sharpest decrease occurring between day LH + 3 and day LH + 4. The largest mean volume of the NCS was found at day LH + 5 in the fertile group and day LH + 6 in the infertile group. The results suggest a delay in the development of this organelle in the infertile women. The present study has documented alterations in the nuclei of uterine glandular cells from infertile patients. In these infertile women, there is also a delayed elaboration of the secretory apparatus and this delay correlates well with the delayed/reduced expression of a luteal-specific glyco-protein. [ABSTRACT FROM PUBLISHER]

Nuclear factor kappa B (NFκB) is a family of transcription factors involved in signalling between IL1 and TNFα 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, IκBα and IκBβ NFκB 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 IκBα and IκBβ 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 IκB 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 NFκB components by the endometrium at the time of implantation. If confirmed, it would suggest that NFκB is involved in the control of factors important in the implantation process. [Copyright &y& Elsevier]