Endometrial Breakthrough Bleeding Research Articles

Endometrial expression of interleukine receptor antagonist (IL-1Ra) is altered during irregular bleeding in progestin-only contraceptive users and influenced by doxycycline therapy

Endometrial expression of interleukine receptor antagonist (IL-1Ra) is altered during irregular bleeding in progestin-only contraceptive users and influenced by doxycycline therapy

Endometrial expression of epithelial neutrophil-activating peptide-78 during the menstrual cycle or in progestin-only contraceptive users with breakthrough bleeding and the influence of doxycycline therapy

Endometrial breakthrough bleeding is characterized by an inflammatory reaction and increased production of proinflammatory mediators, one of which may be epithelial neutrophil-activating peptide-78 (ENA-78), a chemokine with neutrophil-activating properties. We therefore investigated the endometrial expression of ENA-78 in Norplant users as progestin-only contraceptive with various bleeding patterns (n=35) as compared with non-users with a normal menstrual cycle (n=55). The endometrial stromal cells (ESCs) were the major site of ENA-78 expression with the highest levels found during the secretory phase. The expression of ENA-78 was increased in Norplant users with irregular bleeding as compared with those with regular cycles and amenorrhoea. The levels of ENA-78 detected in uterine washes and sera after the use of Norplant for 3-6 months (n=25) increased compared with baseline (P < 0.05). These levels did not significantly change in Norplant users who received doxycycline (Dox) therapy (25 mg/twice daily for 6 months) when measured midway through or at the conclusion of study when compared with the baseline (n=25). Treatments with medroxyprogesterone acetate (MPA) and tumour necrosis factor-alpha (TNF-alpha) (25 ng/ml), but not 17beta-estradiol (E2) or E2 + MPA (10(-8) M), representing endometrium exposed to contraceptive and inflammatory conditions, respectively, increased the levels of ENA-78 production by ESCs, and this was reduced by co-treatments with Dox (25 microg/ml) (P < 0.05). The endometrial production of ENA-78 is altered in progestin-only contraceptive users experiencing breakthrough bleeding and is regulated by MPA and TNF-alpha in ESCs. Although Dox therapy did not alter uterine ENA-78 secretion, its suppression in ESCs suggests that Dox, acting site-specifically and through an anti-inflammatory mechanism, may influence the outcome of breakthrough bleeding in contraceptive users.

Endometrial histology, microvascular density and caliber, and matrix metalloproteinase-3 in users of the Nestorone®-releasing contraceptive implant with and without endometrial breakthrough bleeding

Objective This descriptive study evaluated endometrial histology, microvascular density and caliber, and quantification of matrix metalloproteinase (MMP-3) expression in long-term users of the Nestorone® (NES)-releasing implant who presented or not endometrial breakthrough bleeding (BTB). Methods Endometrial biopsies were obtained from 32 healthy women with unpredictable BTB. The quantitative analysis was performed only in 20 samples. Results The mean duration of use of the implant among the 14 women with BTB was 19.6±1.0 months, and the other six women had used the implant for 17.7±2.3 months (mean±S.E.M.). Histological analysis of the endometrial tissue showed a predominance of progestogenic pattern followed by atrophic and proliferative endometrium in both groups. Mucosal breakdown and glandular pseudostratification were observed in half of the cases. Endometrial vascular density was 73.1±10.0 and 57.5±24.1 vessels/mm 2, and maximum vessel diameter was 923.3±86.0 and 1038.0±404 μm (mean±S.E.M.) in the group with and without BTB, respectively, without significance, and the rate of cells expressing MMP-3×1000 counted stromal cells was 155.8±24.8 and 127.0±19.0 (mean±S.E.M.) in both groups, respectively, without significance. Conclusions This study provides information about some endometrial aspects of women using NES in contraceptive implants. In addition, the endometrium was similar during long-term use of NES-releasing contraceptive implants in women with and without endometrial bleeding.

The causes and management of endometrial breakthrough bleeding

The term breakthrough bleeding (BTB) is rather poorly defined, but essentially describes the symptom of vaginal bleeding occurring with scheduled periods of withdrawal bleeding, in the absence of pelvic pathology in women taking exogenous sex steroids, usually contraceptives or hormone-replacement therapy (HRT). It may also describe occasional bleeding in those who are predominantly experiencing amenorrhoea due to these preparations. Rather confusingly, the term is sometimes used to describe intermenstrual bleeding in women who are not taking sex steroids, when structural or other pathological causes are more likely. In the absence of such pathology intermenstrual bleeding in the normal menstrual cycle is relatively uncommon, suggesting that exogenous sex steroids can profoundly disrupt the tight regulation of endometrial vascular development, function and breakdown. Intermenstrual bleeding also occurs spontaneously in some women and it is possible that this phenomenon has similar mechanisms to that seen in sex-steroid-related breakthrough bleeding.

Steroids and endometrial breakthrough bleeding: future directions for research.

Abnormal endometrial bleeding continues to be a significant problem for women using long-acting gestagens and not only reduces the use of these agents, thus restricting the options available to women, but also reduces the quality of their lives. This symposium addressed a range of basic and clinical studies that have tried over the past 3 years to define the problem. Two areas have been identified. The first is the structure of the blood vessels themselves and the second is the environment of the endometrium in relation to these vessels. This reflects a shift in research interest away from factors that simply control epithelial-mesenchyme interactions. The expression of angiogenic inhibitors and stimulators is being defined and the critical role of the matrix and the immunocompetent cells of the endometrium elucidated. The disappointing results of simple oestrogen supplementation were confirmed. Three broad areas of research were considered to be important for future studies. The first of these is not related to molecular and cellular function but rather to understanding better the attitudes of women to vaginal bleeding in the multicultural diverse situations in which this event occurs. It was recognized that a broader constituency was not interested in this problem with the increasing use of continuous combined regimes of hormone replacement therapy in the developed world. Studies that listen to and educate women about this problem are needed to ensure that these techniques are felt to be better owned by the women themselves. Molecular and cellular studies were identified that attempted to define the factors necessary for the development and maintenance of healthy, non-leaky vessels. These included studies of the pro- and anti-angiogenic agents expressed in endometrium and a definition of their interactions with matrix metalloproteinases in maintaining vessel integrity. Finally there was recognition of the need to understand how neutrophils traffic through the endometrium and to determine how they effect endometrial bleeding.

Perivascular smooth muscle alpha-actin is reduced in the endometrium of women with progestin-only contraceptive breakthrough bleeding.

It has been shown that the endometrium of women using progestin-only contraceptives has increased vascular fragility, although the structural basis for this weakness is unknown, as is its role in breakthrough bleeding (BTB). Perivascular cells such as pericytes and vascular smooth muscle cells surround capillaries during the maturation process following angiogenesis, and act to strengthen and stabilize the vessels. The aim of the present study was to quantify endometrial perivascular smooth muscle alpha-actin (alphaSMA) expression in women using Norplant with and without BTB problems, and compare it to controls. Using immunohistochemical techniques, vessels were classified as level 0, 1 or 2 depending on whether perivascular alphaSMA was absent, discontinuous or continuous. In 15 controls the subepithelial plexus had significantly more level 0 vessels than either the functionalis or basalis (61 +/- 4 versus 31 +/- 6 and 37 +/- 4%, P = 0.0006 and P = 0.0007 respectively). In contrast the functionalis and basalis had significantly more level 2 vessels than the subepithelial plexus (20 +/- 3 and 23 +/- 2 compared to 4 +/- 1%, P = 0.0005 and P = 0.000 respectively). The major finding of the study was that in Norplant users, where the relatively atrophic endometrium cannot be divided into different regions, women with BTB problems (n = 20) had significantly more level 0 vessels than those with reduced bleeding (n = 17) (60 +/- 4 versus 46 +/- 4%, P = 0.0302). Norplant users with BTB problems also had a non-significant reduction in level 2 vessels compared to women without bleeding problems (4 +/- 2 versus 11 +/- 4%, P = 0.0667). These results demonstrate that perivascular alphaSMA is reduced around the endometrial vessels of Norplant users with BTB compared to those with no bleeding problems, and strongly support the concept that reduced vascular structural integrity plays a key role in endometrial BTB.

Endometrial epithelial integrity and subepithelial reticular fibre expression in progestin contraceptive acceptors.

Long-acting progestin contraceptives have been available in many countries for a number of years with a large number of women now using them. Although some improvements in delivery systems have been made, the major problem with progestin-only contraceptives remains unpredictable endometrial breakthrough bleeding (BTB), which is responsible for more than 50% of drop-outs from this form of contraception. Using hysteroscopy, endometrial petechiae and ecchymoses are a common finding among Norplant users, although these features do not always correlate with BTB. It has been postulated that epithelial and subepithelial tissues may provide a barrier to BTB, as long as epithelial integrity is maintained. The aim of this pilot study is to explore structural changes in the endometrial surface epithelium, and subepithelial collagen III fibres. Endometrial biopsies from noresthisterone-enanthate (NetEn) users (n = 6) and controls (n = 6) were assessed using routine haematoxylin and eosin staining and immunohistochemical staining for cytokeratins 8, 18 and 19, and collagen III. A conventional silver impregnation method was also used to identify subepithelial collagen III fibres. Most of the Net-En tissues showed reduced surface epithelial cell height compared controls (P = 0.002). Cytokeratin staining as weaker (P = 0.04) and distributed evenly between basal and apical parts of the cell in Net-En tissue, compared to more apically in controls. Both immunohistochemical and conventional silver staining methods revealed that the subepithelial collagen III meshwork remained unchanged in Net-En compared to control endometrium. Both staining methods identified collagen fibres with equal sensitivity. In conclusion, atrophic changes remain the dominant appearance for progestin-exposed endometrium, with reduced cytokeratin staining, but apparently there is little change in subepithelial collagen III expression.

The role of selective oestrogen receptor modulators in the treatment of endometrial bleeding in women using long-acting progestin contraception.

This paper explores the concept that endometrial breakthrough bleeding results from the stimulatory effects of oestrogen in the endometrium. Though 'progestin-only' contraceptive regimens have long been associated with user dissatisfaction because of unpredictable vaginal bleeding, it is likely that the substantial contribution of endogenous ovarian oestradiol during such treatments predisposes the bleeding problems. Oestrogen causes endometrial proliferation, hyperplasia and neoplasia if unopposed. Oestrogen allows production of growth factors supporting angiogenesis which results in an abundance of dilated or fragile endothelial surface blood vessels, predisposing this tissue to bleeding when these vessels lose competence.

Bcl-2, Fas and caspase 3 expression in endometrium from levonorgestrel implant users with and without breakthrough bleeding.

Women using the progestin-only contraceptive Norplant often suffer from unpredictable bouts of breakthrough bleeding, which usually occurs from a thin atrophic endometrium. The role of cellular apoptosis in the endometrial response to Norplant has not been investigated. The aim of the present study was to use immunohistochemistry to produce semi-quantitative scores for expression of the apoptosis-related proteins Bcl-2, Fas and caspase 3 in endometrium from 16 controls and 42 women using Norplant with minimal or major breakthrough bleeding problems. The results showed no difference in endometrial immunostaining for any of the three proteins between Norplant users with and without breakthrough bleeding. There was also no evidence of endometrial endothelial cell immunostaining for any of the proteins. Bcl-2 was the only protein to show a cyclical pattern, with higher expression in the proliferative compared to secretory glands. All three proteins showed different expression levels in control functionalis versus basalis, with the survival protein Bcl-2 being higher in basalis, and the death receptor Fas and the proteolytic enzyme caspase 3 being higher in the functionalis. Overall, the results suggest that apoptosis is regulated differently in functionalis compared to basalis, and that atrophic Norplant-exposed endometrium appears more like functionalis than basalis with respect to expression of Fas and caspase 3. There was no evidence for a role for apoptosis in the mechanisms that underlie progestin-induced endometrial breakthrough bleeding.

Endometrial vasculature in Norplant users.

Disrupted, prolonged and irregular endometrial bleeding are major unwanted side-effects of progestin-only contraceptives. The aim of this paper is to review current information on steroid control of the microvasculature, microvascular heterogeneity and microvascular fragility, with emphasis on the relevance of these issues to the endometrial microvasculature in women receiving Norplant implant contraception. Subjects were either Indonesian women with between 3 and 12 months exposure to Norplant (n = 191) or Caucasian controls recruited in Melbourne, Australia. Norplant endometrium was always thinner than control endometrium, with a varied histology that usually included a basalis-type appearance, signs of haemorrhage and some dilated and congested subepithelial vessels. Thin-walled vessels were seen which could have been either blood vascular or lymphatics. Steroid control of the vasculature can operate through numerous direct and indirect mechanisms, with up to 30 genes relevant to vascular function having consensus oestrogen response elements in their promoter regions. The vasoactive effects of progesterone are less well documented. However, experimental data for direct effects on the endometrial vasculature are mounting. Progestin-induced endometrial breakthrough bleeding is often focal, suggesting that microvascular heterogeneity may be an important factor in understanding this phenomenon. Increased susceptibility to bleeding may result from increased microvascular fragility, possibly as a consequence of progestins altering the balance of angiogenic promoters and inhibitors in the endometrium, thus leaving the vessels in a permanently weakened state.