Department of Obstetrics and Gynaecology, National University Hospital, National University of Singapore, Singapore
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, National University Hospital, National University of Singapore, Lower Kent Ridge Road, Singapore 119074. e-mail: obgannam{at}nus.edu.sg
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Abstract |
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Key words: angiogenesis/bFGF/LNG-IUS/TGF-1/VEGF
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Introduction |
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The aetiology of aberrant endometrial bleeding associated with progestogen-only contraception is unknown at present. Local changes that occur in the endometrial microvasculature following exposure to long-acting progestogen are believed to be associated with aberrant menstruation (Rogers et al., 1993; Hickey et al., 1998
; 2000). Based on these reports, it is plausible to suggest that the mechanisms controlling endometrial angiogenesis could be altered with the use of long-acting progestogen contraceptives (Hickey et al., 1996
; Hickey and Fraser, 2002
).
It is noteworthy that controversy exists among published reports regarding angiogenesis and vascular density in the natural cycle. Some authors have reported no variations in vascular density in different phases of the natural cycle and have concluded an absence of correlation between vascularity and stromal development (Hourihan et al., 1986; Rogers et al., 1993
). On the other hand, a significant increase in vascular surface area, diameter and total number of capillaries was observed in the secretory phase when compared with the proliferative phase (Ota et al., 1998
).
Recent evidence suggests that exposure to low-dose progestogens may stimulate endometrial angiogenesis characterized by unusual vascular appearances classified as neovascular and mosaic patterns on the endometrial surface (Hickey and Fraser, 2002). Tissue factor plays an important role in endometrial angiogenesis as well as haemostasis, and could therefore contribute to the neovascular patterns observed (Abe et al., 1999
). However, contradictory observations have been made regarding the effect of levonorgestrel on endometrial vascular basement membrane. One group (Palmer et al., 1996
) observed no differences in immunostaining of the three basement membrane components, collagen IV, laminin and heparan sulphate proteoglycan, in endometrial biopsies from 11 women exposed to the subdermal levonorgestrel implant, Norplant®, whereas others (Hickey et al., 1999
) noted a reduction in these basement membrane components. Moreover, a wide variability from one biopsy to the next in the same subject has also been reported (Kelly et al., 1995
). Neither was any clear relationship established between basement membrane components and bleeding patterns, which suggests that other endometrial mechanisms might be involved in breakthrough bleeding. Although subdermal levonorgestrel results in a significant increase in microvascular and endothelial cell density compared with that observed in the normal cycle, the increased vascular density was not related to enhanced endometrial bleeding [Rogers et al., 1993
; Goodger-Macpherson et al., 1994
]. Moreover, no differences in endothelial cell migratory signal production were observed in endometrial explants from Norplant acceptors (Subakir et al., 1995
). These observations were subsequently confirmed by another group (Charnock-Jones et al., 2000
), who observed no association between endometrial bleeding and VEGF, estrogen receptor and progesterone receptor as determined by immunohistochemistry, as well as in the endothelial cell density in endometrium of progestogen acceptors.
The LNG-IUS delivers a high dose of levonorgestrel to the endometrium and induces a rapid decidualization response in the endometrial stroma (Critchley et al., 1998). The irregular appearance of the endometrial surface with polyploidal structures suggest that these micropolyps may contribute to the bleeding patterns observed following exposure to LNG-IUS. This view is supported by the hysteroscopic observations of one group (Brechin et al., 2000
), who reported the presence of polyps in three women exposed to LNG-IUS, and also that the bleeding problems resolved after surgical removal of these polyps. The presence of unscheduled breakthrough bleeding and reduction in menstrual blood loss suggests that the progestogens might affect the endometrial vascular system directly.
Angiogenesis is a process by which the development of new capillaries from pre-existing vessels occurs by sprouting (Risau, 1997). Four mechanisms have been implicated in this process, namely sprouting, intussusception, elongation and incorporation of circulating endothelial progenitor cells into growing vessels (Burri and Tarek, 1990
; Risau, 1997
). A broad range of molecules identified as angiogenic growth factors are present in the endometrium. These polypeptides, which have been implicated in angiogenesis, include vascular endothelial growth factor (VEGF) (Charnock-Jones et al., 1993
), epidermal growth factor (EGF) (Nelson et al., 1991
), basic fibroblast growth factor (bFGF) (Ferriani et al., 1993
) and transforming growth factor-beta (TGF-
1) (Roberts et al., 1986
). These angiogenic factors promote endothelial cell proliferation, migration and tube formation, and also facilitate the changes in extracellular matrix needed to permit new vessel formation. In addition, treatment of isolated human endometrial stromal cells with estradiol-17
and progesterone significantly increased mRNA for VEGF in a dose-dependent manner (Perrot-Applanat et al., 2000
).
VEGF is a heparin-binding, homodimeric glycoprotein with angiogenic activity and is a potent stimulator of microvascular permeability (Dvorak et al., 1987; Ferrara and Davis-Smyth, 1997
). It plays an important role in physiological and pathological neovascularization via its specific receptors Flt-1 and Flk-1 (Terman et al., 1992
; Millauer et al., 1993
). Alternating splicing of the exons encoding the carboxy-terminal portion of the peptide results in the five different mRNAs and peptides of 121, 145, 165, 189 and 206 amino acids respectively (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206). The different isoforms appear to have similar functions, but differ in their binding affinity to the extracellular matrix.
bFGF is mitogenic and chemotactic for endothelial cells. EGF, which is a 6 kDa polypeptide, exerts a variety of biological effects, and in being a potent mitogen causes the proliferation of many cell types including human microvascular endothelial cells (Ushiro et al., 1996). TGF-
1, which is an inhibitor of endothelial cell proliferation, stimulates angiogenesis in vivo. It is possible that TGF-
1 promotes angiogenesis by differentiation of the endothelial cells at the end of the proliferative phase, presumably by inducing the synthesis of extracellular matrix (Wahl et al., 1987
).
The objectives of the present study were to: (i) investigate the effect of LNG-IUS on the release of angiogenic growth factors such as VEGF, bFGF, EGF and TGF-1 by the endometrial tissue and its immunoreactive levels in the plasma; and (ii) investigate whether there exists any correlation between these angiogenic growth factors and whether these factors in the plasma and endometrial tissues correlated with either bleeding/spotting days or/and circulating levels of estradiol-17
and progesterone. In addition, using an RTPCR technique, levels of mRNA transcripts of these angiogenic growth factors before and after contraceptive delivery were monitored.
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Materials and methods |
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Sample collection
Venous blood samples (10 ml) were collected into EDTA tubes, and endometrial biopsies taken from each woman before the insertion of LNG-IUS and at 1, 3, 6 and 12 months post insertion. Blood samples were centrifuged immediately at 1500 g for 10 min at 4°C and the plasma aspirated and stored at 70°C until further analysis. The pre-insertion blood samples and biopsy collected in the secretory phase of the menstrual cycle served as controls. All biopsies were performed in an outpatient setting using a pipelle. Endometrial biopsy samples were collected in phosphate-buffered saline, washed free of blood clots and stored immediately at 70°C. Following insertion of the LNG-IUS, episodes of bleeding or spotting were recorded by the women on a menstrual record card.
Bleeding patterns
Menstrual record card data were analysed using a 90-day reference period (Belsey and Farley, 1988). The reference period was defined as the period of time (in days) to facilitate data analysis. During the present study, the 1-year menstrual diary was divided into four reference periods, each of 90 days. The study was open and non-randomized in design, with each woman acting as her own control for changes in her menstrual pattern following LNG-IUS insertion. The subjects were asked to record the length of menstrual cycles, days of bleeding, and any side effects encountered. Bleeding was defined as any bloody vaginal discharge that required the use of protection such as sanitary pads or tampons, and spotting as any bloody vaginal discharge that was not sufficient to require sanitary protection (Belsey, 1991
). Based on records from the menstrual chart, it was observed that endometrial biopsies did not induce any vaginal bleeding and hence did not interfere in the data analyses.
Preparation of endometrial extracts
Thawed biopsies were added (1:10, weight:volume) to ice-cold cell-lysing buffer (5 mmol/l TrisHCl, pH 7.4, 5 mmol/l NaCl, 1 mmol/l CaCl2, 2 mmol/l EDTA, 2 mmol/l dithiothreitol (DTT) and 25 µg/ml aprotinin). Samples were sonicated on ice with four bursts of 15 s at 50 W, each preceded by a 30 s pause. This suspension was centrifuged in a microfuge at 8650 g for 3 min to remove the nuclear pellet and the supernatant removed and stored at 70°C until further analysis. Protein concentrations of the extract were measured using the Coomassie Blue dye-binding assay (Bio-Rad Laboratories, Hercules, CA, USA). Bovine serum albumin was used as a standard, and lysis buffer as a control.
Measurements of angiogenic growth factors
Sandwich enzyme-linked immunosorbent assay (ELISA) kits were used for the quantitative determination of VEGF, TGF-1, bFGF and EGF in both plasma and endometrial extracts, by employing monoclonal human antibodies specific to VEGF, bFGF, EGF (Quantikine, R&D Systems, Inc., Minneapolis, USA) and TGF-
1 (Bender MedSystems Diagnostics GmbH, Vienna, Austria). The intra- and inter-assay variations were within the range as recommended by the manufacturers. Detectable concentration ranges of standards were as follows: 31 to 2000 pg/ml for VEGF, 10 to 640 pg/ml for bFGF, 3.9 to 250 pg/ml for EGF and 19 to 600 pg/ml for TGF-
1.
Measurement of progesterone and estradiol-17
Progesterone and estradiol concentrations in plasma were determined using a solid-phase 125I radioimmunoassay (Diagnostic Products Corporation, LA, USA).
Extraction of total RNA and semi-quantitative RT-PCR
Total RNA was isolated from the frozen endometrial tissues using the TRIzol reagent following the manufacturers instructions (Gibco-BRL, MD, USA). The integrity of the RNA was checked by 1% denaturing agarose gel electrophoresis and its concentration determined spectrophotometrically. Isolated total RNA was treated with DNase I, RNase-free (Roche Diagnostics) to eliminate genomic DNA before using it for RT-PCR. Human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as the internal control.
The reverse transcription reaction was performed on DNase-treated total RNA, using the SuperScriptTM Preamplification system for first-strand cDNA synthesis and following the manufacturers instructions (Gibco-BRL). Briefly, equal amounts of RNA (2 µg) were used for first-strand cDNA synthesis in a total reaction volume of 20 µl containing 200 IU reverse transcriptase, 2.5 µmol/l oligo dT, 1 mmol/l dNTP, 1x reaction buffer (20 mmol/l TrisHCl, pH 8.4, 50 mmol/l KCl), 8 mmol/l DTT and 2.5 mmol/l MgCl2. A reaction lacking the reverse transcriptase enzyme was used as a negative control. Incubation was continued at 42°C for 1 h, after which the reverse transcription reaction was stopped by heating samples to 75°C. Aliquots of the first-strand cDNA were stored at 20°C until used for amplification.
cDNA was amplified using growth factor-specific primers (see Table I) in a reaction mixture containing Thermus aquaticus (Taq) DNA polymerase (Promega, Madison, USA). For a typical PCR reaction, 2 µl of cDNA was amplified in a 50 µl reaction volume containing 1x PCR buffer (20 mmol/l TrisHCl, pH 8.4, 50 mmol/l KCl), 1.5 mmol/l MgCl2, 0.25 mmol/l dNTPs, 1 U Taq DNA polymerase (Promega) and 0.5 µmol/l growth factor- specific sense and anti-sense primers were added and subjected to PCR thermal cycling (Gene Amp 9600; Perkin Elmer Citus, CA, USA).
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Statistical analysis
Since the concentrations of angiogenic growth factors were not normally distributed, the data were subjected to non-parametric analysis and medians were determined and compared statistically. A Freidman test was used to compare the levels of growth factors and hormone levels before and following the insertion of LNG-IUS at four different time intervals. A Wilcoxon signed rank test with Bonferronis correction was used to identify groups that were significantly different from one another. Spearmans correlation coefficient test was used to investigate whether any correlation existed between these growth factors, and also to determine whether these factors correlated with bleeding/spotting days and plasma steroid concentrations. Analyses were performed using the SPSS version 10, and a P-value < 0.05 was considered statistically significant.
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Results |
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Analysis of menstrual record
The mean (± SD) numbers of bleeding/spotting days after LNG-IUS insertion at the first, second, third and fourth reference periods were 37.8 ± 16.1, 24.5 ± 7.4, 16.6 ± 10.9 and 15.8 ± 7 days respectively. The decrease in number of bleeding/spotting days during the second, third and fourth reference periods, when compared with the first reference period, was noted to be statistically significant (P < 0.007, P < 0.001 and P < 0.001 respectively) (Wilcoxon signed rank test).
Levels of angiogenic growth factors in women using LNG-IUS
Plasma levels
Plasma concentrations of angiogenic growth factors before and after LNG-IUS insertion are listed in Table II. No significant difference was noted in plasma levels of bFGF, EGF and TGF-1 after LNG-IUS insertion, though VEGF was below the detection level in all plasma samples studied.
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Expression of angiogenic growth factors
The expression profiles of VEGF, TGF-1 and bFGF detected using the RT-PCR technique at pre-insertion and at 1, 3 and 6 months after LNG-IUS insertion are shown in Figures 5, 6 and 7 resepectively. bFGF mRNA was expressed in all nine samples studied, there being a 1.2-fold increase in expression at 6 months after LNG-IUS insertion compared with the pre-insertion value (P < 0.01). A 1.1-fold increase in mRNA expression of TGF-
1 occurred at 6 months after LNG-IUS insertion compared with pre-insertion values (P < 0.01). Similarly, the expression of VEGF 121, VEGF 165 and VEFG 189 mRNA isoforms was also observed in all six endometrial extracts taken before and at 6 months after LNG-IUS insertion. However, there was no significant difference in the expression of mRNA levels for VEGF between pre-insertion and at 6 months after exposure to the levonorgestrel device (P < 1, not significant). There was no expression of EGF mRNA in all of the endometrial tissues studied.
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Correlation between angiogenic factors and bleeding pattern
Using the Spearman rank correlation test, a positive correlation was observed in endometrial tissue VEGF concentrations at 6 months after LNG-IUS insertion and the number of bleeding/spotting days during the reference period prior to biopsy (91180 days) (r = 0.705, P < 0.015). In contrast, a negative correlation occurred between levels of TGF-1 in endometrial extracts at 3 months after LNG-IUS insertion and the number of bleeding/spotting days during the reference period before biopsy (190 days) (r = 0.604, P < 0.038). No correlation was found between endometrial extract levels of bFGF, EGF and the number of bleeding/spotting days in the pre-biopsy reference period.
Correlation between angiogenic growth factors and sex steroids
A negative correlation was observed between endometrial extract bFGF levels and plasma estradiol levels at 1 month after LNG-IUS insertion, but no correlation with either estradiol or progesterone was found with the other three angiogenic factors.
No correlation was noted between plasma levels of angiogenic growth factor, bleeding pattern and sex steroids.
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Discussion |
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VEGFs are unique among angiogenic growth factors in that their effects appear to be exclusively on vascular endothelial cells (Shifren et al., 1996). A number of studies have demonstrated the location and distribution of VEGF in the endometrium and its regulation by sex steroids (Charnock-Jones et al., 1993
; Lau et al., 1999
). In the present study, it was noted that VEGF levels in extracts of endometrial tissue increased 2.2-fold at 3 months and 3.8-fold at 6 months after LNG-IUS insertion. These results are in agreement with those of others (Lau et al., 1999
), who have indicated increased immunostaining of endometrial glandular and stromal cells for VEGF in the women receiving Norplant. It is noteworthy that no significant difference in the expression of VEGF mRNA levels occurred at the three different time points after LNG-IUS insertion. Studies using isolated normal human endometrial stromal and epithelial cells however have indicated an up-regulation in VEGF gene expression and protein secretion following exposure to estrogen and progestogen (Zhang et al., 1995
; Shifren et al., 1996
; Lebovic et al., 2000
). In addition, studies on primate endometrium have also reported an increase in the expression of VEGF following exposure to progesterone and levonorgestrel, but down-regulation of the VEGF gene when the endometria are exposed to the anti-progestin, mifepristone. The progestogen-induced increase in VEGF expression is believed to be mediated by progesterone receptors (PR) as its action is blocked by treatment with mifepristone (Greb et al., 1997
; Hyder et al., 1997
). The present study demonstrated a non-proportional change in endometrial VEGF mRNA and protein expression following exposure to intrauterine levonorgestrel. On the basis of these findings, it seems plausible therefore to suggest that the main determinants of the rate of VEGF protein synthesis after exposure to intrauterine levonorgestrel may be attributed to post-transcriptional factors, other than those involving PR, as these receptors are decreased in the endometrium of the women receiving LNG-IUS (Zhu et al., 1999
; Hurskainen et al., 2000
). In addition, VEGF which stimulates plasminogen activators could be involved in the proteolytic degradation of the basement membrane of blood vessels, thereby leading to increased vascular fragility. Superficial vascular dilatation is seen in the endometrium exposed to low-dose progestogens (Hickey et al., 1998
). VEGF stimulates the potent vasodilator, nitric oxide from endothelial cells which would be expected to promote vasodilatation (Papapetropoulos et al., 1997
). In support of the above contention, a hallmark finding of the present study was that a positive correlation occurred between endometrial VEGF levels and the bleeding/spotting days. Moreover, the endometrial tissue VEGF levels were also significantly elevated at 3 and 6 months after LNG-IUS insertion. This observation could reflect the strong endometrial suppression provoked by a high levonorgestrel concentration in the uterine cavity (Pakarien et al., 1995
). The reduction in bleeding episodes during the second, third and fourth reference periods was observed in every woman in the present study. In addition, good counselling regarding the expected bleeding pattern after the insertion of LNG-IUS improved compliance in these women, and no unnecessary removals of this device occurred.
Disturbances in the vaginal bleeding patterns are almost inevitable in users of progestogen devices. It had been reported that in women using LNG-IUS, the PR levels are reduced, thereby rendering the endometrium functionally unresponsive to endogenous and exogenous progestogens (Critchley et al., 1998). A markedly diminished PR level could be attributed to the fact that intrauterine levonorgestrel produces endometrial levels of this progestogen that may be 1000-fold higher than serum concentrations (Pekonen et al., 1992
). This decline in PR may precipitate a chain of events leading to vascular breakdown, perhaps via leukocyte or cytokine activation. In addition, immunohistochemical studies have indicated that breakthrough bleeding arises from capillaries and veins on the endometrial surface (Hickey et al., 1996
; 2000). These small blood vessels are composed only of endothelial cells, their surrounding basement membrane and pericytes, and there is evidence that their integrity is compromised by levonorgestrel. In addition, the first few months of exposure to levonorgestrel (Norplant) results in a reduction in the number of vessels surrounded by the basement membrane components laminin, collagen IV and heparan sulphate proteoglycan (Hickey et al., 1999
). Hysteroscopic observations in Norplant users provide further evidence that exposure to this progestogen alters the superficial endometrial vasculature, and disruption of this microvessel integrity leads to breakthrough bleeding in these women (Hickey and Fraser, 2002
). Further investigations are necessary to confirm the angiogenesis in the endometrium of progestogen acceptors.
Moreover, a recent report on stereological measurements of blood vessels showed that the volume fraction of these vessels, the number of vessel cross-sections per unit area, and the cross-sectional diameters of the largest vascular lumens were significantly increased after treatment with LNG-IUS (McGavigan et al., 2003). Vascular support from pericytes could also be compromised by a reduction in smooth muscle
-actin in these progestogen-exposed endometria, and vascular fragility ensues leading to breakthrough bleeding (Rogers et al., 2000
). In this regard, Norplantwhich interferes with cytokeratin deposition in the endometrial endotheliummay increase the tendency of fragile vessels to leak into the endometrial cavity, thereby causing profuse subepithelial haemorrhages characteristic of bleeding disturbances in these acceptors (Wonodiresko et al., 1996
). However, in contrast to the above reports, others (Gambino et al., 2002
) revealed that angiogenesis in the proliferative phase of ovulating women with menorrhagia occurs due to vessel elongation. Based on stereological data, these authors have suggested that a direct physical stretch stimulus on the endothelial cells from the rapidly growing tissues that surround the blood vessels, or an angiogenic source within the blood vessels, could contribute to endometrial angiogenesis. Whether such a mechanism is operative in LNG-IUS acceptors is however, unknown at present.
Nevertheless, with regard to this potent angiogenic factor, VEGF-A has been immunolocalized in the luminal glandular epithelium and stroma in the proliferative phase of the normal menstrual cycle (Charnock-Jones et al., 1993; Li et al., 1994
), with the expression remaining in epithelial cells but not stroma during the secretory phase (Charnock-Jones et al., 1993
; Torry and Torry, 1997
). Using similar immunohistochemical methods, another group (Lau et al., 1999
) showed that VEGF staining was significantly higher in women on levonorgestrel implants than in normal endometrium. In addition, the expression of VEGF-A, B and C and VEGF receptors 1 to 3 in endometrial blood vessels indicated a highly structured involvement of this potent angiogenic factor in the regulation of angiogenesis in the human endometrium (Mints et al., 2002
). The precise aetiology of menstrual aberration induced by VEGF with progestagen-only contraception remains obscure at present. Nevertheless, a number of factors have been demonstrated to influence VEGF secretion and expression, including estrogen (Charnock-Jones et al., 1993
), hypoxia (Namiki et al., 1995
), tumour necrosis factor-alpha (Yoshida et al., 1997
), gonadotrophins (Christenssen and Stouffer, 1997
), interleukin-1 (Ferrer et al., 1997
) and interleukin-5 (Horiuchi and Weller, 1997
). The higher levels of endometrial VEGF in LNG-IUS acceptors in the present study suggests a positive effect of intrauterine levonorgestrel on VEGF secretion.
Moreover, VEGF has been implicated in blood vessel repair since expression of the VEGF receptor, flt-1 was observed to be elevated in tissue repair (Peters et al., 1993). Therefore, it seems likely that a positive correlation between endometrial VEGF and bleeding/spotting days observed in women in the present study could be indicative of active endometrial angiogenesis with the onset of breakthrough bleeding. Taken together, there is sufficient evidence to suggest that VEGF contributes to vascular permeability, provokes dilatation, and promotes angiogenesis. Increased blood vessel density with reduced stromal support could contribute to vascular fragility and overt bleeding in these women on LNG-IUS. The mechanisms controlling this differential expression and function of VEGF warrant further investigation.
bFGF is an important angiogenic growth factor that is both mitogenic and chemotactic for endometrial cells, and VEGF and bFGF have been shown to exert potent synergistic effects on the induction of angiogenesis in vitro (Pepper et al., 1992). In support of this concept, a statistically significant increase has been observed in the levels of bFGF at 1 and 6 months after LNG-IUS insertion. A similar trend in the up-regulation of bFGF mRNA was also observed in the endometrial tissues from the present subjects exposed to levonorgestrel. In addition, bFGF has also been shown to up-regulate the expression of VEGF in vascular smooth muscle cells, although it was not possible in the present study to demonstrate any association between these two angiogenic factors (Stavri al., 1995
). A similar lack of correlation was also noted between bFGF and bleeding/spotting days in the women using LNG-IUS.
TGF-1, a multifunctional polypeptide which regulates the proliferation and differentiation of various cells with an angiogenic effect in vivo, was noted in the present study to be increased 1.2-fold at 6 months and 1.5-fold after 1 year of LNG-IUS use compared with pre-insertion values. Similar increases in the mRNA level of TGF-
1 were found at 6 months after LNG-IUS insertion. It has been reported that VEGF and bFGF are induced in response to TGF-
1 in fibroblasts and epithelial cells in vitro (Pertovaara et al., 1993
; 1994). In accordance with these reports, the results of the present study also indicated a positive correlation between VEGF and TGF-
1 levels in the endometrium of women receiving LNG-IUS. A negative correlation between the bleeding/spotting days and TGF-
1 was noted. These results suggested that the angiogenic effect of TGF-
1 on endothelial cells may be mediated at least partly by a paracrine induction of VEGF and bFGF in other surrounding cell types. TGF-
1 could also promote angiogenesis by differentiation of the endothelial cells towards the end of the proliferative phase by inducing the synthesis of extracellular matrix. However, further studies are required to confirm the above findings and to investigate the precise role of TGF-
1 in endometrial angiogenesis.
There was no appreciable effect of LNG-IUS on immunoreactive EGF levels in the endometrial tissues from these women. Nonetheless, the question of whether a synergistic effect involving these angiogenic factors resulting in endometrial neovascularization following levonorgestrel delivery needs to be addressed.
There were also no significant changes in the plasma concentration of these angiogenic growth factors in women after exposure to LNG-IUS. The present results are consistent with the lack of the cyclicity observed in the serum concentration of VEGF during the normal menstrual cycle, but appear to contradict the results of another report on increased serum VEGF levels in the mid-luteal phase (Agrawal et al., 1999; Unkila-Kallio et al., 2000
). This may be due to the fact that production and expression of these growth factors is probably a local event at the level of the endometrium that could not be monitored in plasma.
Angiogenesis in the normal cycling human endometrium is believed to be influenced by both endocrine and paracrine factors. Sex steroids have been postulated to modulate the expression of angiogenic growth factors, and in this regard several studies have demonstrated up-regulation of mRNA and protein expression of VEGF and TGF-1 by estradiol and progestins (Arici et al., 1996
; Shifren et al., 1996
). In the present study however, there was an inability to discern any association of endometrial VEGF, TGF-
1 and EGF with sex steroid levels. A similar lack of association was noted between sex steroids and plasma levels of TGF-
1, bFGF and EGF in women with LNG-IUS insertion. It seems plausible to suggest that ovarian steroids, which control the growth cycle of human endometrium, may not be directly implicated in the regulation of expression and secretion of these growth factors.
In summary, the present study has provided evidence for the involvement of more than one angiogenic growth factor that could lead to aberrant angiogenesis in the endometrium exposed to intrauterine levonorgestrel, leading to breakthrough bleeding. Based on these observations, it is suggested that anti-angiogenic compounds be used in women receiving long-acting progestogen-only contraception, with a view to preventing the appearance of abnormal and fragile superficial endometrial blood vessels. Slow-release, local application of such angiogenesis inhibitors directly into the uterine cavity could be helpful in minimizing troublesome bleeding caused by exposure to low-dose progestogens. However, further studies are required to investigate the specific role of angiogenic factors in the endometrium and to elucidate neovascularization under the influence of levonorgestrel. A clarification of the underlying mechanisms might be of benefit in the development of clinical approaches to ameliorate troublesome breakthrough bleeding associated with this LNG-IUS device.
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Acknowledgements |
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References |
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Submitted on September 24, 2002; resubmitted on March 6, 2003; accepted on May 6, 2003.