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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 10 3853-3859
Copyright © 2000 by The Endocrine Society

Original Studies

Effects of Norplant on Endometrial Tissue Factor Expression and Blood Vessel Structure1

Radmila Runic, Frederick Schatz, Livia Wan, Rita Demopoulos, Graciela Krikun and Charles J. Lockwood

Department of Obstetrics and Gynecology, New York University School of Medicine, New York, New York 10016

Address correspondence and requests for reprints to: Frederick Schatz, Ph.D., Department of Obstetrics and Gynecology, New York University School of Medicine, 550 First Avenue, New York, New York 10016.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abnormal uterine bleeding after Norplant administration is primarily responsible for the high discontinuation rate of this safe and effective long-acting implantable progestin-only contraceptive agent. Although tissue factor (TF) is the primary initiator of hemostasis, previous studies indicated that Norplant-associated bleeding persists despite relatively high TF levels in the stromal compartment. Recently, we determined that progestin-enhanced TF expression during decidualization of human endometrial stromal cells involves both the epidermal growth factor receptor and progesterone receptor (PR}. The current study evaluated TF levels in endometrial bleeding (BL) and nonbleeding (NBL) sites obtained by camera-guided hysteroscopy during Norplant contraception. After 1 yr of therapy, immunohistochemical TF levels were unexpectedly higher at BL than at NBL sites. Use of immunohistochemistry and Western blotting indicated that both sites displayed elevated epidermal growth factor receptor levels and that the BL sites exhibited high levels of the PR, as well as the PRA and the PRB isoforms. Microscopic examination of 1-yr biopsies revealed that significantly larger numbers of enlarged, distended vessels were present in BL, compared with NBL sites. Elevated TF levels and abnormally enlarged blood vessels in the BL sites are consistent with the recently discovered angiogenic role of TF. By promoting aberrant angiogenesis, chronic endometrial overexpression of TF could produce fragile vessels, which are at increased risk to bleed. Analysis of endometrial BL and NBL sites, during Norplant contraception, offers the potential of elucidating local mechanisms that control enhanced TF expression, leading to abnormal angiogenesis at specific endometrial sites.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LOW-DOSE, progestin-only contraceptives are specifically recommended when estrogen-containing formulations are contraindicated (e.g. during lactation and for women at risk for thrombosis) (1). Norplant, the archetypal implantable form, is especially long acting and is more convenient to use than oral or injectable progestin-only contraceptives. The Norplant system consists of subdermally implanted Silastic capsules, which steadily release contraceptive blood levels of levonorgestrel for a 5-yr period (2). Contraceptive efficacy (>99%) results from thickening of the cervical mucus, which acts as a barrier to sperm penetration, and suppression or alteration of ovulation. Unfortunately, irregular and prolonged abnormal uterine bleeding is a frequent cause for the discontinuation of this otherwise safe, economical, and effective contraceptive agent (3). The majority of women experience prolonged and irregular breakthrough bleeding, and spotting between cycles. These symptoms account for more than half of the removals during the first year of use and a 30% 5-yr discontinuation rate (4, 5).

Several reports have associated bleeding in endometria exposed to long-term progestin-only contraceptives with compromised endometrial microvessels. Abnormal vascular changes include enlargement and dilatation (6, 7, 8), increased microvascular density (9, 10), capillary endothelial proliferation (11), and endothelial gaps and hemostatic plugs (12). By contrast with the spontaneous focal and transient episodes of hemorrhage from these fragile microvessels, normal menstrual bleeding originates primarily from spiral arterioles in response to withdrawal of circulating progesterone from the estradiol-primed endometrium (13, 14).

In the current study, camera-guided hysteroscopy was used to sample endometrial bleeding (BL) and nonbleeding (NBL) sites, from the same patients, up through 12 months of Norplant contraception. The biopsies were evaluated for expression of tissue factor (TF), the primary initiator of hemostasis (15). Previous studies revealed that TF levels were elevated in human endometrial stromal cells (HESCs) undergoing decidualization in vivo and in vitro (16, 17). Recently, we determined that progestin-enhancement of TF expression during in vitro decidualization of HESCs required costimulation by epidermal growth factor receptor (EGFR) agonists (18). Moreover, EGFR levels were observed to increase in response to progestin exposure in the HESCs (18). Therefore, to assess factors that are likely to regulate endometrial TF expression during Norplant contraception, immunoreactive progesterone receptor (PR) and EGFR levels were also measured in the endometrial biopsies in both BL and NBL sites.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues and bleeding

Written informed consent from patients and approval by the Institutional Board of Research Associates of New York University Medical Center and Bellevue Hospital were obtained before sampling. Endometrial specimens were obtained by blind pipelle biopsy (Unimar, Willon, CT) across normal menstrual cycles before starting Norplant contraception (control group) and by a 5-mm operative hysteroscope (Karl Storz Endoscopy-America Inc., Culver City, CA) connected to a video camera to facilitate separate sampling of BL and NBL (Fig. 1Go) sites after 3 and 12 months of Norplant treatment. Biopsy specimens were full thickness with those displaying significant myometrial components excluded from further analysis. BL sites were ascertained by increased vascularity, and either ecchymosis or overt streaming. Three biopsies each of BL or NBL sites were obtained per patient. In cases where a BL site covered a large area, only a portion was biopsied. During this study, both NBL and BL sites were obtained from the fundus of the uterus to avoid sampling the lower uterine segment near the cervical canal, where trauma resulting from dilating the cervix could cause bleeding artifacts. Control and Norplant-derived biopsies were either fixed in 4% paraformaldehyde and embedded in paraffin or frozen in liquid nitrogen before storage at -80 C. Table 1Go summarizes the bleeding pattern observed after 3 and 12 months of Norplant therapy.



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  		Figure 1. Hysteroscopic biopsy of human endometrial BL and NBL sites after 3 months of Norplant contraception. BL site: TO, Tubal ostium in the uterine fundus; NBL site: the biopsy forceps are shown.

 

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  		Table 1. Total number of bleeding days in norplant patients studied by hysteroscopic biopsy

 
Immunohistochemistry

Five-micron sections were placed on 1% poly-L-lysine-treated slides (Newcomer Supply, Middletown, WI), then deparaffinized and dehydrated with xylene and ethanol. Endogenous peroxidase was quenched with 5% hydrogen peroxide in 100% methanol. Before incubation with primary antibodies, sections were either microwave-heated or, in the case of the EGFR antibody, were untreated. They were then incubated overnight at 4 C with either: 1) 0.3 µg/mL anti-EGFR monoclonal antibody (Oncogene Science, Inc., Cambridge, MA); 2) 1:80 dilution of anti-PR monoclonal antibody (Novocastra Laboratories, Newcastle, UK); 3) 10 mg/mL of anti-PRB isoform monoclonal antibody (clone KC 146) from Dr. G. Greene (University of Chicago, Chicago, IL); 4) 1:500 dilution of antihuman TF rabbit polyclonal antibody from Dr. Y. Nemerson (Mount Sinai School of Medicine, New York, NY); 5) 1:20 dilution anti-EGFR monoclonal antibody (Zymed Laboratories, Inc., San Francisco, CA); or 6) a prediluted preparation of CD-34 monoclonal antibody (BioGenex Laboratories, Inc. San Ramon, CA). Negative controls involved substituting nonimmune mouse or rabbit serum for the primary antibody. Washed sections were treated with antimouse or antirabbit-peroxidase conjugate, and color was developed with the Vectastain ABC kit (Vector Laboratories, Inc. Burlingame, CA). Hematoxylin was used for counterstaining. Luteal-phase specimens were employed as positive controls, because they contain high concentrations of stromal cell TF, PR, and EGFR.

Quantitation of microscopic measurements

At both NBL and BL sites, intensity of immunohistochemical (IHC) staining and measurements of vessel density, lumen width (estimated by red blood cell diameter), endothelial cell width, and vascular smooth muscle thickness were quantitated by two blinded independent observers (R.R. and R.D.). Previously, we described a semiquantitative scoring system (ranging from none, weak, moderate, and strong) to assess relative intensity of IHC staining in endometrial specimens obtained during Norplant contraception (19). Statistical differences between BL and NBL sites were determined using the Wilcoxon/Kruskal-Wallis rank sum test.

Western blotting

Frozen tissues were disrupted in a Dounce homogenizer in 4 vol of ice-cold lysis buffer (25 mmol/L TRIS, 150 mmol/L NaCl, 10 mmol/L EGTA, 2 mmol/L EDTA, 0.5% Nonidet P-40, pH 7.6), containing a protease inhibitor cocktail (16), and were centrifuged at 800 x g for 5 min at 4 C. The resulting supernatant was centrifuged at 100,000 x g for 1 h at 4 C, yielding a cytosolic fraction, which was concentrated using an Ultrafree-4 centrifugal filter with a 30-kDa cutoff (Millipore Corp., Bedford, MA) and a membrane fraction, which was dissolved in lysis buffer. Each fraction was resolved on 7.5% SDS PAGE under reducing conditions and subjected to Western blotting. Incubations were carried out overnight at 4 C with 1:200 dilution of a monoclonal anti-PR antibody (NEOMARKERS, Fremont, CA) for the cytosolic fraction and a 1:100 dilution rabbit polyclonal anti-EGFR antibody (Oncogene Science, Inc.) for the membrane fraction. Detection was carried out with enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway NJ). Densitometry was performed with the Sigmastat program (Jandel Scientific, San Raphael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Endometrial EGFR expression after Norplant treatment

We have now observed that IHC staining for the EGFR is weak in stromal cells of proliferative-phase endometrium, stronger in predecidualized stromal cells localized around blood vessels of periovulatory specimens, and more intense in decidualized stromal cells around blood vessels and adjacent to glands in mid- to late-secretory-phase specimens (results not shown). These findings indicate that, as previously shown for TF expression (16, 17) and for EGFR levels in HESC monolayers (18), EGFR expression increases concomitantly with the progesterone-regulated decidualization reaction.

As expected, EGFR immunostaining in the proliferative-phase control specimen, obtained before Norplant treatment, is weakly diffuse (Fig. 2AGo). By contrast, much more prominent EGFR immunoreactivity, equivalent to that observed for normal secretory endometrium, was evident after 3 months (not shown), and 12 months of Norplant treatment at both BL (Fig. 2BGo) and NBL sites (Fig. 2CGo). The inset shown in Fig. 2BGo indicates that, as is the case for periovulatory- and secretory-phase endometria, IHC staining for the EGFR is intense in perivascular stromal cells. In the Western blot shown in Fig. 3Go: 1) endometrial extracts from both pre-Norplant secretory-phase control and Norplant-derived specimens display a major band at 170 kDa, which corresponds to the electrophoretic mobility of the EGFR previously demonstrated in human endometrial extracts (18, 20); and 2) after Norplant treatment, EGFR levels at 3- and 12-month BL sites and at 3-month NBL sites are comparable with those present in the secretory-phase control specimen, whereas the 12-month NBL sites contain slightly elevated EGFR levels. Thus, the Western blotting results for the effects of Norplant on endometrial EGFR levels (Fig. 3Go) are consistent with the IHC results (Fig. 2Go).



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  		Figure 2. IHC staining for the EGFR in cycling and Norplant-exposed human endometrium (magnification, x200). A, Pre-Norplant proliferative-phase endometrium. Stromal cells (s) show weak staining, and the glands (g) are negative. B, Norplant-exposed endometrium, BL site (12 months). Prominent EGFR staining in the stromal cells. Note that the inset indicates that EGFR staining is concentrated around the vessels (arrows) in the stromal compartment and that the endothelial cells are negative. C, Norplant-exposed endometrium, NBL site (12 months). EGFR staining is similar to that observed in B. Results are typical of 5 preparations.

 


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  		Figure 3. Western blotting for EGFR in pre-Norplant endometrial controls and BL and NBL sites after 3 and 12 months of Norplant. Membrane fractions were prepared from the endometria of the various groups, as described in Materials and Methods. Sixty micrograms protein/lane and molecular weight standards were resolved by 7.5% SDS-PAGE under denaturing conditions. The results are typical of three preparations.

 
PR and its isoforms in Norplant-exposed endometria

Of the two PR isoforms described, PRB is functionally active, whereas PRA can antagonize the actions of PRB (21, 22). However, the PRA isoform may mediate progesterone effects in secretory-phase human endometrial stroma (23). Figure 4Go, A and B, shows PR levels in a day-23 secretory-phase pre-Norplant control endometrial specimen. Prominent staining, using an antibody that recognizes a common epitope on both the PRA and PRB isoforms (Fig. 4AGo) and an antibody specific for the PRB isoform (Fig. 4BGo), is evident in the nuclei of glands and stroma. Total PR was maintained at high levels in the nuclei of glands and stroma of both 3-month BL (Fig. 4CGo) and NBL (Fig. 4DGo) sites and of 12-month BL (4E) sites. At 12 months, lower PR levels were evident in the NBL (Fig. 4FGo) sites. Similar IHC staining results were observed for the presence of PRB at BL and NBL sites after 3 months and 12 months of Norplant treatment (not shown). The IHC staining results for PR and for PRB are typical of five preparations.



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  		Figure 4. IHC staining for total PR and PRB in cycling and Norplant-exposed human endometrium (magnification, x400). A and B, Pre-Norplant secretory-phase control endometrium. Total PR IHC staining (A) is present at high levels in the nuclei of stromal cells and glands. Staining for PRB (B) is comparable with that observed for total PR in the nuclei of both stromal cells and glands. C and D, Norplant-exposed endometrial BL (C) and NBL (D) sites at 3 months. Total PR is maintained at high levels in the nuclei of endometrial stromal cells and glands after 3 months of Norplant treatment at both BL and NBL sites. Comparable levels of staining for PRB were seen (data not shown). E and F, Norplant-exposed endometria BL (E) and NBL (F) sites at 12 months. Maximal levels of total PR were observed after 12 months of Norplant treatment in nuclei of endometrial stromal cells and glands at the BL sites (E). Comparable IHC staining for PRB was seen (data not shown). Note that the endothelial cells of the vessel (v) failed to stain for the PR (E). Results are typical of six specimens.

 
The effects of Norplant contraception on the expression of PRA and PRB were assessed by Western blotting with the antibody recognizing both isoforms. Figure 5Go demonstrates that both PRA (84 kDa) and PRB (116 kDa) were maintained at levels comparable to those shown for the day-21 secretory-phase specimen in BL and NBL sites of the Norplant-derived endometrial extracts.



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  		Figure 5. Western blotting for PRB and PRA isoforms in Norplant-exposed endometria. Cytosolic fractions were prepared from the endometria of the various groups, as described in Materials and Methods. Sixty micrograms protein/lane and molecular weight standards were resolved by 7.5% SDS-PAGE under denaturing conditions. The results are typical of four preparations.

 
Norplant effects on endometrial vascular morphology and TF expression

Studies from our laboratory (19), as well as those of others (6, 7, 8, 9, 10, 11, 12), indicate that Norplant contraception affects the density and integrity of the endometrial microvasculature. The current study extends these microscopic observations to include biopsies from BL and NBL sites after IHC staining with the endothelial cell marker CD-34. As illustrated in Fig. 6Go, after 3 months of Norplant treatment, a trend towards increased microvascular density was discerned in the BL sites (6B). Moreover, after 12 months of Norplant treatment, markedly enlarged endometrial vessels were more prevalent in BL (Fig. 6DGo) vs. NBL (Fig. 6EGo) sites. In 10 endometrial specimens obtained after 12 months of Norplant, a mean 40% increase in the average lumen width was found in vessels from BL vs. NBL sites (P < 0.04). By contrast, no significant differences in vessel density or in vessel wall or endothelial cell thickness were found.



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  		Figure 6. Immunostaining for CD 34 in pre-Norplant and Norplant-exposed endometrial BL and NBL sites (magnification, x400). A, Pre-Norplant secretory-phase endometrium. B and C, Norplant-exposed endometrium (3 months) [BL (B) and NBL (C) site]. Blood vessels (arrows) are delineated by immunostaining with endothelial cell-specific CD 34. D and E, Norplant-exposed endometrium (12 months) [BL (D) and NBL (E) site]. Blood vessels are denoted by arrows, as above. Results are typical of 10 specimens.

 
TF levels in Norplant exposed endometrium

Consistent with our previous report (19), levels of immunoreactive TF were qualitatively reduced after 3 months of Norplant treatment (results not shown), compared with TF levels evident in decidualized stromal cells of secretory-phase endometrial sections. After 12 months of treatment, TF levels were enhanced at BL (Fig. 7BGo), compared with NBL (Fig. 7CGo) sites. These elevated levels were comparable with those of the secretory-phase specimen shown in (Fig. 7AGo). Thus, in apparent contradiction with the classical role of TF as the primary initiator of hemostasis (15), 12 months of Norplant treatment resulted in enhanced TF expression at the BL sites, which also displayed enlarged, distended vessels (Fig. 6DGo).



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  		Figure 7. Immunostaining for TF in pre-Norplant and Norplant-exposed endometrial BL and NBL sites (magnification, x400). After 12 months of Norplant treatment, levels of TF in the stroma of the BL site (B) were comparable with those of the secretory-phase control specimen (A) and were much higher than those of the NBL (C) site. The results are typical of five preparations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The coagulation cascade is initiated by binding of the coagulation factor VII zymogen or its active form VIIa to the transmembrane receptor, TF (15). TF is not normally expressed by cells in contact with circulating blood, such as endothelial cells or monocytes. However, its expression by perivascular cells, such as those in the smooth-muscle layer of large vessels or in the stroma surrounding microvessels, forms a protective hemostatic envelope (24). That TF affects hemostasis in human endometrium was suggested by the results of IHC staining and in situ hybridization, which revealed that TF protein and messenger RNA levels were elevated in decidualized stromal cells of sections of luteal-phase and gestational endometrium (16, 19). During human implantation, trophoblasts breach endometrial blood vessels embedded in a decidual cell matrix. This process provides the embryo with a vital source of oxygen and nutrients, but it risks hemorrhage. Decidual cell-expressed TF can counteract this risk by promoting local hemostasis.

The classical hemostasis-mediating role of TF prompted us to carry out a retrospective study to determine whether altered endometrial stromal cell TF expression contributed to Norplant-derived endometrial bleeding. The results revealed that 3–6 months of Norplant treatment lowered TF messenger RNA and protein levels, compared with pre-Norplant controls (19). Thus, the intense bleeding that is characteristic of this post-Norplant period coincided with reduced local hemostatic capacity secondary to lowered TF levels. However, bleeding continued at a lower frequency at 12 months, on NP, despite restoration of TF expression to similar levels as those found in the secretory phase of normal menstrual cycles. Moreover, these 12-month specimens displayed abnormally enlarged and dilated vessels (19). Thus, continued endometrial bleeding seemed to reflect vessel fragility and not simply impaired hemostasis.

The current study sought to elucidate mechanisms underlying prolonged bleeding on Norplant. To extend our previous retrospective study in which endometria were biopsied at random, camera-guided hysteroscopy was now used to specifically sample endometrial BL and NBL sites up through 12 months of Norplant contraception. In addition to microscopic examination of the blood vessels and assessment of TF expression, the biopsies were evaluated for the presence of the EGFR and PR (as well as the PR) isoforms, PRA and PRB. These endpoints were chosen because of our recent observations that progestin-enhanced TF expression in cultured HESCs required costimulation of the EGFR and that EGFR levels were progestin-enhanced in vitro (18). Recently, Critchley and colleagues (25) assessed the PR isoform status of Norplant-exposed endometria. Given the unavailability of an antibody against the PRA isoform, IHC staining was performed with an antibody against total PR, which recognizes both PR isoforms, and an antibody against the PRB isoform. They then inferred PRA levels by subtracting the intensity of PRB immunostaining from total PR immunostaining. To reduce the subjectivity of such measurements, the current study evaluated PR isoform status by using IHC staining together with Western Blotting.

In the current study, after 12 months of Norplant treatment, microvessels with enlarged lumens were preferentially localized at the BL sites. Unexpectedly, TF levels were selectively up-regulated at the BL sites as well. The BL sites also contained ample levels of EGFR, as well as total PR and PRA and PRB isoforms. The specific coexpression of TF, EGFR, and PR at BL sites is consistent with the absolute requirement for ligand binding to both EGFR and PR for maximal TF expression in endometrial stromal cells (18). Beyond its hemostatic role, TF is now known to mediate angiogenesis (24, 26) and to induce expression of the primary angiogenic agent, vascular endothelial cell growth factor (VEGF) (24, 27, 28). Norplant administration is also reported to increase VEGF levels in endometrial glands and stroma (10). Recently, we determined that decidualization-related transcriptional enhancement of TF expression involves mediation by the Sp1 transcription factor (29, 30). The VEGF gene promoter contains a cluster of Sp1 factor binding sites (14), suggesting that Sp1 may be involved in up-regulation of both TF and VEGF expression. Because bleeding can also increase TF levels, we hypothesize that Norplant administration promotes a feed-forward loop between PR/EGFR-induced elevated TF and VEGF levels and abnormally enlarged vessels. In this model, vessel fragility ultimately overwhelms TF-mediated hemostasis, and bleeding ensues. The use of camera-guided hysteroscopy, to separately biopsy endometrial BL and NBL sites, offers the potential of further elucidating local mechanisms that directly or indirectly lead to fragile, easily disrupted blood vessels.


   Footnotes
 
1 Supported in part by grants from the NIH (5-R01-HD-33937–05; to C.J.L.) and from the General Clinical Research Center (M01-RR-00096). Back

Received December 20, 1999.

Revised May 18, 2000.

Accepted June 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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