Effects of levonorgestrel-releasing intra-uterine system on the expression of vascular endothelial growth factor and adrenomedullin in the endometrium in adenomyosis

J.B. Laoag-Fernandez1, T. Maruo1,5, P. Pakarinen2, I.M. Spitz3 and E. Johansson4

1 Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe, Japan, 2 Department of Obstetrics and Gynecology, University of Helsinki, Finland, 3 Institute of Hormone Research, Shaare Zedek Medical Center, Jerusalem, Israel and 4 Population Council, New York, USA

5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. e-mail: maruo{at}kobe-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The levonorgestrel-releasing intra-uterine system (LNg-IUS) has been used to control menorrhagia, but irregular bleeding during the first 3 months of use was the most notable side effect. Endometrial angiogenesis is believed to be regulated by angiogenic factors. The study aim was to evaluate the effects of LNg-IUS on vascular endothelial growth factor (VEGF) and adrenomedullin (AM) expression in the endometrium. METHODS: VEGF and AM expression were analysed using the avidin-biotin immunoperoxidase method on endometrial curettage specimens from menorrhagic women associated with adenomyosis before and 3 months after LNg-IUS insertion. RESULTS: VEGF expression was abundant both in the endometrial glands and stroma before LNg-IUS insertion, but became scanty 3 months after insertion. No immunostaining for AM was noted in the endometrial glands and stroma before LNg-IUS insertion, whereas AM immunostaining became prominent in the endometrial glands and stroma 3 months after LNg-IUS use. CONCLUSIONS: This is the first study to demonstrate that LNg-IUS insertion results in decreased expression of VEGF and increased expression of AM in the endometrial glands and stroma after 3 months of use. The results obtained suggest that the increase in AM expression in the endometrium may be responsible for the frequent occurrence of irregular bleeding during the initial 3 months of LNg-IUS use.

Key words: adrenomedullin/endometrium/levonorgestrel-releasing intra-uterine system/vascular endothelial growth factor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The levonorgestrel-releasing intra-uterine system (LNg-IUS) has been used as one of the therapeutic modalities for menorrhagia (Kerstin Andersson and Rybo, 1990Go; Barrington and Bowen-Simpkins, 1997Go; Ikomi and Gupta, 1998Go; Fedele et al., 1999Go; Wildemeersch and Schacht, 2001Go) associated with adenomyosis (Fedele et al., 1997Go). It has been noted that LNg-IUS insertion results in a decrease in endometrial proliferation and an increase in apoptosis in endometrial glands and stroma (Maruo et al., 2001Go). This causes the atrophic change of the endometrium and leads to the reduction in menorrhagia. However, LNg-IUS has an unwanted side effect, which is the occurrence of irregular bleeding that is mostly evident during the first 3 months of use (Sivin and Stern, 1994Go; Sturridge and Guillebaud, 1997Go). Irregular bleeding induced by sex steroids has been implicated to be due to aberrant development of the vascularity in the endometrium (Fraser and Lunn, 2000Go; Oehler et al., 2000Go; Gargett et al., 2001Go; Koolwijk et al., 2001Go; Smith, 2001Go). Endometrial angiogenesis is believed to be regulated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) (Gargett and Rogers, 2001Go; Moller et al., 2001Go) and adrenomedullin (AM) (Nikitenko et al., 2000Go).

Although VEGF in the endometrium exists in several isoforms (Charnock-Jones et al., 1993Go; Torry et al., 1996Go), VEGF121 and VEGF165 are predominant (Smith, 1995Go). Although there have been several reports on VEGF expression in the endometrium, the pattern of VEGF expression in the endometrium during the menstrual cycle is not yet established (Li et al., 1994Go; Shifren et al., 1996Go; Lau et al., 1998Go). On the other hand, AM has been implicated to have vital roles in the biology of the endometrium (Michishita et al., 1999Go), uterine cervix (Upton et al., 1997Go; Li et al., 2001Go) and placenta (Moriyama et al., 2002Go). In a previous study on the normal human endometrium involving the proliferative, secretory and post-menopausal endometrial samples, it was also demonstrated that AM expression in the endometrium became apparent in the late proliferative phase and became abundant in the secretory phase, concomitant with angiogenesis of the endometrium (Laoag-Fernandez et al., 2000Go). Nevertheless, several investigators have demonstrated that angiogenesis occurs in three distinct stages during the menstrual cycle (Rogers and Gargett, 1999Go): (i) for the repair of vascular bed during menstruation (Markee, 1978Go); (ii) for rapid endometrial growth in the proliferative phase (Rogers et al., 1993Go) and (iii) for growth and coiling of spiral arterioles which occurs during the secretory phase (Kaiserman-Abramof and Padykula, 1989Go). The present study was conducted to elucidate the effects of LNg-IUS on the expression of angiogenic factors such as VEGF and AM in the endometrium in menorrhagic women associated with adenomyosis after 3 months of use.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Endometrial curettage specimens
Specimens were obtained from 15 women of mean age 32.5 (range 26–39) years. All patients had recurrent menorrhagia associated with adenomyosis diagnosed by magnetic resonance imaging. The LNg-IUS used (Oy Leiras, Turku, Finland) has a T-shaped polyethylene skeleton, the LNg (50% by weight) being located in the cylindrical sleeve and dispersing homogeneously from a polydimethylsiloxane reservoir which is covered by a rate-limiting membrane on the vertical arm of the device. The total amount of LNg in the device was 46 mg, and the rate of release was 20 µg per day. The IUS was inserted in each subject within 7 days of the start of menstrual flow. After having obtained informed consent from each subject, endometrial curettage specimens were obtained before and 3 months after LNg-IUS insertion. Endometrial specimens before and after LNg-IUS insertion were taken from day 4 to day 7 after the onset of menstruation and menstruation-like bleeding respectively. Although irregular spotting was common during the initial 2 months of LNg-IUS use (ranging from 14 to 27 days during the first month and from 7 to 18 days during the second month), irregular bleeding and spotting were not evident in most of the subjects after 3 months of LNg-IUS use. Endometrial specimens taken 3 months after LNg-IUS insertion were from those subjects who had menstruation-like bleeding in their menstrual diaries. The endometrial specimens obtained were fixed in 4% buffered neutral formalin, dehydrated and embedded in paraffin. Sections (4 µm diameter) were deparaffinized and taken for immunohistochemical analysis.

Immunohistochemical analysis
Immunohistochemical staining was carried out using the avidin-biotin immunoperoxidase method, with the polyvalent immunoperoxidase kit (Omnitag, Lipshaw, MI, USA) as described previously (Maruo and Mochizuki, 1987Go). Rabbit polyclonal antibodies against human VEGF (SC 152; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and human AM (Peninsula Laboratories, Belmont, CA, USA) were used as primary antibodies. The anti-VEGF antibody was raised using a peptide corresponding to amino acids 1–20 of the N-terminal region of VEGF; this reacts with VEGF121, VEGF165 and VEGF189. The anti-VEGF and the anti-AM antibodies were diluted 1:200 and 1:500 respectively before use. The first incubation was carried out with the primary antibody, followed by the second incubation with biotinylated polyvalent antibody. The third incubation was carried out with avidin-horseradish peroxidase. Thereafter, the chromogenic reaction was developed by incubating with a freshly prepared solution of 3-amino-9-ethylcarbazole and hydrogen peroxide. The sections were counterstained with Harris’ haematoxylin, mounted in glycerine phosphate buffer solution, and examined microscopically.

The following control procedures were undertaken to assure specificity of the immunological reactions: adjacent control sections were subjected to the same immunoperoxidase method, except that the primary antibodies to VEGF and AM were replaced by non-immune rabbit serum at the same dilution as the specific primary antibody. The replacement of the specific antibody with non-immune rabbit serum resulted in a lack of positive immunostaining.

Images of VEGF and AM staining were randomly captured using a digital camera (Olympus DP50, Japan) mounted onto a microscope and attached to a digital multiscan display (Panasonic, Japan). Four images per section, equivalent to 2.28 mm2 of tissue area were captured. The intensity of immunostaining for VEGF and AM were evaluated by more than two observers, and was graded as: (–) for no immunostaining; (+) for weak but definitely detectable immunostaining; (++) for moderate immunostaining; and (+++) for intense immunostaining. The epithelium and stroma were scored separately and the scoring was carried out in blinded fashion. All samples were scored in one session in order to reduce variability. Sections were examined by two reviewers, and the final score credited to each sample was agreed upon by the two examiners.

Statistical analysis
Analysis of VEGF and AM immunostaining before and 3 months after LNg-IUS insertion was carried out using the Mann–Whitney U-test. A P-value < 0.05 was considered to be statistically significant.

Regression analyses were carried out for VEGF and AM expression levels, the number of spotting or bleeding days during the first 3 months of LNg-IUS use and the duration since the last spotting or bleeding episode.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In all specimens taken before LNg-IUS insertion the endometrium was, histologically, in the early proliferative phase: the endometrium had endometrial glands which appeared as simple tubules and were lined by low columnar epithelium, while the endometrial stroma was dense. Mitotic activity was noticeable in both the endometrial glands and endometrial stroma. By contrast, in all specimens taken 3 months after LNg-IUS insertion the endometrium appeared somewhat different from those taken prior to LNg-IUS insertion, although it was concomitant with the early proliferative phase with regards to the timing of the biopsy. After LNg-IUS insertion, the endometrial glands became atrophic, with evidence of thinning of the mucosa, and the endometrial stroma became swollen with decidualization in some areas.

Before LNg-IUS insertion, VEGF was immunolocalized in the endometrial glands and stroma. The immunohistochemical staining for VEGF was more prominent in the epithelium of the endometrial glands than that in the endometrial stroma (Figure 1A). By contrast, at 3 months after LNg-IUS insertion, immunohistochemical staining for VEGF in the endometrium became scanty, being slightly notable in the epithelium of the endometrial glands and stroma (Figure 1B). The scoring system was based on the randomly taken pictures analysed by the two observers. A statistical comparison showed there were significant differences in VEGF expression levels between biopsies taken before and those after LNg-IUS insertion (epithelium, P = 0.032; stroma, P = 0.025). Adjacent control sections before (Figure 1C) and after (Figure 1D) LNg-IUS insertion revealed negative immunostaining.



View larger version (130K):
[in this window]
[in a new window]
 
Figure 1. (A,B) Immunohistochemical localization of VEGF in the endometrium before (A) and 3 months after (B) LNg-IUS insertion. VEGF was present both in the endometrial glands and stroma, but was more abundant in the endometrial glands before LNg-IUS insertion. VEGF expression became scarcely apparent in the endometrial glands and stroma 3 months after LNg-IUS insertion. (C,D) Adjacent control sections before (C) and after (D) LNg-IUS insertion were negative for immunostaining. E = endometrial gland epithelium; S = endometrial stroma. Scale bar = 10 µm. Original magnification, x400.

 
On the other hand, although there was no appreciable staining for AM in the endometrium before LNg-IUS insertion (Figure 2A), immunohistochemical staining for AM became prominent in both the endometrial glands and endometrial stroma at 3 months after LNg-IUS insertion (Figure 2B). AM expression in the endometrium was more abundant in the epithelium of the endometrial glands than that in the endometrial stroma. Statistical analysis revealed significant differences in levels of AM expression between biopsies taken before and those after LNg-IUS insertion (epithelium, P = 0.024; stroma, P = 0.019). Adjacent control sections before (Figure 2C) and after (Figure 2D) LNg-IUS insertion revealed negative immunostaining. The overall final scores are summarized in Table I.



View larger version (136K):
[in this window]
[in a new window]
 
Figure 2. (A,B) Immunohistochemical localization of AM in the endometrium before (A) and 3 months after (B) LNg-IUS insertion. There was no evident immunostaining for AM in the endometrial glands and stroma before LNg-IUS insertion. AM staining became apparent both in the endometrial glands and stroma 3 months after LNg-IUS insertion, but was more abundant in the endometrial glands. (C,D) Adjacent control sections before (C) and after (D) LNg-IUS insertion were negative for immunostaining. E = endometrial gland epithelium; S = endometrial stroma. Scale bar = 10 µm. Original magnification, x400.

 

View this table:
[in this window]
[in a new window]
 
Table I. Effects of LNg-IUS on vascular endothelial growth factor (VEGF) and adrenomedullin (AM)
 
Regression analyses showed no correlation between the number of spotting or bleeding days during the first 3 months of LNg-IUS use and the expression levels for VEGF [R2 = 0.173, P > 0.1675 (not significant) for epithelium; and R2 = 0.026, P > 0.7054 (not significant) for stroma] and AM [R2 = 0.057, P > 0.5683 (not significant) for epithelium; and R2 = 0.002, P > 0.9104 (not significant) for stroma]. There was a similar lack of correlation between the number of bleeding or spotting days prior to biopsy taken after 3 months of LNg-IUS insertion and the expression levels for VEGF [R2 = 0.078, P > 0.2538 (not significant) for epithelium; and R2 = 0.015, P > 0.3329 (not significant) for stroma] and AM [R2 = 0.023, P > 0.7215 (not significant) for epithelium; and R2 = 0.026, P > 0.3128 (not significant) for stroma].


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study showed that LNg-IUS insertion affected the expression of VEGF and AM in the endometrium. VEGF and AM have been reported to have several similarities, with both of them being induced by hypoxia, increasing vascular permeability and also angiogenic (Shweiki et al., 1993Go; Nagata et al., 1999Go; Hague et al., 2000Go). However, differences exist between the two factors; VEGF in the endometrium has been shown to exhibit estrogen-dependent induction (Zhang et al., 1995Go), while AM in the endometrium could be induced by tamoxifen but not by estrogen (Zhao et al., 1998Go). Moreover, the mitogenic activity of VEGF is restricted mainly in the endothelial cells, while AM exerts widespread effects on benign and malignant tumour cells (Miller et al., 1996Go). Although VEGF and AM both inhibit endothelial cell apoptosis, the effect of AM was not as potent as that of VEGF (Sata et al., 2000Go). It should be emphasized, however, that VEGF and AM are key players in endometrial angiogenesis (Rees et al., 1999Go). Angiogenesis has been demonstrated to occur in three distinct stages during the menstrual cycle.

The results obtained in the present study revealed that LNg-IUS insertion down-regulates VEGF expression in the endometrial glands and stroma after 3 months of use. Similar to the results reported by others (Charnock-Jones et al., 2000Go), an abundant immunostaining intensity for VEGF was also noted in the decidualized cells. A number of reports have shown that endometrial VEGF expression during the menstrual cycle is inconsistent (Lau et al., 1998Go; Sugino et al., 2002Go). Several investigators have shown that glandular VEGF immunoreactivity was lower during the proliferative phase than in the secretory phase, and that stromal VEGF immunoreactivity was low across the menstrual cycle (Shifren et al., 1996Go; Smith, 1996Go; Torry et al., 1996Go). However, others (Li et al., 1994Go) have demonstrated no variation in glandular VEGF immunoreactivity across the menstrual cycle, with higher stromal VEGF immunoreactivity in the proliferative phase than in the secretory phase. By contrast, one group (Nayak and Brenner, 2002Go) have shown a peak in stromal VEGF expression in the proliferative phase with peak glandular VEGF expression during the secretory phase, whereas others (Greb et al., 1997Go) carried out an in-vivo study in monkeys to reveal that VEGF immunostaining in endometrial stromal cells was augmented by progesterone treatment. Another group, using in-situ hybridization, showed that VEGF mRNA signals in the stroma were higher in the proliferative phase than in the secretory phase (Charnock-Jones et al., 1993Go). Differences among the conflicting reports on VEGF expression may be due to the variations in the sex steroid hormone levels at the time of endometrial sampling, or to variations on the areas biopsied (Rogers et al., 1998Go).

The findings of the present study are in agreement with those produced by others (Macpherson et al., 1999Go), wherein Implanon and Mircette—both of which are progestin-containing contraceptives—significantly reduced VEGF expression in the endometrium. These authors also showed that there was a negative correlation between glandular VEGF expression and glandular progesterone receptor expression. In contrast to these findings and those of the present study, another group (Lau et al., 1998Go) reported that VEGF expression in the endometrium in users of a subdermal LNg implant, Norplant, was higher than expression in non-users of Norplant. The reason for the higher expression of VEGF in the endometrium of the former group remains unclear. As endometrial LNg concentrations among LNg-IUS users were considerably higher than when LNg was used as an oral contraceptive (including subdermal implants; unpublished data), the major differences in endometrial LNg concentration when LNg was administered either orally, subdermally or by the intra-uterine route might explain the variation in endometrial VEGF expression during LNg-IUS and Norplant use.

VEGF, which is a key regulator of cyclical angiogenesis, is known to be influenced by multiple factors such as sex steroids and local regulators (Sharkey et al., 2000Go). Although the present study did not include immunostaining of endometrial endothelial cells, several studies (Rogers et al., 1993Go; Goodger Macpherson and Rogers, 1994Go; Macpherson et al., 1999Go) showed the lack of change in the endothelial cell number and intensity of stromal VEGF staining during the menstrual cycle, implicating non-steroidal regulation of these parameters. The use of different progestins resulted in various changes in the endothelial cell number. Neither Implanon nor Mircette use caused any change in endometrial endothelial cell number (Macpherson et al., 1999Go), whereas Norplant increased endometrial endothelial cell number. The use of Norplant might reduce the rate of endothelial cell death (Goodger Macpherson and Rogers, 1994Go; Hickey et al., 1999Go). Treatment with higher doses of norethisterone or medroxyprogesterone acetate significantly decreased endometrial vascular density (Song et al., 1995Go). Furthermore, there seems to be an apparent dissociation between endothelial cell number and endometrial bleeding (Macpherson et al., 1999Go).

Although little is known about the possible relationship between AM expression in the endometrium and progestin use, the present study demonstrated that the use of LNg-IUS up-regulates AM expression in the endometrium. At 3 months after LNg-IUS insertion, AM expression became abundant in endometrial glands and stroma. However, it has been reported (Hague et al., 2002Go) that immunostaining for AM in the endometrium in women without hormonal treatment was positive in the endometrial glands and stroma, but was lessened at 3 years after LNg-IUS insertion. The difference between the present results and those of this group (Hague et al., 2002Go) might be explained by the duration of use of LNg-IUS and the different antibodies used. It has been shown previously that AM expression in the endometrium became abundant in the secretory, progesterone-dominant phase of the normal menstrual cycle (Laoag-Fernandez et al., 2000Go), while AM expression in the ovary became abundant in granulosa lutein cells (Moriyama et al., 2000Go). Progesterone has been implicated to be responsible for the release of AM in the rat (Jerat and Kaufman, 1998Go). These studies suggest that a positive correlation between AM expression and progesterone may exist in the endometrium and corpus luteum.

A previous study revealed abundant expression for AM in the stromal compartment in the post-menopausal endometrium (Laoag-Fernandez et al., 2000Go). The post-menopausal endometrium and the endometrium exposed to LNg for 3 months are apparently similar as far as glandular epithelial lining is concerned. Although both are lined by low columnar inactive epithelium, the stroma of the LNg-exposed endometrium becomes swollen and decidualized (Silverberg et al., 1986Go; Barbosa et al., 1990Go), while the post-menopausal endometrium has a fibrous stroma with diminished cellularity (Herbst et al., 1992Go). On the other hand, it has been reported (Zhao et al., 1998Go) that abnormal uterine bleeding induced by treatment with tamoxifen, an anti-estrogen, might be attributable to the induction of AM in the endometrium. In this connection, up-regulation of AM in cells was noted to be a defence mechanism of the cells against injuries (Shichiri et al., 1999Go; Sata et al., 2000Go; Filippatos et al., 2001Go). Although the exact mechanism is unknown, the increased expression of AM in the endometrium may be responsible for the frequent occurrence of irregular bleeding during the initial 3 months of LNg-IUS use.

In conclusion, the results of the current study demonstrated differences in the expressions of VEGF and AM in the endometrium in LNg-IUS users. LNg-IUS insertion down-regulates VEGF expression in the endometrium, but up-regulates AM expression in the endometrium after 3 months of use. It is possible that VEGF may not be involved in the occurrence of irregular bleeding as opposed to that of AM which may participate in the occurrence of irregular bleeding during the initial 3 months of LNg-IUS use.


    Acknowledgements
 
The authors thank Dr Pekka Lahteenmaki for support and kind donation of the LNg-IUS. This study was supported in part by a grant from the International Committee of Contraceptive Research of the Population Council, New York, USA.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Barbosa, I., Bakos, O., Olsson, S.E., Odlind, V. and Johansson, E.D. (1990) Ovarian function during use of a levonorgestrel-releasing IUD. Contraception, 42, 51–66.[ISI][Medline]

Barrington, J.W. and Bowen-Simpkins, P. (1997) The levonorgestrel intrauterine system in the management of menorrhagia. Br. J. Obstet. Gynaecol., 104, 614–616.[ISI][Medline]

Charnock-Jones, D.S., Sharkey, A.M., Rajput-Williams, J., Burch, D., Schofield, J.P., Fountain, S.A., Boocock, C.A. and Smith, S.K. (1993) Identification and localization of alternately spliced mRNAs for vascular endothelial growth factor in human uterus and estrogen regulation in endometrial carcinoma cell lines. Biol. Reprod., 48, 1120–1128.[Abstract]

Charnock-Jones, D.S., Macpherson, A.M., Archer, D.F., Leslie, S., Makkink, W.K., Sharkey, A.M. and Smith, S.K. (2000) The effect of progestins on vascular endothelial growth factor, oestrogen receptor and progesterone receptor immunoreactivity and endothelial cell density in human endometrium. Hum. Reprod., 15 (Suppl. 3), 85–95.

Fedele, L., Portuese, A., Bianchi, S., Raffaelli, R., Portuese, A. and Dorta, M. (1997) Treatment of adenomyosis-associated menorrhagia with a levonorgestrel-releasing intrauterine device. Fertil. Steril., 68, 426–429.[CrossRef][ISI][Medline]

Fedele, L., Gammaro, L. and Bianchi, S. (1999) Levonorgestrel-releasing intrauterine device for the treatment of menometrorrhagia in the woman on hemodialysis. N. Engl. J. Med., 341, 541.

Filippatos, G., Gangopadhyay, N., Lalude, O., Darameswaran, N., Said, S., Spielman, W. and Uhal, B. (2001) Regulation of apoptosis by vasoactive peptides. Am. J. Physiol. Lung Cell. Mol. Physiol., 281, L749–L761.

Fraser, H.M. and Lunn, S.F. (2000) Angiogenesis and its control in the female reproductive system. Br. Med. Bull., 56, 787–797.[Abstract]

Gargett, C.E. and Rogers, P.A. (2001) Human endometrial angiogenesis. Reproduction, 121, 181–186.[Abstract/Free Full Text]

Gargett, C., Lederman, F., Heryanto, B., Gambino, L. and Rogers, P.A.W. (2001) Focal vascular endothelial growth factor correlates with angiogenesis in human endometrium. Role of intravascular neutrophils. Hum. Reprod., 16, 1065–1075.[Abstract/Free Full Text]

Goodger Macpherson, A.M. and Rogers, P.A.W. (1994) Endometrial endothelial cell proliferation during the menstrual cycle. Hum. Reprod., 9, 1647–1651.[Abstract]

Greb, R.R., Heikinheimo, O., Williams, R.F., Hodgen, G.D. and Goodman, A.L. (1997) Vascular endothelial growth factor in primate endometrium is regulated by oestrogen-receptor and progesterone-receptor ligands in vivo. Hum. Reprod., 12, 1280–1292.[CrossRef][ISI][Medline]

Hague, S., Zhang, L., Oehler, M.K., Manek, S., MacKenzie, I.Z., Bicknell, R. and Rees, M.C.P. (2000) Expression of hypoxically regulated angiogenic factor adrenomedullin correlates with uterine leiomyoma vascular density. Clin. Cancer Res., 6, 2808–2814.[Abstract/Free Full Text]

Hague, S., Mackenzie, I.Z., Bicknell, R. and Rees, M.C.P. (2002) In-vivo angiogenesis and progestogens. Hum. Reprod., 17, 786–793.[Abstract/Free Full Text]

Herbst, A.L., Mishell, D.R., Stenchever, M. and Droegemueller, W. (1992) Comprehensive Gynecology. 2nd edn. Mosby Year-book, St Louis, Missouri, USA.

Hickey, M., Simbar, M., Markham, R., Young, L., Manconi, F., Russell, P. and Fraser, I.S. (1999) Changes in vascular basement membrane in the endometrium of Norplant users. Hum. Reprod., 14, 716–721.[Abstract/Free Full Text]

Ikomi, A. and Gupta, N. (1998) Randomized controlled trial exists in levonorgestrel intrauterine system for menorrhagia. Br. Med. J., 317, 1250.

Jerat, S. and Kaufman, S. (1998) Effect of pregnancy and steroid hormones on plasma adrenomedullin levels in the rat. Can. J. Physiol. Pharmacol., 76, 463–466.[CrossRef][ISI][Medline]

Kaiserman-Abramof, I.R. and Padykula, H.A. (1989) Angiogenesis in the postovulatory primate endometrium: the coiled arteriolar system. Anat. Rec., 224, 479–489.[ISI][Medline]

Kerstin Andersson, J. and Rybo, G. (1990) Levonorgestrel-releasing intrauterine device in the treatment of menorrhagia. Br. J. Obstet. Gynaecol., 97, 690–694.[ISI][Medline]

Koolwijk, P., Kapiteijn, K., Molenaar, B., Van Spronsen, E., Van der Vecht, B., Helmerhorst, F. and Van Hinsbergh, V. (2001) Enhanced angiogenic activity and urokinase-type plasminogen activator expression by endothelial cells isolated from human endometrium. J. Clin. Endocrinol. Metab., 86, 3359–3367.[Abstract/Free Full Text]

Laoag-Fernandez, J.B., Otani, T. and Maruo, T. (2000) Adrenomedullin expression in the human endometrium. Endocrine, 12, 15–19.[CrossRef][ISI][Medline]

Lau, T.M., Affandi, B. and Rogers, P.A.W. (1998) The effects of levonorgestrel implants on vascular endothelial growth factor expression in the endometrium. Mol. Hum. Reprod., 5, 57–63.[ISI]

Li, X.F., Gregory, J. and Ahmed, A. (1994) Immunolocalisation of vascular endothelial growth factor in human endometrium. Growth Factors, 11, 277–282.[ISI][Medline]

Li, Z., Takeuchi, S., Otani, T. and Maruo, T. (2001) Implications of adrenomedullin expression in the invasion of squamous cell carcinoma of the uterine cervix. Int. J. Clin. Oncol., 6, 263–270.[CrossRef]

Macpherson, A.M., Archer, D.F., Leslie, S., Charnock-Jones, D.S., Makkink, W.K. and Smith, S.K. (1999) The effect of etonorgestrel on VEGF, oestrogen, and progesterone receptor immunoreactivity and endothelial cell number in human endometrium. Hum. Reprod., 14, 3080–3087.[Abstract/Free Full Text]

Markee, J.E. (1978) Menstruation in intraocular endometrial implants in the rhesus monkey. Am. J. Obstet. Gynecol., 131, 558–559.[ISI][Medline]

Maruo, T. and Mochizuki, M. (1987) Immunohistochemical localization of epidermal growth factor receptor and myc oncogene product in the human placenta: implication for trophoblast proliferation and differentiation. Am. J. Obstet. Gynecol., 156, 721–727.[ISI][Medline]

Maruo, T., Laoag-Fernandez, J.B., Pakarinen, P., Murakoshi, H., Spitz, I.M. and Johansson, E. (2001) Effects of levonorgestrel-releasing intrauterine system on proliferation and apoptosis in the endometrium. Hum. Reprod., 16, 2103–2108.[Abstract/Free Full Text]

Michishita, M., Minegishi, T., Abe, K., Kangawa, K., Kojima, M. and Ibuki, Y. (1999) Expression of adrenomedullin in the endometrium of the human uterus. Obstet. Gynecol. 93, 66–70.[Abstract/Free Full Text]

Miller, M.J., Martinez, A., Unsworth, E.J., Thiele, C.J., Moody, T.W., Elsasser, T. and Cuttitta, F. (1996) Adrenomedullin expression in human tumor cell lines: its potential role as an autocrine growth factor. J. Biol. Chem., 271, 23345–23351.[Abstract/Free Full Text]

Moller, B., Lindblom, B. and Olovsson, M. (2001) Expression of angiogenic growth factors VEGF, FGF-2, EGF and their receptors in normal human endometrium during the menstrual cycle. Mol. Hum. Reprod., 7, 65–72.[Abstract/Free Full Text]

Moriyama, T., Otani, T. and Maruo, T. (2000) Expression of adrenomedullin by human granulosa lutein cells and its effect on progesterone production. Eur. J. Endocrinol., 142, 671–676.[ISI][Medline]

Moriyama, T., Otani, T. and Maruo, T. (2002) Expression of adrenomedullin by human placental cytotrophoblasts and choriocarcinoma JAr cells. J. Clin. Endocrinol. Metab., 86, 3958–3961.[CrossRef][ISI]

Nagata, D., Hirata, Y., Suzuki, E., Kakoki, M., Hayakawa, H., Goto, A., Ishimitsu, T., Minamino, N., Ono, Y., Kangawa, K. et al. (1999) Hypoxia-induced adrenomedullin production in the kidney. Kidney Int., 55, 1259–1267.[CrossRef][ISI][Medline]

Nayak, N. and Brenner, R. (2002) Vascular proliferation and vascular endothelial growth factor expression in the rhesus macaque endometrium. J. Clin. Endocrinol. Metab., 87, 1845–1855.[Abstract/Free Full Text]

Nikitenko, L.L., MacKenzie, I.Z., Rees, M.C.P. and Bicknell, R. (2000) Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium. Mol. Hum. Reprod., 6, 811–819.[Abstract/Free Full Text]

Oehler, M.K., Rees, M.C. and Bicknell, R. (2000) Steroids and the endometrium. Curr. Med. Chem., 7, 543–560.[ISI][Medline]

Rees, M., Hague, S., Oehler, M.K. and Bicknell, R. (1999) Regulation of endometrial angiogenesis. Climacteric, 2, 52–58.[Medline]

Rogers, P.A.W. and Gargett, C.E. (1999) Endometrial angiogenesis. Angiogenesis, 2, 287–294.[CrossRef]

Rogers, P.A.W., Au, C.L. and Affandi, B. (1993) Endometrial microvascular density during the normal menstrual cycle and following exposure to long-term levonorgestrel. Hum. Reprod., 8, 1396–1404.[Abstract]

Rogers, P.A., Lederman, F. and Taylor, N. (1998) Endometrial vascular growth in normal and dysfunctional states. Hum. Reprod. Update, 4, 503–508.[Abstract/Free Full Text]

Sata, M., Kakoki, M., Nagata, D., Nishimatsu, H., Suzuki, E., Aoyagi, T., Suguira, S., Kojima, H., Nagano, T., Kangawa, K. et al. (2000) Adrenomedullin and nitric oxide inhibit endothelial cell apoptosis via a cyclic GMP-independent mechanism. Hypertension, 36, 83–88.[Abstract/Free Full Text]

Sharkey, A.M., Day, K., McPherson, A., Malik, S., Licence, D., Smith, S.K. and Charnock-Jones, D.S. (2000) Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia. J. Clin. Endocrinol. Metab., 85, 402–409.[Abstract/Free Full Text]

Shichiri, M., Kato, H., Doi, M., Marumo, F. and Hirata, Y. (1999) Induction of max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis. Mol. Endocrinol., 13, 1353–1363.[Abstract/Free Full Text]

Shifren, J.L., Tseng, J.F., Zaloudek, C.J., Ryan, I.P., Meng, Y.G., Ferrara, N., Jaffe, R.B. and Taylor, R.N. (1996) Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J. Clin. Endocrinol. Metab., 81, 3112–3118.[Abstract]

Shweiki, D., Itin, A., Neufeld, G., Gitay-Goren, H. and Keshet, E. (1993) Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. J. Clin. Invest., 91, 2235–2243.[ISI][Medline]

Silverberg, S.G., Haukkamaa, M., Arko, H., Nilsson, C.G. and Luukkainen, T. (1986) Endometrial morphology during long-term use of levonorgestrel-releasing intrauterine devices. Int. J. Gynecol. Pathol., 5, 235–241.[ISI][Medline]

Sivin, I. and Stern, J. (1994) Health during prolonged use of levonorgestrel 20 mg/d and the copper Tcu 380 Ag intrauterine contraceptive devices: a multicenter study. Fertil. Steril., 61, 70–77.[ISI][Medline]

Smith, S.K. (1995) Angiogenic growth factor expression in the uterus. Hum. Reprod. Update, 1, 162–172.[ISI]

Smith, S.K. (1996) Vascular endothelial growth factor and the endometrium. Hum. Reprod., 11 (Suppl. 2), 56–61.

Smith, S.K. (2001) Regulation of angiogenesis in the endometrium. Trends Endocrinol. Metab., 12, 147–151.[CrossRef][ISI][Medline]

Song, J.Y., Markham, R., Russell, P., Wong, T., Young, L. and Fraser, I.S. (1995) The effect of high-dose medium- and long-term progesterone exposure on endometrial vessels. Hum. Reprod., 10, 797–800.[Abstract]

Sturridge, F. and Guillebaud, J. (1997) Gynaecological aspects of the levonorgestrel-releasing intrauterine system. Br. J. Obstet. Gynaecol., 104, 285–289.[ISI][Medline]

Sugino, N., Kashida, S., Karube-Harada, A., Takiguchi, S. and Kato, H. (2002) Expression of vascular endothelial growth factor (VEGF) and its receptors in human endometrium throughout the menstrual cycle and in early pregnancy. Reproduction, 123, 379–387.[Abstract/Free Full Text]

Torry, D.S., Holt, V.J., Keenan, J.A., Harris, G., Caudle, M.R. and Torry, R.J. (1996) Vascular endothelial growth factor expression in cycling human endometrium. Fertil. Steril., 66, 72–80.[ISI][Medline]

Upton, P.D., Austin, C., Taylor, G.M., Nandha, K.A., Clark, A.J., Ghatei, M.A., Bloom, S.R. and Smith, D.M. (1997) Expression of adrenomedullin (ADM) and its binding sites in the rat uterus: increased number of binding sites and ADM messenger ribonucleic acid in 20-day pregnant rats compared to non-pregnant rats. Endocrinology, 138, 2508–2514.[Abstract/Free Full Text]

Wildemeersch, D. and Schacht, E. (2001) Treatment of menorrhagia with novel ‘frameless’ intrauterine levonorgestrel-releasing drug delivery system: a pilot study. Eur. J. Contracept. Reprod. Health Care, 6, 93–101.[ISI][Medline]

Zhang, L., Rees, M.C.P. and Bicknell, R. (1995) The isolation and long term culture of normal human endometrial epithelium and stroma: expression of mRNAs for angiogenic polypeptides basally and on oestrogen challenge. J. Cell Sci., 108, 323–331.[Abstract/Free Full Text]

Zhao, Y., Hague, S., Manek, S., Zhang, L., Bicknell, R. and Rees, M.C.P. (1998) PCR display identifies tamoxifen induction of the novel angiogenic factor adrenomedullin by a non-oestrogenic mechanism in human endometrium. Oncogene, 16, 409–415.[CrossRef][ISI][Medline]

Submitted on July 29, 2002; resubmitted on November 5, 2002; accepted on January 7, 2003.