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Alterations in Endometrial Stromal Cell Tissue Factor Protein and Messenger Ribonucleic Acid Expression in Patients Experiencing Abnormal Uterine Bleeding While Using Norplant-2 Contraception¹

Radmila Runi, Frederick Schatz, Lewis Krey, Rita Demopoulos, Stephen Thung, Livia Wan and Charles J. Lockwood

Departments of Obstetrics and Gynecology (R.R., F.S., L.K., S.T., L.W., C.J.L.), Cell Biology (L.K.), and Pathology (R.D.), New York University School of Medicine, New York, New York 10016

Address all correspondence and requests for reprints to: Dr. Charles J. Lockwood, Department of Obstetrics and Gynecology, New York University School of Medicine, 550 First Avenue, New York, New York 10016.

	Abstract

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A high incidence of irregular uterine bleeding is the primary patient complaint limiting the utility of long term, progestin-only contraceptive agents such as Norplant. The onset of hemorrhage requires both inadequate hemostasis and impaired vascular integrity. Thus, we first tested whether Norplant-associated endometrial bleeding was accompanied by altered expression of perivascular stromal cell tissue factor (TF), the primary initiator of hemostasis. Norplant effects on TF messenger ribonucleic acid (mRNA) and protein expression by endometrial stromal cells were assessed by in situ hybridization and immunohistochemical examination of endometrial biopsies obtained from normally cycling control women (n = 14) and from patients experiencing Norplant-induced abnormal uterine bleeding (n = 24). TF mRNA and protein expression was increased 150% in secretory vs. proliferative phase endometrial specimens. By contrast, endometrial TF mRNA and protein levels were reduced during 1–6 months of Norplant treatment by about 2-fold (P < 0.05 for protein) compared to the values for control secretory phase specimens. These changes were consistent with observations that patients on Norplant begin to bleed during this interval. Further reductions of TF mRNA and protein levels to 2- and 3-fold of those in secretory phase control specimens were observed in endometria obtained after 6–12 months of Norplant therapy (P < 0.05 and P < 0.01, respectively). A modest rebound in TF mRNA and protein expression was observed after 12 months of Norplant therapy, which occurred commensurate with reduced patient complaints of abnormal uterine bleeding. Pathologically enlarged venous sinusoids were ubiquitous in endometrial specimens obtained after Norplant therapy. The combination of fragile blood vessels and reduced TF expression may account for bleeding in patients receiving Norplant therapy.

	Introduction

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OF APPROXIMATELY 3 million unplanned pregnancies occurring each year in the United States, about 45% end in legal abortion (1). Unintended pregnancies are most common among adolescents and women in reduced socioeconomic circumstances. Their associated burdens, therefore, fall disproportionately on those with the least resources (1, 2). Implantable forms of progestin-only contraceptive agents offer a practical solution to this public health dilemma because they are long acting, inexpensive, reversible, and require no daily compliance or reliance on a partner’s cooperation.

Norplant-2 consists of two subdermally implanted SILASTIC brand rods (Dow Corning, Midland, MI) that provide 5 yr of contraceptive activity via the release of levonorgestrel (LNG) (3). Norplant’s more than 99% contraceptive efficacy results primarily from LNG-induced thickening of the cervical mucus, which inhibits sperm penetration. However, LNG-induced endometrial changes may also form a barrier to implantation (3). Prolonged uterine bleeding as well as irregular breakthrough bleeding and spotting between "cycles" are the major causes of the high rate of discontinuation of Norplant therapy (4). Such abnormal uterine bleeding was distinguished from menstrual bleeding by Alexander and d’Arcangue, who noted that "during normal menstruation, bleeding emanates from endometrial spiral arterioles, whereas breakthrough bleeding probably originates from capillaries and seems to be limited to patchy areas of the endometrial surface" (5).

Our laboratory linked decidualization (DZ) of human endometrial stromal cells with enhanced expression of tissue factor (TF) (6), the primary initiator of hemostasis (7). Thus, immunohistochemical (IH) levels of TF were elevated in decidualized stromal cells of secretory phase and gestational endometrium (6). Moreover, progestins increased TF messenger ribonucleic acid (mRNA) and protein levels in human endometrial stromal cell monolayers; estradiol (E₂) augments these effects despite a lack of response to E₂ alone (6). This differential steroid response, which is reported for several DZ-related end points (as reviewed in 8 , mimics in vivo events in which E₂ primes the endometrium for the decidualizing effects of progesterone by enhancing progesterone receptor (PR) levels (9). As shown for several other DZ markers (8), withdrawal of progestins from cultured stromal cells reverses progestin-enhanced TF expression (10), suggesting that DZ-associated TF expression requires continuous progestin stimulation. The hypothesis underlying the current study is that abnormal uterine bleeding during Norplant contraception reflects the effects of chronic LNG release on endometrial stromal/decidual cell TF expression.

	Materials and Methods

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Endometrial tissue collection

Control endometrial specimens were obtained from cycling women not using hormonal contraception or intrauterine devices. These included 12 women attending the family planning clinic at Bellevue Hospital in New York and 2 women undergoing hysterectomy for myomas. Specimens were also obtained from 24 women experiencing moderate to heavy breakthrough uterine bleeding during Norplant-2 contraception (The Population Council, New York, NY). All biopsies were obtained by endometrial suction curettes (Unimar, Willon, CT). This study was undertaken after obtaining written informed consent and approval by the Institutional Board of Research Associates of New York University Medical Center and Bellevue Hospital. Patient biopsies from control and Norplant patients were fixed in 10% formalin and embedded in paraffin. Some biopsies from the control groups were frozen in OCT compound (Baxter Scientific Products, Edison, NJ) in cold 2-methylbutane (Sigma Chemical Co., St. Louis, MO) and were cryosectioned.

IH staining

Five-micron tissue sections were applied to poly-L-lysine-treated glass slides (Newcomer Supply, Middleton, WI) and deparaffinized for 2 h at 58 C before dehydration with xylene and ethanol. Endogenous peroxidase was quenched for 5 min using 5% hydrogen peroxide in 100% methanol. Sections were microwave heated for 10 min to unmask TF epitopes (11), then washed in PBS and incubated overnight at 4 C with 5 µg/mL monoclonal mouse anti-TF antibody (American Diagnostica, Greenwich, CT). Negative control slides were preabsorbed with a 7-fold molar excess of recombinant human TF (Genentech, San Francisco, CA) for 2 h at room temperature. Treatment with antimouse peroxidase conjugate and color development with diaminobenzidine were carried out using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Samples were counterstained with hematoxylin.

In situ hybridization

Five-micron endometrial specimens were deparaffinized before hybridization or were processed as cryosections by fixing with 4% paraformaldehyde for 20 min. Results obtained by paraffin sections were comparable to those obtained with cryosections. In situ hybridization (ISH) studies used ³⁵S-labeled TF 49-mer oligonucleotides prepared as follows. All templates were synthesized by Genosys (Woodlands, TX). The antisense and sense templates were 49 nucleotides long, and primers were 9-mers. The antisense template corresponded to position 592–640 of the TF mRNA sequence (12). The antisense primer sequence was 5'-CCCGGAGGC-3', and sense primer was 5'-GATGAACGG-3'. Slides were prehybridized for 4 h at 45 C in 50% deionized formamide, 1 mM ethylenediaminotetraacetic acid (pH 8.0), 0.6 M sodium chloride (NaCl), 1 x Denhardt’s solution, 10% dextran sulfate, 20 mM dithiothreitol, 0.5 mg/mL transfer RNA, and 100 µg/mL denatured salmon sperm DNA in 10 mM Tris-HCl, pH 8.0. The slides were hybridized overnight at 42 C in this buffered solution containing oligoprobe (13), then washed with 1 x sodium saline citrate at 56 C (four times) for 15 min and at room temperature (twice) for 1 h. After graded dehydration in ethanol, slides were exposed to x-ray film overnight, dipped in NTB Kodak emulsion (Eastman Kodak, Rochester, NY), and exposed for 2 weeks. After developing, slides were counterstained with hematoxylin, and dark- and lightfield pictures were taken on a Zeiss Axiophot microscope (Reinhardt Instruments, Woodbury, NY).

Scoring of IH and ISH results

A semiquantitative scoring system ranging from none (0), weak (1), moderate (2), strong (3), to intense (4) was used to assess the relative intensity of IH staining or ISH signal for both the control and the Norplant-exposed endometria. IH staining was evaluated in at least 30 microscopic fields (x100), whereas the ISH autoradiographic signal was assessed over the entire surface area of each specimen. The specific TF signal was obtained by subtracting the value of the sense (negative) control signal from that of the antisense signal. Measurements were performed blindly by two persons. A weighted measure was used to compare the interobserver correlation for the IH results (r = 0.63) and for the ISH results (r = 0.42) as well as the intraobserver variation for the IH results (r = 0.72 and r = 0.59) and the ISH results (R = 0.84 and R = 0.82). The Mann-Whitney rank sum test was used to compare the results presented in Figs. 2 and 4.

View larger version (21K): [in this window] [in a new window]   Figure 2. Levels of stromal TF protein in control and Norplant-treated groups. Bars show the mean ± SEM for IH results; the ordinate shows the relative intensity of TF IH staining (see Materials and Methods for details); the abscissa shows control and treatment groups: P, proliferative; S, secretory; 1–6 m, 1–6 months of Norplant; 6–12 m, 6–12 months of Norplant; 1yr+, more than 1 yr of Norplant. Statistical results determined by Mann-Whitney rank sum test showed: *, proliferative vs. secretory control, P < 0.01; **, 1–6 months of Norplant vs. secretory control, P < 0.05; ***, 6–12 months of Norplant vs. proliferative control (P < 0.05) or secretory control (P < 0.01); ****, more than 1 yr of Norplant vs. secretory control, P < 0.01.

View larger version (20K): [in this window] [in a new window]   Figure 4. Quantitation of TF mRNA levels in the control and Norplant groups. Bars show the mean ± SEM for ISH results; the ordinate shows the relative intensity of the autoradiographic ISH signal (see Materials and Methods). Statistical comparisons by Mann-Whitney rank sum test yielded: proliferative vs. secretory control, P < 0.05 (*); and 6–12 months of Norplant vs. secretory control, P < 0.05 (**).

	Results

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Of 24 Norplant-treated patients, 21 reported heavy and virtually continuous bleeding from 14–244 days before biopsy, 2 reported prolonged menstrual bleeding, and one reported postcoital bleeding. Norplant-exposed specimens were distributed among three groups based on duration of Norplant therapy: less than 6 months, 6–12 months, and greater than 12 months.

Norplant effects on endometrial morphology

Figure 1 compares endometrial morphological changes induced by Norplant with the morphology of endometria from cycling women. Typical features exhibited by the latter include elongated glands with mitotic figures and dense stroma (proliferative phase endometrium; Fig. 1A), and dilated, convoluted glands with edematous stroma (secretory phase endometrium; Fig. 1B). Norplant contraception elicited several morphological changes, which we categorized as proliferative type, stromal-gland asynchrony, and atrophic type. Of 24 endometria from Norplant-treated women, Table 1 indicates that 15 displayed a proliferative-type morphology. Of these, 13 showed areas of sloughing, which is typical of premenstrual and menstrual phase tissues but not of proliferative phase tissues. An area of sloughing, present in Fig. 1E but absent from Fig. 1D, was characterized by highly condensed stroma, nuclear debris, and fibrin thrombi. Table 1 also indicated that 5 of the 24 specimens from the Norplant group showed stromal-gland asynchrony. One specimen of the latter also showed evidence of sloughing. stromal-gland asynchrony was distinguished by the presence of a few small simple glands and decidualized stroma (Fig. 1F). Three of the 24 Norplant-treated patients exhibited an atrophic type morphology, characterized by small simple glands devoid of mitotic figures, and dense stroma (Fig. 1G). Table 1 shows that only one of these specimens displayed sloughing. The occurrence of an atrophic type morphology appeared to require approximately twice the duration of Norplant treatment necessary to induce the appearance of the other morphological types. Despite significant morphological differences in the glands and stroma, all the groups displayed greatly enlarged capillary-venous sinusoids (Fig. 1, F and G), which were absent in the control specimens. The abnormal vessels were particularly prevalent with prolonged Norplant administration.

View larger version (114K): [in this window] [in a new window]   Figure 1. IH localization of TF in endometria obtained from women receiving Norplant and from normal cycling women. In control specimens levels of TF IH staining were lower in proliferative phase (A) than secretory phase endometrial sections (B) and were absent after preabsorption with a 50-fold molar excess of TF antigen (C). D, Levels of TF IH after 3 months of Norplant treatment. Glands (g) and stroma (s) are morphologically similar to those in the proliferative control in A. Heterogeneous IH staining for TF shown in D (arrowhead, moderate staining; arrow, weak staining) compares with the more intense and homogeneous IH staining for TF present in the secretory phase of B. The presence of erythrocytes (Er) denotes areas of bleeding, which stain nonspecifically. An endometrial specimen obtained 6 months after Norplant therapy (E) displays extensive sloughing (*) and minimal TF IH staining (arrow). Erythrocytes (Er) are present between the sloughing and the nonsloughing areas. Six months after Norplant therapy (F), stromal-glandular asynchrony is evident; the glands (ag) are atrophic, and the stroma (s) is decidualized. Note virtually no IH staining for TF in this specimen. An endometrial specimen after 2 yr of Norplant therapy (G) displays atrophic glands (ag) and more intense heterogeneous stromal TF IH staining (arrowhead, stronger staining; arrow, weaker staining). The presence of enlarged venous sinusoids (v) is characteristic of endometria obtained after Norplant therapy (F and G; x340).

In addition to morphological changes, Fig. 1 also displays levels of TF IH staining in both normal cycling endometria and endometria obtained from women receiving Norplant therapy; Fig. 2 indicates the relative intensity of IH staining for TF in the stromal compartments of the various endometrial specimens. Consistent with our reported results (6), more intense IH staining was seen in secretory (Fig. 1B) than proliferative phase control endometria (Fig. 1A). Figure 2 indicates that these differences were significant (P < 0.01). A biphasic temporal pattern of Norplant-associated endometrial stromal cell TF staining was evident. Thus, there was a decline in TF protein after 1–6 months of Norplant use (P < 0.05), with a further decline in TF IH at 6–12 months. Indeed, endometria from this Norplant group displayed a 3-fold lower intensity of IH for TF compared with specimens derived from secretory phase controls (P < 0.01) and a 2-fold reduction compared with proliferative phase controls (P < 0.05). However, the intensity of IH for TF rebounded upward after more than 1 yr of therapy to levels comparable to those found at less than 6 months of therapy and in proliferative phase controls (Fig. 2), but this TF IH remained at 50% of the levels found in secretory phase controls (P < 0.01; Fig. 2).

Norplant effects on endometrial TF mRNA expression

In endometria of both cycling and Norplant-treated women, changes in the abundance and localization of TF mRNA as ascertained by ISH corresponded to levels of TF protein determined by IH studies. The autoradiographic signals for TF mRNA are illustrated in Fig. 3, and the results are quantified in Fig. 4. The grain density for TF mRNA was significantly lower (P < 0.05) in proliferative phase (Fig. 3A) compared with secretory phase control endometrium (Fig. 3C). Scrutiny of Fig. 3C reveals accentuated expression of TF mRNA adjacent to or in stromal cells just below the luminal epithelium corresponding to sites of initial DZ. Analogous to the IH staining for TF protein, grain density for endometrial TF mRNA reached a nadir after 6–12 months of Norplant use (P < 0.05) compared with that in the secretory phase controls (Fig. 3, E and C) and rebounded upward after 1 yr of Norplant use to levels seen after less than 6 months of use (Figs. 3G and 4). Despite clear morphological features of pseudodecidualization, as exemplified by stromal-gland asynchrony, TF mRNA levels remained suppressed in Norplant-treated endometria (Fig. 3E). Moreover, Norplant-derived specimens exhibiting proliferative morphology tended to display lower levels of TF mRNA expression, as seen in proliferative controls. The intensity of the TF mRNA signal in areas of sloughing did not differ from those in nonsloughing areas.

View larger version (124K): [in this window] [in a new window]   Figure 3. In situ localization of TF mRNA in endometria from women treated with Norplant and from normal cycling women. TF mRNA is denoted by the presence of white grains in the darkfield photomicrographs (upper panels). Morphology is illustrated by lightfield views (lower panels) of sections seen in darkfield. For control specimens note that the grain density of the secretory phase (C) is much higher than that of its respective sense (-) control (C') and is higher than that of the proliferative phase (A and B). The arrowheads of the darkfield photomicrographs correspond to the areas visualized in the lightfield views as just below the luminal epithelium (e). The darkfield illumination of C indicates more abundant TF mRNA in stromal cells than in glandular epithelial cells (arrow, C). The latter corresponds to glands present in the lightfield photomicrograph (D). For Norplant groups the grain density of TF mRNA of the specimen 1 year after Norplant (G and H) was about twice that seen in specimen after 6 months of Norplant (E and F). The rebound increase seen in G has almost attained the level of TF mRNA expression evident in the secretory phase specimen (C; x230).

	Discussion

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As enhanced stromal cell TF expression in the cycling human endometrium requires continuous progestational stimulation (6), reduced TF expression secondary to Norplant therapy shown in the current study appears to be paradoxical. However, prolonged exposure to a high progestational, low estrogenic hormonal milieu profoundly down-regulates endometrial PR levels (19), suggesting that Norplant treatment creates a functionally hypoprogestational state. Evidence supporting this hypoprogestational hypothesis includes the lower rates of abnormal uterine bleeding associated with combined E₂/progestin oral contraceptive therapy vs. those with progestin-only contraceptives (20); the increase in abnormal uterine bleeding resulting from a switch to an oral contraceptive with lower progestin for a given estrogen dose (21); the reduction in such bleeding and spotting accompanying intermittent ethinyl estradiol treatment in Norplant, depo-Provera, and low dose combined oral contraceptive users (18, 21); and the reduction in irregular bleeding associated with increasing ovulatory activity in Norplant users (20).

In contrast to the above arguments that chronic LNG treatment invokes a paradoxically hypoprogestational state, Critchley et al. (22) reported that endometria from Norplant-treated patients manifest elevated IH for PR. One solution to this apparent endocrinological conundrum (i.e. the presence of increased PR content but decreased progestin effects) may lie in differences in the activities of the various PR isoforms in modulating progestationally regulated transcription. Two predominant isoforms of the PR exist, a higher molecular mass form (120 kDa), PR_B, and a second N-terminus-truncated, lower molecular mass form, PR_A (94 kDa) (23, 24). Transfection studies have demonstrated that the presence of PR_A can inhibit PR_B-modulated transcription at physiological levels of progesterone (25). Moreover, the dominant negative action exerted by PR_A on PR_B-stimulated transcription is both cell type specific as well as promoter specific and can occur in the absence of DNA binding by PR_A (26). Thus, assessing levels of functionally active PR in a given cell type requires ascertaining not only total PR content, but the relative distributions of the various isoforms. Differences in promoter activity regulation by the two PR isoforms appear to result from variable TAF1 functions (27, 28) due to their differing N-terminal sequences. Intriguingly, expression of the two PR isoforms may be regulated by E₂, with increased levels of E₂ enhancing the progestationally agonistic PR_B, while E₂ withdrawal enhances the PR_A antagonistic isoform (28).

In this retrospective study of endometrial specimens derived from Norplant patients, we observed reductions in the expression of endometrial stromal cell TF mRNA and protein in Norplant users experiencing bleeding within the first 6 months of therapy, with a nadir in TF expression occurring at 6–12 months corresponding to peak bleeding episodes (29). The frequency of bleeding disturbances decreased significantly after the first year of use (25), when we observed a rebound increase in TF expression. The latter is likely to be involved in mediating this reduction in bleeding, as vascular integrity appeared to be further compromised during this 6- to 12-month period.

	Acknowledgments

 
We acknowledge the technical assistance of Ms. Rebeca Caze and Dr. Graciela Krikun with the IH procedures, Mr. David Ziegler for help in preparing artwork for this manuscript, Dr. Seth Guller for advice and reading the manuscript, and Edward Kuczynski for advice on carrying out statistical measures.

	Footnotes

 
¹ This work was supported by NIH Grant HD-33937–02 (to C.J.L.) and Grant 6315628 (to The Population Council).

Received October 8, 1996.

Revised January 27, 1997.

Accepted February 25, 1997.

	References

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