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
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Abstract |
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Key words: adrenomedullin/endometrium/levonorgestrel-releasing intra-uterine system/vascular endothelial growth factor
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Introduction |
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Although VEGF in the endometrium exists in several isoforms (Charnock-Jones et al., 1993; Torry et al., 1996
), VEGF121 and VEGF165 are predominant (Smith, 1995
). 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., 1994
; Shifren et al., 1996
; Lau et al., 1998
). On the other hand, AM has been implicated to have vital roles in the biology of the endometrium (Michishita et al., 1999
), uterine cervix (Upton et al., 1997
; Li et al., 2001
) and placenta (Moriyama et al., 2002
). 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., 2000
). Nevertheless, several investigators have demonstrated that angiogenesis occurs in three distinct stages during the menstrual cycle (Rogers and Gargett, 1999
): (i) for the repair of vascular bed during menstruation (Markee, 1978
); (ii) for rapid endometrial growth in the proliferative phase (Rogers et al., 1993
) and (iii) for growth and coiling of spiral arterioles which occurs during the secretory phase (Kaiserman-Abramof and Padykula, 1989
). 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.
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Materials and methods |
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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, 1987). 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 120 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 MannWhitney 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.
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Results |
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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.
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Discussion |
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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., 2000), 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., 1998
; Sugino et al., 2002
). 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., 1996
; Smith, 1996
; Torry et al., 1996
). However, others (Li et al., 1994
) 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, 2002
) 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., 1997
) 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., 1993
). 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., 1998
).
The findings of the present study are in agreement with those produced by others (Macpherson et al., 1999), wherein Implanon and Mircetteboth of which are progestin-containing contraceptivessignificantly 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., 1998
) 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., 2000). Although the present study did not include immunostaining of endometrial endothelial cells, several studies (Rogers et al., 1993
; Goodger Macpherson and Rogers, 1994
; Macpherson et al., 1999
) 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., 1999
), whereas Norplant increased endometrial endothelial cell number. The use of Norplant might reduce the rate of endothelial cell death (Goodger Macpherson and Rogers, 1994
; Hickey et al., 1999
). Treatment with higher doses of norethisterone or medroxyprogesterone acetate significantly decreased endometrial vascular density (Song et al., 1995
). Furthermore, there seems to be an apparent dissociation between endothelial cell number and endometrial bleeding (Macpherson et al., 1999
).
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., 2002) 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., 2002
) 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., 2000
), while AM expression in the ovary became abundant in granulosa lutein cells (Moriyama et al., 2000
). Progesterone has been implicated to be responsible for the release of AM in the rat (Jerat and Kaufman, 1998
). 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., 2000). 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., 1986
; Barbosa et al., 1990
), while the post-menopausal endometrium has a fibrous stroma with diminished cellularity (Herbst et al., 1992
). On the other hand, it has been reported (Zhao et al., 1998
) 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., 1999
; Sata et al., 2000
; Filippatos et al., 2001
). 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.
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Acknowledgements |
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References |
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Submitted on July 29, 2002; resubmitted on November 5, 2002; accepted on January 7, 2003.