Department of Obstetrics & Gynecology, Chang-Gung Medical College, Chang-Gung Memorial Hospital, Lin-Kou Medical Center, Taipei, Taiwan, Republic of China
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Abstract |
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Key words: endometrium/hormone replacement therapy/IGF/IGFBP-1/progesterone
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Introduction |
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IGFBP-1 mRNA is not detectable in the proliferative endometrium but is present in stromal cells of the secretory endometrium (Zhou et al., 1994; Rutanen et al., 1997
). In addition, progesterone induces production of IGFBP-1 by proliferative phase endometrium and stimulates its secretion from secretory phase endometrium (Rutanen and Pekonen, 1991
; Wang and Chard, 1999
). Collectively, IGFBP-1 expression is believed to be only in secretory phase endometrium. On the other hand, IGF receptors are present in human endometrium throughout the menstrual cycle (Rutanen and Pekonen, 1991
). It has been shown that IGFBP-1 inhibits receptor binding of IGF-I to endometrial membranes, suggesting a role as a paracrine inhibitor of IGF action (Rutanen and Pekonen, 1991
). Recent studies have also demonstrated that the endometrium expresses IGF, and both the growth factors and their receptors in the endometrium are stimulated by oestrogen (Murphy and Ghahary, 1990
).
Hormone replacement therapy (HRT) relieves climacteric syndromes in perimenopausal women and benefits postmenopausal women through prevention of cardiovascular diseases and osteoporosis, having been accepted as a treatment to improve the health of menopausal women. Addition of progesterone into HRT regimens has been shown to protect the endometrium from hyperplasia and carcinoma (Wood, 1994; Writing Group, 1995). Immunoreactive IGFBP-1 has been detected in the endometrium after treatment with a combination of oestrogen and progesterone (Suvanto-Luukkonen et al., 1995
). In addition, it has previously been reported that treatment with progesterone increases serum concentrations of IGFBP-1 (Wang and Soong, 1996
). However, the mechanism of protective effects on the endometrium by progesterone and the role of IGFBP-1 in postmenopausal women undergoing HRT are not completely clear.
To investigate further the possible regulatory roles of IGF and IGFBP-1 on postmenopausal endometrium after HRT, expression of mRNA for IGF-I, IGF-II, type 1-IGF receptor, IGFBP-1, oestrogen receptor (ER) and progesterone receptor (PR) in the endometrium was explored by a semiquantitative reverse transcriptionpolymerase chain reaction (semiquantitative RT-PCR). In addition, serum concentrations of IGFBP-1, oestradiol, progesterone, follicle stimulating hormone (FSH) and sex hormone-binding globulin (SHBG) were also determined by radioimmunoassays and immunofluorometric assays.
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Materials and methods |
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Collection of samples
Samples of blood were collected at the time of endometrial sampling (before HRT, and 6 and 12 months after HRT). Serum samples were stored at 20°C until assays for circulating oestradiol, progesterone, SHBG and FSH. Collection of endometrial samples was performed following cervical dilatation under general anaesthesia (i.v. injection of fentanyl and propofol). A part of endometrial tissue was sent to the pathology department for histological evaluation. The rest of the endometrial tissue was isolated from contaminated red blood cells (RBC) by density gradient centrifugation in 50% Percoll at 800 g for 15 min followed by immediate weighing and RNA extraction.
Assays for oestradiol, progesterone, FSH and SHBG
To verify the postmenopausal hormone status, serum concentrations of oestradiol and progesterone were determined by immunofluorometric assays (IFMA) (Pharmacia, Turku, Finland). The detection limit of the oestradiol assay was 13.6 pg/ml. The intra-assay coefficients of variation for oestradiol were 5.7% at 36 pg/ml and 3.1% at 91 pg/ml (n = 10). The inter-assay coefficients of variation for oestradiol were 8.4% at 36 pg/ml and 5.6% at 91 pg/ml (n = 6). The minimum detection limit of the assay for progesterone was 0.1 ng/ml. The intra-assay coefficients of variation for progesterone were 2.5% at 1.5 ng/ml and 6.8% at 11 ng/ml (n = 10). The inter-assay coefficients of variation for progesterone were 5.9% at 1.5 ng/ml and 9.3% at 11 ng/ml (n = 6).
Serum concentration of FSH was measured by radioimmunoassays using a commercial kit (Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). The lowest measurable concentration for FSH was 0.5 mIU/ml. The intra-assay coefficients of variation for FSH were 4.5% at 2.5 mIU/ml and 8.7% at 6.9 mIU/ml (n = 12). The inter-assay coefficients of variation for FSH were 8.7% at 2.5 mIU/ml and 13.3% at 6.9 mIU/ml (n = 22).
Concentrations of SHBG were determined by an immunoradiometric assay (IRMA) using a commercially available kit (Diagnostic Systems Laboratories, Inc., Webster, TX, USA). The minimum detection limit for SHBG was 3 nmol/l. The intra-assay coefficients of variation for SHBG were 4.2% at 60 nmol/l and 8.8% at 130 nmol/l (n = 8). The inter-assay coefficients of variation for SHBG were 9.4% at 60 nmol/l and 13.3% at 130 nmol/l (n = 12).
Extraction of RNA
Total RNA in the endometrium was isolated using a guanidium thiocyanate-phenol-chloroform procedure (Chomczynski and Sacchi, 1987). Briefly, homogenized endometrial tissue was lysed in 0.5 ml denaturing solution (4 mol/l guanidium thiocyanate, 25 mmol/l sodium citrate, 0.5% sarcosyl, 0.1 mol/l 2-mercaptoethanol) followed by the addition of 0.05 ml of 2 mol/l sodium acetate, 0.5 ml of phenol and 0.1 ml of chloroform/isoamyl alcohol (49:1). After vigorous shaking, the tubes were incubated on ice for 15 min and centrifuged at 10 000 g at 4°C for 20 min. The aqueous layer was collected followed by an addition of equal volume of cold isopropanol. RNA was pelleted, re-suspended in 0.3 ml of denaturing solution and re-precipitated with isopropanol. The RNA pellet was then washed twice with 75% ethanol and dissolved in diethyl pyrocarbonate (DEPC)-treated water. The amount of RNA was quantified spectrophotometrically and stored at 70°C until analysis.
Semiquantitative RT-PCR with multiple primers
In semiquantitative RT-PCR, two sets of primers were used simultaneously in each tube (one for target mRNA and the other for ß-actin mRNA used as an internal control) (Table I). Aliquots of 2 µg of total RNA from each case were used to run reverse transcription using a Gene Amp RNA PCR kit (Perkin-Elmer Cetus, AT, USA). In each tube, total RNA was reversely transcribed with murine leukaemia virus (MuLV) reverse transcriptase (50 IU) as well as the downstream primers for both ß-actin mRNA and one of target mRNA in a final volume of 40 µl. Using a DNA thermal cycler (Perkin-Elmer Cetus), reagents were incubated at 42°C for 15 min, heated to 99°C for 1 min to denature the MuLV reverse transcriptase, rapidly cooled to 4°C and stored at 4°C.
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Following amplification, 15 µl of the reaction products mixed with 3 µl of loading dye [0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol and 30% (v/v) glycerol in water] was applied to a 2% agarose (Promega Corporation, Madison, WI, USA) gel containing a minimal amount of ethidium bromide. Molecular weight standards included 1 µg of `100 bp DNA Ladder®' (Fermentas Ltd, Vilnius, Lithuania). At the end of electrophoresis, the intensity of RT-PCR products was visualized on a UV box and analysed by using an image analyser (Gel Doc 1000, Bio-Rad Laboratories, Hercules, CA, USA).
Statistical analysis
Changes in serum hormonal concentrations and intensity of PCR products were statistically analysed using Student's t-test (two-tailed) for paired and unpaired data. In all cases, P values <0.05 were considered to indicate significance.
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Results |
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Elevated serum oestradiol concentrations were found 6 and 12 months after HRT in comparison with those before HRT (P < 0.0005). Similarly, circulating concentrations of SHBG 6 and 12 months after HRT were higher than those before HRT (P < 0.02 and P < 0.01 respectively). In addition, substantial elevation in serum concentrations of progesterone was found 6 and 12 months after HRT (6.7 ± 2.3 and 7.3 ± 2.8 ng/ml; mean ± SD). By contrast, decreased serum FSH was detected 6 and 12 months after HRT (P < 0.01 and P < 0.005 respectively) (Table II).
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After amplification with semiquantitative RT-PCR, a band at 838 bp on the agarose gel represented the expression of ß-actin mRNA (Figures 1 and 2). In the endometrium obtained from postmenopausal patients with abnormal uterine bleeding before HRT, expression of IGFBP-1 mRNA (bands at 417 bp) was not detected using semiquantitative RT-PCR, whereas substantial expression of IGFBP-1 mRNA was detected after HRT (Figures 1, 2 and 3
). In contrast, expression of progesterone receptor (PR) mRNA (bands at 630 bp) was abundant before HRT while it became undetectable after HRT (Figures 1, 2 and 3
). In addition, elevated expression of IGF-II mRNA (bands at 538 bp) and decreased expression of IGF-I mRNA (bands at 514 bp) by semiquantitative RT-PCR were observed after HRT (P < 0.0005 and P < 0.00001 respectively) (Figure 3
). In the endometrium from both controls and postmenopausal women undergoing HRT for 12 months, findings after semiquantitative RT-PCR were similar to those 6 months after HRT (Figure 2
).
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Discussion |
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There is evidence that expression of IGF-I is prominent in proliferative phase endometrium (Zhou et al., 1994), and that the cyclic changes of IGF-I mRNA in the proliferative phase of the endometrium are coincident with serum oestradiol concentrations (Giudice et al., 1993
). In addition, IGF binds to both cell membrane receptors and soluble IGFBP-1 with similar affinity in the endometrium (Giudice et al., 1993
). In postmenopausal women after HRT, expression of IGFBP-1 was substantially increased and that of IGF-I was decreased in the endometrium, whereas there was no change in type 1-IGF receptor (Figure 3
). In postmenopausal women enrolled in the present study, symptoms of abnormal uterine bleeding subsided and thickness of the endometrium was significantly suppressed after HRT. Thus, it is plausible that the protective effects of progesterone from possible endometrial overgrowth or hyperplasia induced by unopposed oestrogens might, in addition to direct inhibition of expression of IGF-I mRNA, be indirectly mediated through neutralizing the actions of IGF-I by increasing the expression of IGFBP-1 without affecting the type 1-IGF receptor (IGFR-1) in the endometrium. Furthermore, the data presented here suggest that detection of IGFBP-1 mRNA in the endometrium by semiquantitative RT-PCR may be a useful assessment of the progestogen effects on the endometrium during HRT.
In human endometrium, IGF-I expression is substantially higher during the proliferative than the secretory phase, whereas the converse is true for IGF-II (Zhou et al., 1994; Gao et al., 1995
). In the present study, expression of IGF-I was decreased while that of IGF-II was enhanced after HRT (Figure 3
). This further confirms previous studies that gene expression of IGF-I is prominent in the presence of oestrogen (proliferative endometrium) and abundant IGF-II gene expression is found in the presence of both oestrogen and progesterone (secretory endometrium) (Giudice et al., 1993
). Furthermore, the decrease in expression of IGF-I and the preferential expression of IGF-II and IGFBP-1 mRNA in secretory endometrium suggests that the differentiation of the endometrium might also be mediated by IGF-II under the regulation of progesterone.
Serum concentrations of progesterone and oestradiol were substantially increased in women after HRT (Table II). The principal metabolite of medroxyprogesterone acetate (MPA, Provera®) is a 3-enol form of MPA glucuronide. Thus, the progesterone antibodies in the immunofluorometric assay (IFMA) used to determine serum progesterone concentrations in the present study might have partial cross-reaction with the 3-enol form of MPA glucuronide. Using RT-PCR, expression of IGFBP-1 mRNA was detected only in the endometrium from postmenopausal women after HRT, but not before HRT (Figures 1 and 2
). None of the women investigated complained of abnormal uterine bleeding after HRT. Histologically, no simple hyperplasia of the endometrium was observed in the studies women after HRT. These clinical observations suggest that medroxyprogesterone acetate (Provera®, 5 mg per day) may elicit a protective effect on the endometrium even in the milieu of relatively high concentrations of oestrogen in postmenopausal women undergoing HRT (Table II
). Thus, it is possible that medroxyprogesterone acetate (Provera®, 5 mg per day) exerts anti-oestrogenic effects through regulation of the endometrial IGF/IGFBP system.
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Acknowledgments |
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Notes |
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References |
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Submitted on April 19, 1999; accepted on August 17, 1999.