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Menopausal Hormone Therapy and Irregular Endometrial Bleeding: A Potential Role for Uterine Natural Killer Cells?

School of Women’s and Infants’ Health (M.H., J.C., D.D.) and Department of Clinical Immunology and Biochemical Genetics (J.P.G.), Royal Perth Hospital and School of Surgery and Pathology, University of Western Australia, Perth 6008, Western Australia; Department of Clinical Immunology and Biochemical Genetics (C.S.W., F.T.C.), Royal Perth Hospital, Perth 6847, Western Australia; Sydney Centre for Reproductive Health Research (I.S.F.), Family Planning Association Health and Department of Obstetrics and Gynaecology, University of Sydney, Sydney 2006, Australia; and Prince Henry’s Institute of Medical Research (L.A.S.), Melbourne, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Martha Hickey, Associate Professor, School of Women’s and Infants’ Health, King Edward Memorial Hospital, Subiaco, Perth 6008, Western Australia. E-mail: mhickey{at}obsgyn.uwa.edu.au.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: Irregular bleeding affects many users of combined menopausal hormone therapy (HT) and commonly leads to invasive and expensive investigations to exclude underlying malignancy. In most cases no abnormality is found.

Objective: The main objective of this study was to explore the role of uterine natural killer (uNK) cells and their regulatory cytokine IL-15 in irregular bleeding in HT users.

Design: This was a prospective observational study conducted between 2002 and 2004.

Setting: The study was conducted in a tertiary referral menopause clinic at King Edward Memorial Hospital, Western Australia.

Patients: Patients included 117 postmenopausal women taking combined HT.

Interventions: Outpatient endometrial biopsies were taken during and outside bleeding episodes.

Main Outcome Measures: The relationship between endometrial uNK cells (CD56+) and bleeding patterns was measured. We also addressed the impact of HT exposure on uNK cell populations, the relationship between endometrial IL-15 expression and uNK cell populations, and killer Ig like receptor genotype in subjects with irregular bleeding.

Results: Endometrial CD56+ uNK cells were significantly increased in biopsies obtained during bleeding episodes (P < 0.001), compared with HT users with no bleeding. The highest level of IL-15 expression was also seen in biopsies taken during bleeding. No clear relationship between killer Ig like receptor genotype and bleeding on HT was observed.

Conclusions: Little is known about the mechanisms underlying irregular bleeding in HT users. This is the first report of uNK cells and their association with regulating cytokines in postmenopausal endometrium and demonstrates a possible mechanism by which HT may induce irregular bleeding.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
HORMONE THERAPY (HT) is used by peri- and postmenopausal women for the relief of menopausal symptoms. Estrogen provides the therapeutic benefit, but progestogen is also required for those women who retain their uterus to prevent endometrial hyperplasia and adenocarcinoma (1). Up to 60% of HT users suffer from unpredictable, unscheduled vaginal bleeding and spotting (2), which leads to discontinuation of therapy in up to one in three users (3). Irregular bleeding was the reason for 40% unblinding in the recent Women’s Health Initiative study (4). Irregular bleeding tends to settle over the first year of use, but because many national guidelines now advise restricting use of HT to less than 5 yr, irregular bleeding is likely to continue to be a clinical problem.

The management of HT-associated bleeding problems is often unsatisfactory because there are no established methods of regulating or reducing bleeding. None of the HT preparations currently available can guarantee either regular bleeding or amenorrhea, suggesting that common mechanisms underlie these phenomena, independent of the precise quantities and timings of hormone provision (5). Unscheduled bleeding is common with both cyclic and continuous combined HT (6). Erratic bleeding in peri- or postmenopausal women may be a presenting symptom of pelvic malignancy; therefore, current management protocols often include invasive and costly investigations. More than 30% of cyclic HT users and nearly half of all continuous combined HT users make at least one visit to their gynecologist with irregular bleeding (6). In the majority of cases, no pathology is found (7).

Relatively few studies have addressed the possible mechanisms of HT-induced bleeding, and the underlying mechanisms are poorly understood. Bleeding patterns do not correlate well with endometrial histology or the type or dose of HT used (8). Individuals vary widely in their response to the same HT, and it is not known whether this variation reflects endometrial, systemic, or other factors.

Normal menstrual bleeding shows many similarities to inflammation, with a tightly regulated influx of leukocytes and a significant increase in the number of uterine natural killer cells (uNK), preceding endometrial breakdown and bleeding (9). uNK cells are a subset of the leukocyte population displaying a unique phenotype (CD56^bright, CD16^dim, CD3^dim), which distinguishes them from peripheral blood NK cells (CD56^dim, CD16^bright, CD3^–). In normal cycling endometrium, the presence and distribution of uNK cells is tightly regulated by ovarian steroids (10). They are minimal during the early proliferative phase but increase toward midcycle, reaching a maximum in early pregnancy, at which time uNK cells represent up to 70% (11) of the total stromal cell population and are in close contact with the endometrial vasculature (12). Their function in the nonpregnant endometrium is not fully understood, but they are thought to be regulated indirectly by progesterone and to play a key regulatory role in controlling whether endometrial shedding occurs or preparation for implantation and pregnancy (10, 11). uNK cells are thought to proliferate in utero (13, 14) under the influence of chemokines. IL-15 is a cytokine known to regulate uNK cell differentiation and is up-regulated in the endometrial epithelium, perivascular endothelial cells, and decidua of early pregnancy when uNK cell populations are increased (15, 16). In vitro studies have demonstrated that uNK cells will proliferate in response to stromal cell contact and stromal cell-derived IL-15 (17), and IL-15 may be the principal paracrine mediator of uNK cell proliferation, differentiation, and maturation.

uNK cells also express high levels of killer Ig like receptors (KIR). The genes that give rise to KIRs are classified by the number of extracellular Ig-like domains they encode (two or three) and the presence of long or short cytoplasmic domains. Various KIRs recognize specific HLA-C allotypes and HLA-Bw4. In general, long-tailed KIRs are considered to mediate inhibitory signals, whereas short-tailed KIRs mediate activating signals. These receptors play an important role in the regulation of natural killer (NK) cell function and are thought to influence uNK cell function (18). The KIR genes are highly polymorphic in the population in both terms of gene content and the alleles present. Such polymorphism has been shown to influence susceptibility to several disease (19) and in an increased predisposition to preeclampsia (18). In addition, an increase in the frequency of NK cells expressing KIR2DL1 has been reported in endometriosis (20). However, no studies have examined their importance in endometrial bleeding. We have therefore investigated whether any KIR genotype is associated with high uNK cell densities or irregular bleeding patterns.

The role of uNK cells in HT-induced irregular bleeding has not previously been explored in postmenopausal endometrium. The aim of this study was to investigate the role and regulation of uNK cells in postmenopausal HT users and hence to further understand the endometrial mechanisms that may lead to irregular bleeding.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
One hundred seventeen postmenopausal HT users were recruited from the menopause clinic at King Edward Memorial Hospital (Perth, Western Australia). A detailed medical history was obtained at recruitment, including bleeding patterns and current and previous HT use. Bleeding diaries were completed over a 90-d reference period. The 90-d reference period is a standard methodology for describing bleeding patterns in users of long-acting hormonal contraceptives in which bleeding patterns may be erratic (21) and was hence chosen as a suitable method for describing bleeding patterns in HT users. The study was restricted to those using oral and transdermal HT (Table 1). In those subjects using cyclic HT, all biopsies were collected during the estrogen and progestogen phase of treatment. Subjects were classified according to their bleeding patterns as having either amenorrhea or irregular bleeding. Subjects with irregular bleeding were asked to contact the research nurse during bleeding episodes so that paired biopsies could be obtained both during and outside bleeding episodes. Irregular bleeding in HT users was investigated according to standard hospital protocols. In brief, a Pap smear was obtained and transvaginal pelvic ultrasound arranged. If the endometrial thickness was less than 6 mm, an endometrial biopsy was obtained to assess histology. If the endometrium was greater than 6 mm in thickness, hysteroscopy and endometrial biopsy were performed. Most biopsies were obtained from women with an endometrial thickness less than 6 mm.

Biopsies were fixed immediately in 10% formalin for 18 h, and tissue was then embedded in paraffin blocks using a standard dehydration protocol. Sections were cut at 5 µm on an RM2135 microtome (Leica, Heidelberg, Germany) onto SuperFrostPlus glass slides (Menzel-Glaser, Strasburg, Germany). The sections were dried at 37 C overnight. All biopsies had a standard hematoxylin and eosin section submitted to an experienced histopathologist for classification according to criteria of Noyes et al. (22) and identification of any pathological features.

Immunohistochemical labeling

Five-micrometer sections were rehydrated through three changes of Histoclear (National Diagnostics, Atlanta, GA) and descending concentrations of ethanol to distilled water followed by 0.01 M PBS (pH 7.4). Antigen retrieval was carried out by microwaving the sections in 0.01 M trisodium citrate buffer (pH 6.0) at a temperature greater than 100 C for 10 min. Endogenous peroxidase was blocked by immersing the sections in 3% hydrogen peroxide in PBS for 10 min. After thorough rinsing in three changes of PBS, the slides were incubated in a blocking solution containing 1% nonspecific horse serum (Vector Laboratories, Burlingame, CA), 1% BSA (Sigma, St. Louis, MO), and 0.01% Tween 20 (Sigma). After PBS rinsing, the tissue sections were incubated overnight at 4 C in primary antibody at the following concentrations: CD56 at 10 µg/ml (Zymed Laboratories Inc., South San Francisco, CA) and IL-15 at 2.5 µg/ml (R&D Systems, Minneapolis, MN).

Primary antibody binding was localized using biotinylated horse antimouse IgG (Vector Laboratories) followed by Vector ABC reagent (avidin-biotin-peroxidase) as prescribed by the manufacturers. Visualization was achieved using 5 mg 3,3-diaminobenzidine (DAB; ICN Biomedicals Inc., Aurora, OH) in water 1:2 (wt/vol) with 0.03% hydrogen peroxide for 5 min at room temperature to give a brown signal. Sections were counterstained in Gill’s hematoxylin and dehydrated through graded alcohols and Histoclear to DPX (ProSciTech, Thuringowa, Queensland, Australia). Negative controls were included in each run substituting the primary antibody for an iso-type nonspecific Ig at the same concentration as the primary antibody.

uNK cell counts were obtained by photographing each immunolabeled biopsy section using an Olympus BX51 light microscope fitted with a SPOT RT Slider digital camera (Diagnostic Instruments, SciTech, Preston, Victoria, Australia). Images were obtained from five separate fields at x400 magnification wherever possible. In the few cases in which this was not possible, this was due to small sample size (8). Areas were selected for cell counting from fields with maximum labeled cell density and at least 90% stroma (maximum < 10% gland or epithelium present). All images were analyzed for specific labeled cells using the image analysis program Image-Pro Plus (Media Cybernetics Inc., Silver Spring, MD). Individual labeled cells were counted within each measured field (200 x 250 µm, 0.05 mm²) using a grid overlay on each image. Stromal cell density was also recorded using the same images by counting all hematoxylin stained nuclei in five separate fields (50 x 50 µm), including those nuclei that crossed two sides only of each selected area, as specified for stereologic counting. Mean stromal cell density per square millimeter was calculated.

IL-15 was evaluated by scoring each immunolabeled biopsy section in a blinded, anonymous method and repeated the following day, using the following system as a guide: 0, nil-labeled cells; 1, occasional positive-labeled cells present; 2, moderate number of positive cells present; and 3, high numbers of labeled cells present.

KIR genotyping

Venous blood samples (10 ml) were collected from participating women, and the buffy coat used for the extraction of cDNA. The KIR gene repertoire for this cohort was determined by sequence-specific priming (23) with the following changes; positive control primers for human GH were included in all reaction tubes. The primers for KIR3DL1 were those described by Hsu et al. (24). The primers for KIR2DL5 were the forward primers for KIR2DL5.1 (24), and the reverse primer (GGG GTC ACA GGG CCC ATG AT) detected only the transcribed alleles of KIR2DL5. KIR2DS4 alleles 001 and 002 were detected using the primers of Hsu et al. (24), and the KIR2DS4*003 allele, which has a 22-bp deletion and does not code for a membrane receptor, was detected separately (24). The Uhrberg forward primer for KIR2DL2 was replaced with the Fa538 primer (25) to detect all KIR2DL2 alleles. KIR2DL1 alleles *001, 002, 003, 005, and KIR2DL1*004 were detected separately (25). KIR2DS5 was detected using the Uhrberg forward primer (23) and the reverse primer described by Norman et al. (26). Genotyping of the KIR2DL4 transmembrane region was performed by single-stranded conformational polymorphism as previously described (27). Population controls for the KIR repertoire frequencies of each KIR gene were derived from 69 subjects randomly selected from the Busselton Population survey (http://bsn.uwa.edu.au/). For the KIR2DL4 transmembrane genotype, population frequencies were derived from 46 blood donors.

Statistical analysis of CD56⁺ (uNK) cell counts

CD56-positive cell data were expressed as mean cell count per square millimeter. Descriptive statistics used medians and ranges to describe the data. Analysis of group differences was performed on log-transformed cell counts due to heterogeneity of distribution in raw counts. Repeated ANOVA was used to compare groups according to their HT use and bleeding patterns. The categories were: no HT, HT and no bleeding in the last 90 d, HT and bleeding in the last 90 d, and HT plus bleeding during the biopsy as a fixed effect and individual patients as random effects. All biopsies per woman were used in the analysis. They were suitably weighted so that under each HT/bleeding condition, the data for each woman had an equal contribution to the overall mean effect. The cell counts were summarized using estimated means and 95% confidence intervals.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
One hundred seventeen women were recruited, and endometrial biopsies of adequate quality for analysis were obtained from 56 (52%) of these women. Of the 56 subjects with usable biopsies, 46 were using continuous combined HT (82%) and 10 were using cyclic HT (18%) with progestogens for 12 d of every 28. All biopsies from sequential HT users were obtained during the combined estrogen and progestogen phase. Additional biopsies were obtained from eight comparison subjects before commencing HT, of which five subsequently contributed post-HT biopsies of adequate quality for longitudinal study analysis.

Single biopsies were obtained from 36 women and on more than one occasion from 20 subjects (13 women gave two biopsies and seven women gave three biopsies), generating a total of 83 usable biopsies. In 10 cases, paired biopsies were obtained during and outside a bleeding episode in subjects with irregular bleeding. The median age of subjects was 53 yr (range 41–64 yr) and the median duration of HT use was 34 months.

As anticipated, subjects were using a wide range of HT products (Table 1). All subjects in the longitudinal study were commenced on the same oral HT preparation (Kliovance, Novo Nordisk, New South Wales, Australia, containing 1 mg estradiol and 0.5 mg norethisterone acetate).

Investigation of irregular bleeding

Pap smears were performed if a normal Pap smear was not available for the previous 12 months. All results were negative. All subjects with irregular bleeding were investigated with transvaginal ultrasound and hysteroscopy, if ultrasound thickness of the endometrium exceeded 6 mm (15 subjects). In eight cases hysteroscopy was normal, but a further seven subjects were excluded because of endometrial pathology that may have contributed to the bleeding (polyps or pedunculated submucous fibroids). No endometrial cancers were identified.

Endometrial histology

Tissue was classified as atrophic or weakly proliferative in more than half of all the biopsies. Occasional biopsies showed signs of decidualization and secretory type morphology. There was no relationship between the histological appearance and bleeding patterns or to the type of estrogen or progestogen in the HT used. No differences were observed between cyclic and continuous combined HT users in endometrial histological appearance.

uNK cells

uNK cells were identified by the surface-expressed CD56 protein. This was visualized as a surface ring of antibody-specific labeling, around a small circular cytoplasm approximately 8–10 µm in diameter containing a relatively large nucleus approximately 5–6 µm in diameter (Fig. 1E). uNK cells are differentiated from circulating NK cells by virtue of the fact that they strongly express this CD56 surface protein, whereas it is only very weakly expressed in the circulating NK cells. Endometrial stromal NK cells were observed in all the biopsies examined, although their number and distribution varied markedly between subjects dependent on bleeding patterns (Fig. 1, A–D). No distinct localization of uNK cells around either blood vessels or glands was observed, although they were sometimes seen in clusters below the surface epithelium.

View larger version (149K): [in this window] [in a new window]   FIG. 1. Representative fields used for counting immunohistochemical labeled CD56+ uNK cells (DAB, brown label; counterstained with hematoxylin, blue) in endometrial biopsies collected from no HT and no bleeding in the last 3 months (A), amenorrhea after more than 3 months HT (B), after more than 3 months HT and irregular bleeding episodes (C), after more than 3 months HT and collected during a bleeding episode (D) (uNK cell density, 4480 cells/mm2). E, Immunolabeled CD56+ cell x1000. epi, Epithelium; bv, blood vessel; gl, gland. Magnification, x400. Scale bar, 50 µm, A–D; 20 µg; E.

View larger version (11K): [in this window] [in a new window]   FIG. 2. Scatter graph of CD56+ cell density/mm2 within groups. Group 1, before HT, no bleeding for more than 9 months; group 2, HT amenorrhea; group 3, HT with irregular bleeding; group 4, HT with irregular bleeding and bleeding at time of biopsy.

Of the five women who contributed a biopsy before commencement of HT and again 3 months after HT, three developed irregular bleeding and two gave biopsies during a bleeding episode, one between bleeding episodes. Two women did not develop irregular bleeding after 3 months HT. Figure 3A shows that four of these five women commencing HT had an increase in mean uNK cell density from less than 100/mm² before commencing HT, rising to 180–660/mm² at the time of repeat biopsy.

View larger version (17K): [in this window] [in a new window]   FIG. 3. A, Comparing uNK cell densities in paired biopsies before and after more than 3 months HT (n = 5). B, Paired biopsies comparing uNK cell densities between bleeding episodes and during bleeding episodes (n = 10).

Immunolabeling for IL-15

Immunolabeling of adjacent serial 5-µm sections demonstrated the presence of IL-15 in the cytoplasm of both endometrial glandular and surface epithelium as well as in occasional stromal cells in all biopsies (Fig. 4). The greatest expression of IL-15 was seen in the superficial endometrial stromal cells and particularly in the surface epithelium (Fig. 4). IL-15 was widely expressed in the stromal cells of decidualized endometrium (Fig. 4B), although less obviously in the epithelium and not by uNK cells (Fig. 4, A and B, adjacent sections). Endometrial expression of IL-15 was related to both uNK cell numbers and bleeding patterns in HT users. In subjects not using HT, moderate to strong immunoreactive IL-15 was seen in only 25% of biopsies, whereas in HT users IL-15 staining was moderate to strongly expressed in 46% of biopsies. The greatest IL-15 expression was in subjects with irregular bleeding in which IL-15 was expressed in more than 50% of biopsies (Fig. 5).

View larger version (154K): [in this window] [in a new window]   FIG. 4. Representative fields of IL-15 immunolabeled biopsies from different groups (DAB, brown label; counterstained with hematoxylin, blue). A and B, From a decidualized HT user collected during a bleeding episode, labeled for CD56 uNK cells localized to blood vessels (A) and adjacent section to A, labeled for IL-15 showing strong expression in decidualized stromal cells (B, grade 2.5). C, From a HT user with amenorrhea grade less than 1. D, From a HT user collected between bleeding episodes and adjacent section to Fig. 1C, grade 1. E, From a HT user during a bleeding episode and adjacent section to Fig. 1D, grade 2. epi, Epithelium; bv, blood vessel; gl, gland. Magnifications, x400. Scale bar, 50 µm.

View larger version (8K): [in this window] [in a new window]   FIG. 5. The percentage of biopsies in bleeding and nonbleeding groups expressing IL-15 at moderate to high levels (grade > 1–3). Group 1, Nil HT, nil bleed; group 2, HT, nil bleed; groups 3 and 4, HT with irregular bleed; and group 4, also bleeding at the time of biopsy.

The frequency of each of the KIR genes is shown in Table 2. There were no significant differences in the frequency of each KIR gene between normal controls and HT users and no relationship with bleeding patterns. There were also no significant differences in the total number of KIR or the number of activating or inhibitory KIRs.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

uNK cell proliferation in premenopausal endometrium is thought to be regulated indirectly by progesterone (15). Most subjects in this study were using continuous combined HT, which has a predominantly progestogenic endometrial effect (28), potentially mimicking the secretory phase of the normal menstrual cycle preceding endometrial breakdown. However, unlike the normal menstrual cycle, the tightly regulated sequential pattern of ovarian steroid production is absent. The mechanism of progestogen regulation of uNK cells is not fully understood. uNKs do not express progesterone receptor, only estrogen receptor-ß1 receptors (29), suggesting nongenomic effects or paracrine influences. The positive association between stromal uNK cell density and irregular bleeding suggests that these activated leukocytes may be contributing to endometrial breakdown. Vascular breakdown in normal menstrual cycles is preceded by an inflammatory cascade of leukocytes producing cytokines and proteases capable of breaking down the extracellular matrix and initiating bleeding (30). In premenopausal women with breakthrough bleeding using progestogen-only contraception, dysregulation of leukocytes associated with increased endometrial protease expression is seen and may account for this abnormal bleeding (31).

Leukocyte migration into the endometrium is mediated by chemokines that act locally and in concert (32). Stimulation of uNK cells by endometrial IL-15 results in production of the cytokines interferon- and IL-10, a process that can be blocked by TGFß1 (33). Interferon- can stimulate cell proliferation and is known to contribute to vascular remodeling (34). Previous studies by the authors have suggested that HT may induce vascular remodeling and abnormal angiogenesis (8). Our observation that IL-15 as well as uNK cells are associated with irregular bleeding suggests a regulatory pathway for vascular breakdown in HT users. uNK cells also express mRNA for vascular endothelial growth factor-C and angiopoietin-2, factors known to play a critical role in endometrial angiogenesis and shown to be perturbed in irregular bleeding associated with long-acting progestogen-only contraception (35, 36). Recently it has been shown that mice lacking uNK cells show deficient endometrial vascular modeling and decidualization (14, 37). uNK cells possess a varying number of membrane-bound cytoplasmic granules, which contain cytolytic molecules such as granzyme and perforin and a variety of matrix metalloproteinases; all of these may augment endometrial breakdown (11). Hence, uNK cells may act via a variety of local mechanisms to alter the integrity and function of endometrial blood vessels, leading to irregular bleeding.

KIR genotype is known to affect uterine vascular function (18) perhaps through NK-matched modification of uterine blood vessels. In this study we postulated that KIR genotype may be a useful marker of the vascular response to hormonal therapy. Although there were no significant associations between irregular bleeding and KIR genotype, the number of samples in each patient group was relatively small, and a small effect of KIR genotype cannot be excluded. For instance, KIR2DS2 and KIR2DL2, which are in strong linkage disequilibrium, tended to be more common among HT users with irregular bleeding than population controls and were less common among HT users with amenorrhea than population controls. Larger study groups may show a significant difference, but it is unlikely that such small differences in frequencies would be of prognostic utility.

Subjects in this study were exposed to a wide range of HT preparations, and this may have influenced the endometrial cellular response. However, because irregular bleeding is common to all combined HT preparations, it is also reasonable to propose that common mechanisms may underlie this response. A large number of endometrial biopsies were too small to be analyzed, particularly in the HT users with no bleeding, and was probably due to the presence of atrophic endometrium. This may have introduced a selection bias into our studies. In premenopausal progestogen users, subjects with absent or insufficient tissue from outpatient endometrial biopsy differed in circulating sex steroid levels from those in whom adequate tissue was obtained (38). However, one reason so few studies have addressed the mechanisms of irregular bleeding in HT users is that endometrial tissue from these subjects is extremely difficult to obtain. Relatively few HT users undergo hysterectomy in our busy unit, and we rely on outpatient biopsies for assessment. Obtaining substantial numbers of these from older women, particularly during bleeding episodes, is challenging.

In summary, we have demonstrated that activated uNK cells and IL-15 are present in postmenopausal endometrium and that their numbers relate to bleeding patterns in HT users. It is therefore likely that these uNK cells are activated by the increased IL-15 in their local microenvironment within the postmenopausal endometrium of HT users, providing a mechanism by which HT commonly induces irregular bleeding.

	Footnotes

 
This work was supported by the National Health and Medical Research Council (NHMRC) of Australia. J.C. and L.A.S. are supported by the NHMRC of Australia (Fellowship Grants 254645 and 143798).

First Published Online July 26, 2005

Abbreviations: DAB, 3,3-Diaminobenzidine; HT, hormone therapy; KIR, killer Ig like receptor; NK, natural killer; uNK, uterine NK.

Received March 30, 2005.

Accepted July 20, 2005.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Beresford SA, Weiss NS, Voigt LF, McKnight B 1997 Risk of endometrial cancer in relation to use of oestrogen combined with cyclic progestogen therapy in postmenopausal women. Lancet 349:458–461[CrossRef][Medline]
2. al-Azzawi F, Habiba M 1994 Regular bleeding on hormone replacement therapy: a myth? Br J Obstet Gynaecol 101:661–662[Medline]
3. Limouzin-Lamothe MA 1996 What women want from hormone replacement therapy: results of an international survey. Eur J Obstet Gynecol Reprod Biol. 64:S21–S24
4. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J 2002 Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 288:321–333[Abstract/Free Full Text]
5. Thomas AM, Hickey M, Fraser IS 2000 Disturbances of endometrial bleeding with hormone replacement therapy. Hum Reprod 15:7–17
6. Ettinger B, Li DK, Klein R 1998 Unexpected vaginal bleeding and associated gynecologic care in postmenopausal women using hormone replacement therapy: comparison of cyclic versus continuous combined schedules. Fertil Steril 69:865–869[CrossRef][Medline]
7. Elliott J, Connor ME, Lashen H 2003 The value of outpatient hysteroscopy in diagnosing endometrial pathology in postmenopausal women with and without hormone replacement therapy. Acta Obstet Gynecol Scand 82:1112–1119[CrossRef][Medline]
8. Hickey M, Pillai G, Higham JM, Sullivan M, Horncastle D, Doherty D, Stamp G 2003 Changes in endometrial blood vessels in the endometrium of women with hormone replacement therapy-related irregular bleeding. Hum Reprod 18:1100–1106[Abstract/Free Full Text]
9. Salamonsen LA, Lathbury LJ 2000 Endometrial leukocytes and menstruation. Hum Reprod Update 6:16–27[Abstract/Free Full Text]
10. Kelly RW, King AE, Critchley HO 2002 Inflammatory mediators and endometrial function—focus on the perivascular cell. J Reprod Immunol 57:81–93[CrossRef][Medline]
11. Trundley A, Moffett A 2004 Human uterine leukocytes and pregnancy. Tissue Antigens 63:1–12[CrossRef][Medline]
12. King A 2000 Uterine leukocytes and decidualization. Hum Reprod Update 6:28–36[Abstract/Free Full Text]
13. Jones RK, Bulmer JN, Searle RF 1998 Phenotypic and functional studies of leukocytes in human endometrium and endometriosis. Hum Reprod Update 4:702–709[Abstract/Free Full Text]
14. Croy BA, Esadeg S, Chantakru S, van den Heuvel M, Paffaro VA, He H, Black GP, Ashkar AA, Kiso Y, Zhang J 2003 Update on pathways regulating the activation of uterine natural killer cells, their interactions with decidual spiral arteries and homing of their precursors to the uterus. J Reprod Immunol 59:175–191[CrossRef][Medline]
15. Kitaya K, Yasuda J, Yagi I, Tada Y, Fushiki S, Honjo H 2000 IL-15 expression at human endometrium and decidua. Biol Reprod 63:683–687[Abstract/Free Full Text]
16. Dunn CL, Critchley HO, Kelly RW 2002 IL-15 regulation in human endometrial stromal cells. J Clin Endocrinol Metab 87:1898–1901[Abstract/Free Full Text]
17. Okada H, Nakajima T, Sanezumi M, Ikuta A, Yasuda K, Kanzaki H 2000 Progesterone enhances interleukin-15 production in human endometrial stromal cells in vitro. J Clin Endocrinol Metab 85:4765–4770[Abstract/Free Full Text]
18. Hiby SE, Walker JJ, O’Shaughnessy KM, Redman CW, Carrington M, Trowsdale J, Moffett A 2004 Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J Exp Med 200:957–965[Abstract/Free Full Text]
19. Rajagopalan S, Long EO 2005 Understanding how combinations of HLA and KIR genes influence disease. J Exp Med 201:1025–1029[Abstract/Free Full Text]
20. Maeda N, Izumiya C, Oguri H, Kusume T, Yamamoto Y, Fukaya T 2002 Aberrant expression of intercellular adhesion molecule-1 and killer inhibitory receptors induces immune tolerance in women with pelvic endometriosis. Fertil Steril 77:679–683[CrossRef][Medline]
21. Belsey EM 1991 Menstrual bleeding patterns in untreated women and with long-acting methods of contraception. Task Force on Long-Acting Systemic Agents for Fertility Regulation. Adv Contracept 7:257–270[CrossRef][Medline]
22. Noyes RW, Hertig AT, Rock J 1975 Dating the endometrial biopsy. Am J Obstet Gynecol 122:262–263[Medline]
23. Uhrberg M, Valiante NM, Shum BP, Shilling HG, Lienert-Weidenbach K, Corliss B, Tyan D, Lanier LL, Parham P 1997 Human diversity in killer cell inhibitory receptor genes. Immunity 7:753–763[CrossRef][Medline]
24. Hsu KC, Liu XR, Selvakumar A, Mickelson E, O’Reilly RJ, Dupont B 2002 Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes, each with multiple subsets. J Immunol 169:5118–5129[Abstract/Free Full Text]
25. Gomez-Lozano N, Vilches C 2002 Genotyping of human killer-cell immunoglobulin-like receptor genes by polymerase chain reaction with sequence-specific primers: an update. Tissue Antigens 59:184–193[CrossRef][Medline]
26. Norman PJ, Stephens HA, Verity DH, Chandanayingyong D, Vaughan RW 2001 Distribution of natural killer cell immunoglobulin-like receptor sequences in three ethnic groups. Immunogenetics 52:195–205[CrossRef][Medline]
27. Witt CS, Whiteway JM, Warren HS, Barden A, Rogers M, Martin A, Beilin L, Christiansen FT 2002 Alleles of the KIR2DL4 receptor and their lack of association with pre-eclampsia. Eur J Immunol 32:18–29[CrossRef][Medline]
28. Wells M, Sturdee DW, Barlow DH., Ulrich LG, O’Brien K, Campbell MJ, Vessey MP, Bragg AJ 2002 Effect on endometrium of long term treatment with continuous combined estrogen/progestogen replacement therapy; follow-up study. BMJ 325:239–242[Abstract/Free Full Text]
29. Henderson TA, Saunders PT, Moffett-King A, Groome NP, Critchley HO 2003 Steroid receptor expression in uterine natural killer cells. J Clin Endocrinol Metab 88:440–449[Abstract/Free Full Text]
30. Salamonsen LA, Zhang J, Brasted M 2002 Leukocyte networks and human endometrial remodelling. J Reprod Immunol 57:95–108[CrossRef][Medline]
31. Vincent AJ, Salamonsen LA 2000 The role of matrix metalloproteinases and leukocytes in abnormal uterine bleeding associated with progestin-only contraceptives. Hum Reprod. 15(Suppl 3):135–143
32. Jones RL, Hannan NJ, Kaitu’u TJ, Zhang J, Salamonsen LA 2004 Identification of chemokines important for leukocyte recruitment to the human endometrium at the times of embryo implantation and menstruation. J Clin Endocrinol Metab 89:6155–6167[Abstract/Free Full Text]
33. Eriksson M, Meadows SK, Wira CR, Sentman CL 2004 Unique phenotype of human uterine NK cells and their regulation by endogenous TGF-ß. J Leukoc Biol 76:667–675[Abstract/Free Full Text]
34. Ashkar AA, Di Santo JP, Croy BA 2000 Interferon contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. J Exp Med 192:259–270[Abstract/Free Full Text]
35. Charnock-Jones DS, Macpherson AM, Archer DF, Archer DF, Leslie S, Makkink WK, Sharkey AM, Smith SK 2000 The effect of progestins on vascular endothelial growth factor, oestrogen receptor and progesterone receptor immunoreactivity and endothelial cell density in human endometrium. Hum Reprod. 15(Suppl 3):85–95
36. Krikun G, Critchley H, Schatz F, Wan L, Caze R, Baergen RN, Lockwood CJ 2002 Abnormal uterine bleeding during progestin-only contraception may result from free radical-induced alterations in angiopoietic expression. Am J Pathol 161:979–986[Abstract/Free Full Text]
37. Greenwood JD, Minhas K, di Santo JP, Makita M, Kiso Y, Croy BA 2000 Ultrastructural studies of implantation sites from mice deficient in uterine natural killer cells. Placenta 21:693–702[CrossRef][Medline]
38. Hadisaputra W, Affandi B, Witjaksono J, Rogers PA 1996 Endometrial biopsy collection from women receiving Norplant. Hum Reprod. 11(Suppl 2):31–34

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