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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-0724
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 9 3633-3638
Copyright © 2006 by The Endocrine Society

Long-Term Progestin-Only Contraceptives Result in Reduced Endometrial Blood Flow and Oxidative Stress

M. Hickey, G. Krikun, P. Kodaman, Frederick Schatz, C. Carati and C. J. Lockwood

School of Women’s and Infants’ Health (M.H.), University of Western Australia and Women and Infants Research Foundation, King Edward Memorial Hospital, Subiaco 6008, Western Australia; Department of Obstetrics, Gynecology and Reproductive Sciences (G.K., P.K., F.S., C.J.L.), School of Medicine, Yale University, New Haven, Connecticut 06520; and Department of Anatomy and Histology (C.C.), Flinders University of South Australia, Adelaide 5001, South Australia

Address all correspondence and requests for reprints to: Dr. Graciela Krikun, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University, School of Medicine, 333 Cedar Street, New Haven, Connecticut, 06520-8063. E-mail: graciela.krikun{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Because of their safety and efficacy, long-term progestin-only contraceptives (LTPOCs) are well-suited for women with restricted access to health care. However, abnormal uterine bleeding (AUB) causes half of all users to discontinue therapy within 12 months. Endometria of LTPOC-treated patients display aberrant angiogenesis with abnormally enlarged, thin-walled, fragile blood vessels, inflammation, and focal hemorrhage. In this study, similar effects were observed with a new third-generation implantable LTPOC.

Objective: We hypothesized that LTPOC reduces uterine and endometrial blood flow, leading to hypoxia/reperfusion, which triggers the generation of reactive oxygen species. The latter induce aberrant angiogenesis, causing AUB.

Design: Endometrial perfusion was measured by laser-Doppler fluxmetry in women requesting LTPOCs. Endometrial biopsies were obtained for in vivo and in vitro experiments.

Setting: The study was conducted in the Yale University School of Medicine and Family-Planning Center in Western Australia.

Patients: Seven women 18 yr or older requesting implantable LTPOCs were recruited in Western Australia.

Intervention: Women received etonorgestrel implants.

Main Outcome: LTPOC treatment resulted in reduced endometrial perfusion and increased endometrial oxidative damage.

Conclusions: We propose that LTPOCs result in hypoxia reperfusion, which leads to aberrant angiogenesis resulting in AUB.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
CONTINUOUS, LOW-DOSE PROGESTINS offer safe, discrete, and highly effective contraception. Currently available forms include Mirena (Schering, Berlin, Germany), an intrauterine device that releases levonorgestrel for a period of 5 yr; Depo-Provera (Pharmacia/Upjohn Company, Kalamazoo, MI), an injectable form of medroxyprogesterone acetate that persists for 3 months; and Implanon (Organon, Oss, NL), a newly available, single implantable rod providing continuous release of etonogestrel over a 3-yr period. Implanon is a highly effective and popular contraceptive in Europe, Asia, and Australasia because it is easy to insert and remove, making it ideally suited for use by women with limited access to trained medical personnel. These agents can be used safely during lactation and among patients with contraindications to estrogen use. Unfortunately, long-term progestin-only contraceptives (LTPOCs) lead to irregular uterine bleeding in the majority of users. Bleeding disturbances are the main reason for discontinuation of therapy and dissuade others from initiating these contraceptives (1, 2). Therapeutic options are largely restricted to use of supplemental estrogens that are only beneficial for the duration of concomitant use (3).

Endometria from LTPOC-treated patients display dilated fragile vessels that are irregularly distributed across the endometrial surface (4, 5, 6). Camera-guided hysteroscopic biopsies of endometria after 12 months of LTPOC therapy displayed a statistically significant increase in mean lumen width at bleeding compared with those in nonbleeding sites (7). The key regulators of endometrial angiogenesis are vascular endothelial growth factor (VEGF) (8) and angiopoietin-2 (Ang-2) (9). Each molecule has been shown to be up-regulated in endometria treated with LTPOC (9, 10, 11). Because both VEGF and Ang-2 are induced by hypoxia and reactive oxygen species (ROS) (9, 12, 13, 14, 15), we posit that LTPOC induces aberrant angiogenesis by reducing endometrial blood flow, inducing hypoxia, and increasing endometrial oxidative stress (ROS).

Numerous studies have demonstrated that uterine perfusion is regulated by endogenous and exogenous sex steroids (16). However, endometrial perfusion cannot be ascertained reliably from measurements of total uterine perfusion, because the uterine artery also supplies the ipsilateral ovary, the fallopian tube, and the upper vagina as well as the myometrium (17). External Doppler ultrasound measurements of uterine blood flow cannot be extrapolated to the level of the endometrium, because these two vascular beds have been shown to vary independently (18). Laser-Doppler fluxmetry (19, 20) is a relatively noninvasive technique allowing dynamic quantification of perfusion in microvascular beds. This technique provides a relatively simple and highly sensitive method of measuring localized endometrial perfusion, and it appears to be well tolerated. Thus, the aim of this study was to assess the effects of exposure to the LTPOC Implanon on endometrial perfusion as measured by laser fluxmetry and to examine the relationship between perfusion and the expression of endometrial markers of ROS.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Seven women requesting Implanon for contraception were recruited through Family-Planning Western Australia. Subjects were excluded if they had hypertension, vascular disease, coagulopathies, pelvic pathology, myomas, polyps, Asherman’s syndrome, endometrial hyperplasia or carcinoma, or if they used vasoactive drugs or cyclooxygenase inhibitors. All subjects gave informed consent, and the study was approved by the institutional ethics committee of King Edward Memorial Hospital. For the experiments involving cell culture studies, tissue collection was approved by the human investigation committee of the Yale University School of Medicine.

The average age of the subjects was 30 yr (range 21–41 yr). All had been off hormonal contraception for at least 1 month; none had breastfed in the last 3 months. Subjects were advised to use barrier methods of contraception in the control cycle. Their mean weight and height were 67.5 kg (range 64–71 kg) and 1.66 m (range 1.6–1.75 m). Blood pressures were all less than 140/90 and more than 100/60 mm Hg; the patients’ pulse rates were less than 120 and more than 80 bpm. All had normal complete blood counts. Only subjects with regular menstrual cycles confirmed by menstrual diaries were recruited. All endometrial perfusion measurements were made during the mid to late luteal phase.

Perfusion measurements before and after Implanon

Endometrial perfusion was measured by laser-Doppler fluxmetry in the secretory phase of the normal (control) cycle, as well as 4 wk and 6 months after insertion of Implanon. In all cases, Implanon was inserted during the first 3 d of the onset of menses in the following cycle. A TSI Laserflo BPM 403A laser-Doppler instrument (Vasamedics, St. Paul, MN) was used, operating from a laser diode at a wavelength of 780 ± 20 nm, delivering 2 mW at the probe tip. This was equipped with a 2-m long and 2.1-mm diameter flexible endoscopic probe measuring at 90° to the probe axis (TSI PR-436). After the perineum and vagina were prepped with an iodine solution, a sterile speculum was placed in the vagina, and the probe was aseptically introduced into the endocervical and endometrial canals. Perfusion was measured for approximately 5 min at each of five to six sites on the endometrial surface. Measurements were systematically made of anterior, posterior, fundal, right, and left uterine cavity surfaces.

Endometrial biopsies

Two endometrial biopsies were taken from seven subjects. The first was obtained from the secretory phase of the cycle, via pipelle before treatment with LTPOC. The second was obtained hysteroscopically after the perfusion measurement, 1 month after insertion of Implanon. Portions of each specimen were either snap frozen and later used for ELISA measurements or formalin fixed and paraffin embedded for immunohistochemistry evaluation.

Cell culture

Telomerase immortalized human endometrial endothelial cells (HEEC) were cultured in endothelial cell basal medium-2 and supplemented with endothelial cell growth medium-2 (EGM-2) MV (Cambrex Bio Science, Walkersville, MD) and then grown to confluence as previously described (21). For the nitrotyrosine experiments, confluent HEECs were incubated for 48 h in EGM-2 MV under normoxic or hypoxic conditions. For the experiments involving hypoxia, cultures were placed in humidified sealed chambers containing a portable gas oxygen analyzer. The chambers were purged with 5% CO2, 95% N2 for 15 min after the O2 analyzers read 2% O2 (12–14 mm Hg). Sealed chambers were placed in a standard 37 C incubator for 48 h, and the cells were harvested as previously described (21).

Immunoassays

Nitrotyrosine. Nitrotyrosine expression was detected by ELISA in HEEC-lysates as per the manufacturer’s instructions (Bioxytech-Oxis, Portland, OR).

8-Isoprostane. Whole endometrial samples were obtained as described in Endometrial biopsies, sonicated in 0.1 M Tris (pH 7.4) and diluted 1:5 in eicosanoid affinity buffer (Cayman Chemical Company, Ann Arbor, MI) and 8-isoprostane was detected by ELISA as per the manufacturer’s instructions.

Immunohistochemistry

Peroxidase staining was carried out as previously described by our laboratory (9) on formalin-fixed, paraffin-embedded tissues obtained at the time of perfusion as described above. Nitrotyrosine was detected with an antibody from Sigma-Aldrich (St. Louis, MO) and 8-hydroxydeoxyguanosine (8-OHdG) with an antibody from JaICA (Fukuroi City, Japan). Negative control slides were carried out with isotype matched preimmune serum.

Statistics

For laser fluxmetry, Fast Fourier Transforms were performed with the Chart 5 program (AD Instruments, New Castle, Australia), and the Power Density Spectrum was calculated for representative areas of the recording (~60 sec) per patient and group averages calculated. All other statistical analyses were carried out with the SigmaStat Program (SPSS Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Endometrial perfusion studies

Marked (up to 8-fold) reductions in endometrial perfusion were observed after Implanon insertion. Figure 1Go provides fluxmetry data and spectral analysis for a representative patient. Note that, before insertion, the power spectrum displays a wide range of frequencies (i.e. red blood cell velocities) and high amplitudes (i.e. volume of blood flow). However, after Implanon insertion, power spectrum analysis detected decreases in both the range of frequencies and amplitude of the Doppler spectra. Implanon insertion significantly decreased the power density spectrum of endometrial blood flow. Consistent with significantly reduced endometrial blood flow after LTPOC administration, median (range) values before insertion were 455.5 (70.9–1725.6) ml/min/102 compared with 57.1 (0.43–792.6) ml/min/102 and 21.2 (5.2–109.1) ml/min/102 at 1 and 6 months postinsertion, respectively (ANOVA P = 0.01). This reduction could reflect either lower velocity and volume of flow or fewer vessels. However, given the increased density of dilated vessels in LTPOC patients, these findings are strongly suggestive of reduced uterine and/or conduit artery flow.


Figure 1
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  		FIG. 1. Laser Doppler endometrial perfusion. Measurements are shown before Implanon insertion and after 1 month (second visit) and 6 months (third visit) of treatment. Top panel, Representative blood flow measurements in milliliters per minute per 10 mm2 (60 sec each); bottom panel, their representative power density spectra (power = milliliters per minute per 10 mm2 vs. frequency in hertz).

 
As expected, bleeding patterns were variable in the first month after Implanon insertion with two of seven women having either amenorrhea or infrequent bleeding/spotting (<5 d of 30), two of seven exhibited an average number of bleeding/spotting days (≤7 d), and three of seven reported frequent or prolonged bleeding/spotting (>7 d). The subject with the most prolonged and frequent bleeding after Implanon also displayed the most marked decrease in perfusion (88%).

ROS generation and lipid peroxidation studies

Abnormal surface vascular appearances were seen in all subjects after Implanon insertion (Fig. 2Go). Immunohistochemistry qualitatively demonstrated the induction of nitrotyrosine and 8-OHdG (Fig. 3Go) after Implanon insertion. These are both specific markers of ROS generation.


Figure 2
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  		FIG. 2. Hysteroscopy. Images illustrate superficial dilated vessels with focal hemorrhage in Implanon users manifesting frequent and prolonged bleeding. These abnormal vascular patterns suggest aberrant angiogenesis.

 

Figure 3
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  		FIG. 3. Immunostaining for nitrotyrosine and 8-OHdG in control and LTPOC-treated endometrium. Immunostaining for nitrotyrosine in human control (A) endometrium, and that exposed to high dose LTPOC (B). Similar staining was conducted for 8-OHdG in control (C) and LTPOC-treated endometria. Brown peroxidase staining identifies antigens.

 
LTPOC induces lipid peroxidation

The isoprostanes are members of the prostanoid family of eicosanoids produced by the nonenzymatic, free-radical-induced peroxidation of arachidonic acid and tissue phospholipids (22, 23). Several members of this family, in particular 8-isoprostane, display biological activity that correlates with the pathophysiological induction of inflammation and postischemic reperfusion (23). Figure 4Go indicates that, compared with samples obtained before LTPOC, endometrial specimens obtained 1 month after Implanon treatment contained markedly elevated levels of 8-isoprostane, indicating that Implanon treatment enhances endometrial lipid peroxidation.


Figure 4
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  		FIG. 4. Implanon affects endometrial production of 8-isoprostane. 8-Isoprostane was measured as described in Subjects and Methods in homogenized hysteroscopic sections before and after 1 month of Implanon treatment. Mean ± SEM, n = 7 patients, P < 0.001.

 
In vitro studies: effects of hypoxia on nitrotyrosine expression on cultured HEECs

This study demonstrates that culturing HEECs under hypoxic conditions significantly induced nitrotyrosine levels (Fig. 5Go). Nitrotyrosine is formed from the direct interaction of the peroxinitrite anion radical with tyrosine residues. These findings indicate that hypoxia results in oxidative damage to endometrial endothelial cells.


Figure 5
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  		FIG. 5. Effects of hypoxia on the production of nitrotyrosine by HEECs. HEECs were grown to confluence and incubated for 48 h, and nitrotyrosine expression was detected by ELISA as described in Subjects and Methods (n = 3, P < 0.02). C, Control; HX, hypoxia.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Implanon is a long-acting implantable contraceptive that has proven to be highly effective in both developed and developing countries (24, 25). The current study explores the mechanisms of irregular bleeding resulting from LTPOC treatment by analyzing women before and after Implanon insertion. We now show that the LTPOC Implanon significantly reduces endometrial perfusion and induces oxidative damage. The latter is indicated by elevated levels of endometrial lipid peroxidation, 8-OHdG expression, a marker of oxidative DNA damage, and nitrotyrosine, a marker of oxidative protein damage in endometrial specimens obtained 1 month after Implanon insertion. We propose that these changes play an integral role in producing abnormally distended, fragile vessels that are the source of abnormal uterine bleeding (AUB) associated with LTPOC use. Furthermore, we demonstrate that, in vitro, hypoxia induces the expression of nitrotyrosine, a marker of peroxinitrite anion generation in cultured endometrial microvascular endothelial cells.

The results of the current study, together with previous findings from our laboratory (9), are strongly suggestive that oxidative stress is a critical component in the genesis of endometrial pathophysiology observed in LTPOC users.

Previously, we hypothesized that hypoxia/reperfusion induces free radical production that inhibits endometrial expression of Ang-1, a vessel-stabilizing factor, leaving unopposed the effects of endothelial Ang-2, a vessel-branching and permeability factor. Immunohistochemical studies confirmed selective decreases in stromal cell Ang-1 in LTPOC-exposed endometrium (9). Additionally, we indirectly assessed the possibility of oxidative damage after LTPOC treatment by demonstrating that the stress-activated kinases stress-activated protein kinase/c-Jun N-terminal kinase and p38 were phosphorylated in tissues. The current study indicates that exposure to Implanon profoundly reduces endometrial perfusion and induces ROS, and lipid peroxidation, DNA, and protein damage. The resulting hypoxic environment results in an imbalance in angiogenic factors Ang-1 and Ang-2 as previously described (9).

Alterations in the production or distribution of VEGF have been proposed to be related to LTPOC-induced AUB. One study demonstrated that the endometrial glandular and stro-mal cell VEGF staining indices were significantly greater in endometria after LTPOC treatment than in untreated endometria; however, no correlation was found between the VEGF staining index and endometrial microvascular density (10). In contrast, in women treated with Implanon, only endometrial stromal cell VEGF immunostaining was found to be significantly elevated compared with controls with a positive correlation observed between stromal-VEGF immunoreactivity and endothelial cell density (26). It is well known that the regulation of VEGF is exquisitely sensitive to oxygen concentrations as well as ROS (27, 28). Although physiological VEGF levels promote angiogenesis, overexpressed VEGF induces endothelial vascular "leakiness", bleeding, and perivascular extracellular matrix dissolution (29). Thus, our findings of reduced endometrial perfusion, hypoxia, and ROS generation after Implanon placement provide a clear mechanism for increased endometrial VEGF production in LTPOC users.

The aberrant vascular patterns and bleeding profiles associated with LTPOC treatment stand in contrast to the only physiological state in which the endometrium is exposed to native progesterone for prolonged periods, to wit, pregnancy. However, in pregnancy, trophoblast modification of uterine spiral arteries maintains adequate endometrial blood flow. Moreover, in pregnancy there are abundant levels of circulating estrogens, as is the case with the luteal phase. Although synthetic progestins may mimic many of the effects of native progesterone, progestins may react differently with progesterone receptors or via alternate mechanisms. Indeed, a study by Simoncini et al. (30) demonstrated that, although native progesterone significantly increased nitric oxide synthesis on rat aortas via transcriptional and nontranscriptional mechanisms, medroxyprogesterone acetate was devoid of such effects. Moreover, the authors found that, when used together with physiological estradiol concentrations, native progesterone potentiated, whereas medroxyprogesterone acetate impaired, estrogen signaling via MAPK and phosphatidylinositol-3 kinase pathways. Thus, it is possible that the endometrial vascular changes observed after LTPOCs reflect biological differences among native progesterone vs. synthetic progestins.

In summary, the present study strongly demonstrates that endometrial hypoxia with or without reperfusion and generation of ROS accompanies LTPOC use. These perturbations likely drive the enhanced angiogenesis noted in these patients, leading to aberrant vessels and bleeding. Figure 6Go represents a schematic of this hypothesis.


Figure 6
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  		FIG. 6. Central scheme. LTPOCs result in hypoxia/reperfusion (HX/RP), ROS generation, and altered angiogenic factor expression leading to damaged vessels and AUB. ?, Other pathways not yet elucidated may exacerbate this mechanism.

 


   Footnotes
 
This work was supported in part by a grant from the National Institutes of Health (RO1HD33937-06 to C.J.L.).

First Published Online June 6, 2006

Abbreviations: Ang-2, Angiopoietin-2; AUB, abnormal uterine bleeding; HEEC, human endometrial endothelial cell; LTPOC, long-term progestin-only contraceptive; 8-OHdG, 8-hydroxydeoxyguanosine; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

Received April 4, 2006.

Accepted May 31, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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