The correlation of autoantibodies and uNK cells in women with reproductive failure.

There is conflicting evidence on the role of autoimmune disorders in reproductive failure, including recurrent miscarriage (RM) and recurrent implantation failure (RIF), after in vitro fertilisation (IVF). Several commonly studied autoimmune markers in women with reproductive failure include antiphospholipid antibodies (APAs), thyroid peroxidase antibodies (TPA) and uterine natural killer (uNK) cells. However, there have not been any studies that have examined the correlation of these markers in women with reproductive failure. To determine if women who tested positive for autoantibodies (APA and thyroid peroxidase antibodies) have significantly higher uNK cell numbers than women who tested negative for these antibodies, the percentage of stromal cells that stained positive for CD56 was identified by immunocytochemistry in endometrial biopsies from 42 women with unexplained RM (29 women tested negative for autoantibodies and 13 women tested positive for autoantibodies) and 40 women with unexplained RIF (30 women tested negative for autoantibodies and 10 women tested positive for autoantibodies). Biopsies were obtained on days LH+7 to LH+9. There was no significant difference in uNK cell numbers between women with unexplained RM who tested negative and those who tested positive for autoantibodies. Similarly, there was no significant difference in uNK cell numbers between women with unexplained RIF who tested negative and those who tested positive for autoantibodies. In women with reproductive failure the presence of autoantibodies does not appear to affect the numbers of uNK cells in the endometrium around the time of implantation.

Characterization of invasive trophoblasts generated from human embryonic stem cells.

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 hCGß 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.

A preliminary study comparing the endometrial expression of inhibin, activin and follistatin in women with a history of implantation failure after IVF treatment and a control group.

The aim of this study was to evaluate the expression of activin: beta A and beta B subunit and follistatin in endometrium of women with implantation failure (n = 10) and compare it with a fertile control group (n = 7). Immunohistochemical staining intensity for follistatin in the endometrial glandular epithelium from women with implantation failure were significantly lower than that in control women (P = 0.03). The decreased expression of follistatin in epithelial cells in the endometrium of women with implantation failure after in vitro fertilisation (IVF) may suggest that follistatin may play a role in the implantation process.

Does free androgen index predict subsequent pregnancy outcome in women with recurrent miscarriage?

BACKGROUND: Several studies have investigated plasma androgen levels in women with recurrent miscarriage (RM) with conflicting results on whether an association between hyperandrogenaemia and RM exists. However, none of these studies included sensitive androgen measurements using a large data set. We therefore investigated the free androgen index (FAI) in a large number of women with RM in order to ascertain whether hyperandrogenaemia is a predictor of subsequent pregnancy outcome. METHODS: We studied 571 women who attended the Recurrent Miscarriage Clinic in Sheffield and presented with > or =3 consecutive miscarriages. Serum levels of total testosterone and sex hormone-binding globulin were measured in the early follicular phase and FAI was then deduced. RESULTS: The prevalence of hyperandrogenaemia in RM was 11% and in a subsequent pregnancy, the miscarriage rate was significantly higher in the raised FAI group (miscarriage rates of 68% and 40% for FAI > 5 and FAI < or = 5 respectively, P = 0.002). CONCLUSIONS: An elevated FAI appears to be a prognostic factor for a subsequent miscarriage in women with RM and is a more significant predictor of subsequent miscarriage than an advanced maternal age (> or =40 years) or a high number (> or =6) of previous miscarriages in this study.

Prognostic value of the measurement of uterine natural killer cells in the endometrium of women with recurrent miscarriage.

BACKGROUND: Studies in mice suggest that CD56 + uterine natural killer (uNK) cells play an important role in implantation. Studies in humans have described an increase in the number of uNK cells in the non-pregnant mid-secretory endometrium of women with unexplained recurrent miscarriage (RM). However, the predictive value of uNK cell number in the maintenance of pregnancy is controversial. METHODS: A blind retrospective study was undertaken. The percentage of stromal cells positive for CD56 was identified by immunocytochemistry in endometrial biopsies from 10 normal control women and 87 women with unexplained RM, of whom 51 became pregnant following biopsy. Biopsies were obtained on days LH + 7 to LH + 9. RESULTS: As in previous studies, the number of uNK cells in the 87 women with RM (mean 11.2% range 1.1-41.4%) was significantly higher (P = 0.013) than in the control women (mean 6.2% range 2.2-13.9%). No significance difference in uNK numbers was observed between 19 women who miscarried (mean 9.6% range 1.7-25.0%) and 32 women who had a live birth (mean 13.3% range 1.1-41.4%) in a subsequent pregnancy. CONCLUSIONS: In this study numbers of uNK cells in the peri-implantation endometrium of women with unexplained recurrent miscarriage did not predict subsequent pregnancy outcome.

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.

Menstrual cycle-dependent changes of Toll-like receptors in endometrium.

BACKGROUND: Rapid innate immune defences against infection usually involve the recognition of invading pathogens by specific pattern recognition receptors recently attributed to the family of Toll-like receptors (TLRs). Reports from our laboratory and others have demonstrated the existence of TLRs 1-6 in the female reproductive tract. However, little has been done to identify TLRs 7-10 in the female reproductive tract, particularly in the uterus. Also little information exists regarding variation in TLRs in the female reproductive tract during the menstrual cycle. METHOD: The distribution of TLR7-10 protein was detected by immunostaining in timed endometrial biopsies from normal women. RT-PCR was used to show the existence of TLR1-10 genes in endometrial tissue and real-time PCR analysis to investigate the relative expression of these genes during the menstrual cycle in normal human endometrium. RESULTS: TLR7-10 proteins were detected in endometrial epithelium and stroma. TLR1-10 genes were expressed in human endometrial tissue, and the mean relative expression of TLR2-6, 9 and 10 genes was significantly higher during the secretory phase compared with other phases of the menstrual cycle. CONCLUSIONS: TLR7-10 localization is not limited to endometrial epithelium but is also present in the stroma of the endometrial tissue. Endometrial TLR2-6, 9 and 10 genes are cyclically expressed during the menstrual cycle.

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.

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.

Markers of endometrial function in women with unexplained recurrent pregnancy loss: a comparison between morphologically normal and retarded endometrium.

BACKGROUND: Endometrial defect, usually described as luteal phase defect (LPD), is associated with recurrent miscarriage. Recurrent miscarriage has also been associated with the abnormal expression of various molecules by endometrial cells. The aim of this study was to determine if any of these molecules or cells could be used to distinguish LPD from in-phase endometrium. METHODS: Immunocytochemistry was used to compare endometrial expression of CD45+, CD56+, CD3+ and CD4+ cells, leukaemia inhibitory factor, interleukin-6 and estrogen and progesterone receptors in precisely timed endometrial biopsies obtained between days LH+6 and LH+11 from recurrent miscarriage women with in-phase and retarded endometrium. RESULTS: In all samples there was a positive correlation between the number of CD45+ cells and LH day and a negative correlation between progesterone receptor and LIF expression and LH day. A significantly lower number (P<0.05) of CD56+ cells in peri-implantation endometrium and a decreased mid-cycle estrogen level (P<0.05) was seen in women with LPD compared to in-phase endometrium when single analysis was carried out. However, these differences were not significant after application of the Bonferroni correction for multiple analysis. CONCLUSIONS: The results are in line with previous associations observed between estrogen levels and LPD and suggest that the number of CD56+ cells is different in LPD and in-phase endometrium, although this could be due to delayed endometrial development in women with LPD. Interpretation must be cautious because these differences could have arisen by chance.

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.

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.

Recurrent miscarriage: aetiology, management and prognosis.

Recurrent miscarriage (RM) is a heterogeneous condition. A large number of studies has recently been published, yet many of them have conflicting conclusions. The various aetiological factors, management, prognostic features and outcomes of a subsequent pregnancy in women with RM are reviewed.

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 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.

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.

Use of human menopausal gonadotropins in the treatment of endometrial defects associated with recurrent miscarriage: preliminary report.

OBJECTIVE: To test the hypothesis that controlled ovarian stimulation by gonadotropins, which enhances estrogen priming, is of beneficial value in the treatment of endometrial defects associated with recurrent miscarriage. DESIGN: A retrospective, observational, nonrandomized study. SETTING: A regional recurrent miscarriage clinic in a teaching hospital. PATIENT(S): Twenty-one subjects with otherwise unexplained recurrent miscarriage who had retarded endometrial development in the mid-luteal phase. Endometrial biopsies were timed by the luteinizing hormone surge. INTERVENTION(S): Controlled ovarian stimulation using human menopausal gonadotropins and repeat endometrial biopsy in the treatment cycle in 13 subjects. MAIN OUTCOME MEASURE(S): Histological dating of endometrial biopsy in treatment cycles and miscarriage rate in treatment and nontreatment cycles. RESULT(S): Eleven (85%) of the 13 biopsies in the treatment cycle were found to be normal. The miscarriage rate in the treatment group, 2 of 13, was significantly lower than that in the nontreatment group (7/12) (chi2 5.0, P<.05). CONCLUSION(S): In this small series, preliminary experience suggests that controlled ovarian stimulation by human menopausal gonadotropins in the follicular phase is an effective treatment for luteal phase defect associated with recurrent pregnancy loss. There is now a case for a prospective, controlled study to confirm the value of such a treatment.

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.

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.

Endocrinological and endometrial factors in recurrent miscarriage.

OBJECTIVE: To investigate the endocrinological and endometrial factors in women with unexplained recurrent miscarriage DESIGN: Prospective, case study SETTING: Recurrent miscarriage clinic, Jessop Hospital for Women, Sheffield PARTICIPANTS: One hundred and forty-four women with unexplained recurrent (> or =3) miscarriages METHODS: A blood sample was obtained in early follicular phase (day 3-5) to measure follicle stimulating hormone, luteinising hormone, prolactin, androgens and thyroid function; daily blood/urine samples were obtained from mid-follicular phase to measure luteinising hormone until the luteinising hormone surge was identified; endometrial biopsy and a further blood sample for progesterone measurement were obtained in the mid-luteal phase. A transvaginal ultrasonography was performed to evaluate ovarian morphology. RESULTS: Hypersecretion of luteinising hormone or ultrasonographic features of polycystic ovarian disease was present in 8% and 7.8% of women, respectively. The free androgen index was elevated in 14.6% of subjects. In the mid-luteal phase, low progesterone level was found in 17.4% and delayed endometrial development was noted in 27.1% of women. Although women with recurrent miscarriage women and delayed endometrium had significantly lower progesterone levels than those with normal endometrial development, only 8/24 had mid-luteal progesterone levels below 30 nmol/L. Recurrent miscarriage was not associated with hyperprolactinaemia or abnormal thyroid function test. CONCLUSIONS: Endocrinological and endometrial abnormalities are present in about a quarter of women with unexplained recurrent miscarriage.