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Angiogenic Effects of Norplant Contraception on Endometrial Histology and Uterine Bleeding

Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143

Address all correspondence and requests for reprints to: Robert N. Taylor, M.D., Ph.D., Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, 513 Parnassus, Health Sciences West 1656, University of California, San Francisco, San Francisco, California 94143-0556. E-mail: taylorr{at}obgyn.ucsf.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Norplant, a sc contraceptive device, releases levonorgestrel in a sustained fashion. Its effectiveness is offset by irregular bleeding patterns. Because vascular endothelial growth factor (VEGF) is stimulated by synthetic progestogens in vitro and in vivo, we postulated that correlations between this angiogenic factor and uterine bleeding patterns might exist. Twenty-eight women who were exposed to Norplant and 13 control women were prospectively followed for 6–8 months. Bleeding diaries were collected, hysteroscopies were performed, endometrial biopsies were obtained for standardized histological evaluation, and VEGF histochemical immunostaining (H)-scores were assigned. Cluster determination-34 (CD34) staining was also performed to quantify the number of endometrial blood vessels per high-power field. Irregular uterine bleeding was common among women using Norplant devices. Endometrial VEGF H-scores were greater in women using Norplant than in control women. New findings of this study show that vessel density did not correlate with epithelial VEGF H-scores but was highly associated with the intensity of stromal and perivascular VEGF. VEGF expression in the latter regions correlated significantly with hysteroscopic abnormalities and irregular bleeding. The expression of this angiogenic protein, particularly in the stromal and perivascular compartments, correlated with microvascular density, hysteroscopically documented hypervascularity, and uterine bleeding profiles. Irregular bleeding with Norplant use appears to reflect paracrine-mediated effects on vascular function by angiogenic factors, such as VEGF.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
NORPLANT IS A sc, implantable contraceptive device that releases levonorgestrel in a sustained fashion, providing long-term protection against unwanted pregnancy. It is one of the most effective forms of birth control, with less than 0.5 pregnancies per 100 woman-years of use (1). However, since its Food and Drug Administration approval and clinical introduction in 1990, less than 1.5% of reproductive-age women desiring contraception chose implantable progestogen devices such as Norplant (2).

The discrepancy between the high efficacy of the device and its lack of popularity among women seeking contraception can be attributed to the side effects of Norplant. At least 40% of users experience irregular uterine bleeding. Although the flow is light in the majority of women, bleeding is unpredictable. It can be manifested as lengthened bleeding intervals, irregular spotting between cycles, or a mixture of both patterns (3). This irregular bleeding is more common in the first 2 yr of Norplant use, when levonorgestrel blood levels are highest and ovulation is less frequent. Irregular bleeding is the most common reason that women discontinue use of the implantable contraceptives (1). The irregular bleeding is postulated to be attributable to effects on the vessels within the endometrium of Norplant users. Reported changes include increased microvascular density (4, 5, 6), increased capillary endothelial proliferation (7), and enlargement and dilatation of superficial vessels (8, 9, 10, 11). Increased vascular fragility has also been implicated as a basis for the bleeding (12), possibly associated with vascular apoptosis and decreased perfusion, hypoxia, and thrombin generation (13).

In a previous study of long-term Norplant users, we observed a correlation between unpredictable bleeding and altered endometrial histology. Irregular bleeding patterns were more common in women with morphological evidence of proliferative changes in the endometrium, which were characterized using a semiquantitative histological endometrial index (EI) (14). Because endometrial histology is dependent on the establishment of its vasculature (15), in the current analysis we evaluated two markers of vascular development: expression of a key angiogenic factor and microvascular density. Vascular endothelial growth factor (VEGF) is the angiogenic factor predominantly responsible for the cyclical growth of new blood vessels in the endometrium, which appear to arise through a combination of vascular sprouting, elongation, and intussusception (15, 16, 17). VEGF also modulates vascular permeability. In some studies, VEGF has been shown to correlate with endometrial microvascular density (18, 19), and VEGF gene expression is responsive to natural ovarian sex steroids and synthetic progestogens (17, 19, 20, 21, 22). Therefore, VEGF is an excellent candidate as a mediator of the aberrant vessel function seen with Norplant use. We hypothesized that endometrial VEGF is increased in women exposed to Norplant compared with normally cycling women and that VEGF expression correlates with microvascular density. We also surmised that correlations might exist among VEGF, vascular density, endometrial morphology, and clinical uterine bleeding patterns.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Menstrual calendars were collected, and hysteroscopy was performed in 28 women using the Norplant contraceptive method and in 13 normal, ovulatory women who used barrier contraception and did not receive exogenous hormones as controls. All women were recruited contemporaneously from the University of California, San Francisco (UCSF) Family Planning Clinic. They were of reproductive age and, for this study, were required to have a history of regular menstrual cycles. Consenting subjects were evaluated randomly at routine clinic visits, which were unrelated to complaints of uterine bleeding. Controls, likewise, were sampled randomly, relative to their ovulatory cycle. Endometrial biopsies were collected by Pipelle de Cornier (Unimar, Danbury, CT) aspiration after hysteroscopy, as described below in Hysteroscopy. Written informed consent was obtained under a protocol approved by the UCSF Committee on Human Research.

Uterine bleeding pattern evaluation

The evaluation of menstrual cycles was based on the definitions for menstrual regularity established by Schneider et al. (23). In brief, cycles were categorized as regular (21–45 d, with 7 d of cycle-to-cycle variation), mildly irregular (21–45 d, with >7 d of cycle-to-cycle variation), moderately irregular (25% of cycles were out of the 21- to 45-d range), severely irregular (26–50% of cycles were out of the 21- to 45-d range), or extremely irregular (>50% of cycles were out of the 21- to 45-d range). All of the control cycles fell within the regular category.

Hysteroscopy

We performed hysteroscopy after applying povidone iodine solution to the cervix and administering a paracervical block using 5–10 ml 1% chloroprocaine. A 5-mm, 12-degree hysteroscope was inserted without dilation of the cervix, if possible, using normal saline as the distention medium. Hysteroscopic photographs representative of the endometrial cavity were obtained, and these were later evaluated for vascularization and hyperemia of the endometrial surface by an independent observer (R.N.T.) who was blinded to the contraceptive practice of the subject. The vascularity score was based on a scale of 0–4 (0 = absent or rare vessels observed; 4 = prominent and diffuse submucosal vessels or active bleeding from the endometrial surface). The hysteroscope was removed, and endometrial biopsies were collected using the Pipelle device directed toward the most vascular regions of the endometrium based on the hysteroscopic findings.

Biopsy specimen preparation

After the biopsies, endometrial tissues were fixed immediately at room temperature in a non-cross-linking reagent (Histochoice, Amresco Inc., Solon, OH) and embedded in paraffin within 3 d. Five-micrometer serial sections were then cut and mounted on silane-treated glass slides. Hematoxylin and eosin staining was performed using standard methods, and the histological patterns were evaluated blindly.

EI

Using sections from the biopsy material of each participant, a single section stained with hematoxylin and eosin was evaluated microscopically in a blinded fashion by two independent reviewers (I.P.R. and C.J.Z.) and given an EI score. The EI is a standardized scoring system that allows the endometrium to be rated according to its normal cyclical proliferative or secretory properties, based on 10 histomorphometric criteria: 1) volume of tissue; 2) gland configuration; 3) gland nuclear localization; 4) percentage of ciliated cells; 5) mitotic figures in glands and stroma; 6) cytoplasmic eosinophilia; 7) secretory activity of glands; 8) compaction of stroma; 9) stromal hemorrhage; and 10) stromal lymphocytes. The mean composite scores from the two observers, based on the aforementioned histomorphometric categories, were used to derive the final EI score. An EI score of 1.0 was consistent with a specimen in the mid-late proliferative phase of the cycle, whereas a score of 0.0 was consistent with a specimen in the midsecretory phase. This current scoring system represented an expanded modification of the EI described previously, which segregated normal and abnormal bleeding patterns in a pilot study of 18 long-term Norplant users (14). Repeated evaluation of the same slides was performed to assure consistency within and between the observers. Inter- and intraobserver coefficients of variation of the final EI score were less than 10%.

Immunohistochemistry and H-scoring for VEGF and cluster determination-34 (CD34)

On successive cuts through each imbedded specimen, immunohistochemistry was performed using antibodies against VEGF and CD34 (an endothelial antigen to identify microvessel density), previously optimized for endometrial tissue (17). Briefly, the method is as follows: after mounting, the samples were deparaffinized in xylene and rehydrated in graded concentrations of ethanol. The slides were then exposed to 20 min of 3% hydrogen peroxide and 0.1% saponin in 0.05 M Tris-buffered saline (TBS) to quench endogenous hydrogen peroxide activity. Each sample was then washed in TBS and preincubated in 3% horse serum in TBS for 20 min. Primary antibodies were applied, and the samples were incubated overnight at 4 C.

Rabbit polyclonal antihuman VEGF-A antibodies were used for VEGF immunohistochemistry at a concentration of 10 µg/ml (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Vascular endothelial CD34 antigen was detected using mouse monoclonal antihuman CD34 antibodies at a concentration of 2 µg/ml (Zymed, South San Francisco, CA). An avidin-biotin peroxidase reaction was then performed (Elite Vectastain ABC, Vector Laboratories, Inc., Burlingame, CA), followed by a diaminobenzidine-black substrate reaction (Zymed). Biotinylated universal reagents (recognizing both mouse and rabbit antibodies) were used as secondary antibodies.

Positive control tissues were included in each experiment: ovary and lung for VEGF-A and CD34, respectively, to assure reliability and reproducibility of the technique. Negative controls comprised identical concentrations of nonimmune rabbit sera and an irrelevant mouse monoclonal IgG (antisynaptophysin) to control for nonspecific effects. An H-score (24) was assigned to each sample after VEGF and CD34 immunohistochemistry was performed. The H-score is a semiquantitative method to assess distribution and intensity of antigen, according to the equation: H = (% cells stained) (intensity of staining + 1). Each tissue section was evaluated blindly, in five random areas at 40x magnification, by two investigators (E.A.P. and M.D.M.) to establish the percentage of stained cells per field and given scores of 0–3, based on the intensity of immunostaining. The mean H-scores of the two observers were assigned for each section. Random sampling was instituted to minimize the effects of varied endometrial gland/stroma ratios among the different subjects. Endometrial glands, stroma, and perivascular regions (defined as regions surrounding CD34-positive vessels in adjacent sections) were scored separately. H-scores ranged from 0–4. Vessel density was determined by counting the number of CD34-positive endothelium-lined vascular structures per high-power field, i.e. over a standardized area. Repeated evaluation of the same slides was performed to assure consistency within and between the observers. Inter- and intraobserver coefficients of variation of the final EI score were less than 15%.

Statistics

Descriptive data are presented as mean ± SE. Conservative, nonparametric statistics, Mann-Whitney U and Spearman rank () correlation tests were used to compare groups and outcomes. A P value of less than 0.05 was considered significant for two-tailed tests. Dunn’s corrections were applied for multiple comparisons.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The ages of the participants ranged from 19–45 yr and were not different between Norplant (27 ± 1 yr) and control (31 ± 2 yr) groups (P = 0.23, Mann-Whitney U test). Duration of Norplant use at study completion ranged from 7–88 months (those women reaching 60 months of device use had new Norplants inserted). Not surprisingly, bleeding patterns were irregular in the Norplant users. The median pattern in our group of Norplant subjects, based on the criteria of Schneider et al. (23), was severely irregular. This differed significantly (P < 0.01) from the regular median pattern of the normal controls, but it is important to note that a history of regular menses was an inclusion criterion for the study subjects. Hysteroscopic evidence of endometrial hyperemia also was significantly higher in the Norplant group (2.1 ± 0.2) compared with the control group (0.6 ± 0.2; P < 0.01).

Endometrial morphology among women using Norplant was variable with a mean ± SE EI of 0.51 ± 0.04, reflecting mixed histology as described previously (14). Given the variable histological patterns observed with Norplant, we elected to compare VEGF H-scores and microvascular density to those in ovulatory controls sampled randomly throughout the cycle. As expected, biopsies from normally ovulating controls showed either predominantly proliferative or secretory histological patterns; hence, EIs were segregated at either end of the score spectrum but not significantly weighted in either direction (² = 0.06; P = 0.81). The mean ± SE EI in controls was 0.47 ± 0.12 and did not differ significantly from the Norplant group (P = 0.58, Mann-Whitney U test). However, the increased variance in the control group is consistent with the bimodal distribution of biopsy EI scores.

VEGF H-scores were significantly higher in glands, stroma, and perivascular spaces in women using Norplant (3.32 ± 0.05, 2.84 ± 0.05, and 2.90 ± 0.07, respectively), compared with normoovulatory controls (2.46 ± 0.17, 1.73 ± 0.18, and 0.52 ± 0.19, respectively; P 0.01 for all locations, Mann-Whitney U tests). In Norplant users, VEGF H-scores were not correlated with length of Norplant use. Epithelial VEGF H-scores did not correlate with vessel counts ( = –0.06; P = 0.76), but stromal and perivascular VEGF H-scores were highly correlated with vessel density as quantified by CD34-positive endothelia/high-power field ( = 0.49, P < 0.01; and = 0.50, P < 0.01, respectively) (Figs. 1–3). Good correlations were noted between stromal and perivascular VEGF H-scores and clinical bleeding patterns and hysteroscopic hyperemia scores ( 0.40; P < 0.01), whereas epithelial VEGF H-scores did not correlate significantly with bleeding or hysteroscopic findings ( = 0.06 and = 0.04, respectively; P 0.66).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Scatter plot of VEGF H-scores for glandular epithelium compared with vessel density per high-power field (magnification, x40) ( = –0.06; P = 0.76).

View larger version (27K): [in this window] [in a new window]   FIG. 2. Scatter plot of VEGF H-scores for stroma compared with vessel density per high-power field (magnification, x40) ( = 0.49; P < 0.01).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Scatter plot of VEGF H-scores for perivascular tissue compared with vessel density per high-power field (magnification, x40) ( = 0.50; P < 0.01).

Despite our previous observation that EI and uterine bleeding pattern appeared associated (14), EI scores in the present Norplant study failed to correlate significantly with VEGF H-scores ( = 0.10, P = 0.52; = –0.08, P = 0.62; = 0.17, P = 0.31, for glands, stroma, and perivascular spaces, respectively; Spearman rank tests), microvascular density ( = –0.07; P = 0.72), bleeding pattern ( < 0.01; P = 0.99), or hysteroscopic hyperemia ( = 0.17; P = 0.22). Representative examples of vascular density (CD34) and VEGF immunostaining in Norplant biopsies are shown in Fig. 4, and representative photographs of hysteroscopic findings in Norplant subjects and controls are shown in Fig. 5.

View larger version (133K): [in this window] [in a new window]   FIG. 4. Photomicrographs of endometrial biopsies from Norplant subjects stained with antihuman CD34 antibodies (left panels) and antihuman VEGF antibodies (right panels) (magnification, x20). A and B, EI for this sample was 0.1, consistent with predominantly secretory activity. Note the serpentine glands and scattered dilated vessels throughout the stroma. C and D, EI for this sample was 0.4, consistent with an intermediate score, partially proliferative, and partially secretory. The luminal epithelium appears more strongly positive for VEGF than the endometrial glands, which are relatively simple. The stromal density is varied with vessels of variable diameter. E and F, EI for this sample was 0.8, consistent with predominantly proliferative characteristics. Luminal epithelium VEGF is more prominent than that in the glandular epithelium. The glands are small and sparse, and the stroma is dense and regular. The endometrial capillaries are simple and straight. Scale bar, 100 µm.

View larger version (45K): [in this window] [in a new window]   FIG. 5. Hysteroscopic photographs from a representative Norplant subject (A) (hyperemia score = 2.5) and a representative control subject (B) (hyperemia score = 0.5).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In the present study, in situ VEGF expression, as quantified by endometrial biopsy H-scores, was increased in the endometrium of women using Norplant compared with controls. Lau et al. (27) previously made the same observation. Although epithelial cells appear to have the highest intensity and production of VEGF among the resident endometrial cells (Fig. 4, B, D, and F; and Refs. 17 , 18 , and 28), the direction of secretion is luminal rather than abluminal (29). By contrast, new findings from our study indicate that stromal and, particularly, perivascular cell production of VEGF correlated significantly with vascular density (Figs. 2 and 3). There is some logic to this compartmentalized observation, because the latter is the anatomic microenvironment in closest proximity to growing endometrial capillaries, and this may explain why previous studies of global endometrial VEGF expression have not correlated well with angiogenesis (28). The recent work of Nayak and Brenner (30) in rhesus macaques also suggests that stromal VEGF is most directly associated with endometrial angiogenesis. The precise cellular source(s) of perivascular VEGF expression has not been fully delineated, but we showed by dual immunoenzymatic labeling that extravasating neutrophils, in addition to perivascular stromal cells, have highly concentrated VEGF expression in the endometrium (31). This observation has been confirmed by others (32).

Our results are consistent with the hypothesis that sc levonorgestrel implants promote structural and functional vascular remodeling through paracrine actions of endometrial stromal and perivascular cells. Most investigators have shown that maximal endometrial VEGF production occurs in the secretory phase of the menstrual cycle in vivo (18, 21, 33) and progestogens increase VEGF mRNA accumulation in vitro (20, 21, 22). The latter effect appears to be mediated by transcriptional regulation of the promoter of this gene (34). To date, attempts to ameliorate the irregular bleeding pattern associated with Norplant using vitamin E and low-dose aspirin have not been effective (35). We speculate that inhibitors of VEGF and other progestogen-modulated endometrial angiogenic factors [e.g. IL-8 (13), angiopoietin 1 (36), adrenomedullin (37), and basic fibroblast growth factor (38)] might reduce the bleeding associated with Norplant and other continuous progestogen-only contraceptives. Notwithstanding the need for careful monitoring to prevent their potential teratogenic effects (39), such antiangiogenic agents currently are undergoing phase III clinical trials as adjuvants for cancer therapy, and these agents could potentially enhance the acceptability of highly effective progestogen-only contraceptive devices.

	Acknowledgments

 
We thank Ana Alvarado for her tireless contribution to subject recruitment and Evelyn Garrett for expert assistance with immunohistochemistry.

	Footnotes

 
This work was supported by National Institutes of Health Grants R01-HD33238 (to R.N.T., J.L.S., C.J.Z., A.P.K., and P.D.D.) and T32-HD07263 (to E.A.P., I.P.R., D.I.L., and J.L.S.).

Present address for E.A.P.: Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Wisconsin School of Medicine, Madison, Wisconsin 53792.

First Published Online December 28, 2004

Abbreviations: CD34, Cluster determination-34; EI, endometrial index; TBS, Tris-buffered saline; VEGF, vascular endothelial growth factor.

Received August 23, 2004.

Accepted December 16, 2004.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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