1 Centre for Reproductive Biology, Chancellors Building, 49 Little France Crescent, Edinburgh EH16 4SB, 2 Department of Oncology, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK and 3 Division of Reproductive Sciences, Oregon Regional Primate Centre, 505 NW 185th Avenue, Beaverton, OR 97006, USA
4 To whom correspondence should be addressed. e-mail: Hilary.Critchley{at}ed.ac.uk
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Abstract |
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Key words: androgen receptor/endometrium/17-hydroxysteroid dehydrogenase type 2/levonorgestrel
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Introduction |
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The temporal and spatial distributions of sex steroid receptors such as estrogen receptor (ER), progestin receptor (PR) and AR in endometrium have been established. AR has been spatially localized to the endometrial stroma with up-regulation in the estrogen-dominated proliferative phase followed by down-regulation in the late secretory phase (Mertens et al., 2001; Slayden et al., 2001
). ER and PR are also up-regulated in glandular and stromal cells in the proliferative phase. In the secretory phase, a down-regulation of ER is noted in both glands and stroma. PR is also down-regulated in the glands but receptor expression persists in stromal tissues (Garcia et al., 1988
; Lessey et al., 1988
; Snijders et al., 1992
; Critchley et al., 1993
).
LNG, delivered locally by the LNG-IUS, is known to down-regulate ER and PR expression in the human endometrium (Critchley et al., 1998). This alteration in sex steroid receptor expression may affect endometrial cytokine release (Jones et al., 2000
). The cellular and molecular expression of the androgen receptor in LNG-IUS-treated endometrium has not been previously reported and AR may play an important role in endometrial physiology (Slayden et al., 2001
; Brenner et al., 2002
).
In reproductive tissues, the local actions of sex steroids are modulated by hydroxysteroid dehydrogenases (HSD). The human 17HSD family has six known members, each being a separate gene from a different chromosome with distinct properties in terms of substrates and redox direction. The type 2 isoform (17
HSD2) has a major role in the inactivation of estradiol to estrone. 17
HSD2 is also the enzyme responsible for converting androgens to less potent forms while also activating progesterone. The 17
HSD2 isoform is expressed in endometrial glandular epithelium, and is up-regulated by progesterone (Maentausta et al., 1993
). Its activity decreases when progesterone concentrations decline pre-menstrually or following antiprogestin administration (Maentausta et al., 1993
; Mustonen et al., 1998
). LNG binds avidly to the AR (Kloosterboer et al., 1988
). The availability of other androgenic ligands to bind to the AR may be influenced by the availability of the enzyme 17
HSD2.
We have thus evaluated expression of both AR and 17HSD2 in a longitudinal study of endometrial samples from naturally cycling women and from women treated with LNG-IUS over a 12 month time frame. The results provide new data on the expression patterns of these two molecular components that may be relevant to the bleeding patterns seen during intrauterine treatment with the LNG-IUS.
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Materials and methods |
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The first group (control group) consisted of fertile women (n = 24) with regular menstrual cycles lasting between 25 and 35 days who had not been using hormonal preparations for the preceding 3 months. Samples were obtained at the time of hysterectomy for benign indications. Subjects with significant uterine pathology (for example, fibroids) were excluded. Endometrial tissue was collected from women during the proliferative phase (n = 6), early secretory (n = 6), mid secretory (n = 6) and late secretory phase (n = 6) of the menstrual cycle. After the uterus had been removed, a full thickness biopsy of the uterus was obtained, extending from the lumen to the myometrial layer that included the superficial and basal endometrium in addition to the myometrium. The stage of the menstrual cycle was consistent with the patients reported last menstrual period and was confirmed by histological dating, according to the criteria of Noyes et al. (1950). Furthermore, serum samples collected at the time of endometrial tissue collection for determination of circulating estradiol and progesterone concentrations by radioimmunoassay were consistent with the designated cycle stage, and a significant decline in progesterone concentrations was apparent between biopsies taken in the mid and late secretory phase of the cycle (P < 0.01).
Endometrial biopsies were fixed overnight in 4% paraformaldehyde, then embedded in paraffin for immunohistochemical analysis. Additional biopsies from the endometrium of the same patients were frozen immediately at the point of tissue collection in liquid nitrogen, then stored at 80°C. Frozen biopsies were later used to obtain RNA (see below).
The second group consisted of fertile women (n = 12) with regular menstrual cycles lasting between 25 and 35 days who had not been using hormonal preparations for the preceding 6 months. The patients were to have a LNG-IUS inserted as a method of long-term hormonal contraception. All patients underwent a pre-insertion biopsy in either the proliferative (n = 5) or secretory (n = 5) phase of the cycle, immediately after which a LNG-IUS (Mirena; Schering HC, UK) was inserted as an outpatient procedure. The stage of the cycle prior to insertion of the LNG-IUS was determined by reference to serum sex steroid concentrations and histological dating by the criteria of Noyes et al. (1950). Further endometrial samples were collected at 1 (n = 2), 3 (n = 5), 6 (n = 5) and 12 months (n = 5) following insertion of the LNG-IUS. All specimens were fixed in 10% neutral-buffered formalin at 4°C and embedded in paraffin for immunohistochemical analysis.
The third patient group included women attending a menstrual problems clinic for management of unscheduled bleeding with the LNG-IUS. Endometrial samples were collected from patients attending the clinic with an LNG-IUS in utero for 3 months, 6 months, or >1 year. Samples were frozen immediately upon collection in liquid nitrogen and stored at 80°C.
RNA extraction and real time quantitative RTPCR
Frozen samples of endometrium stored at 80°C were first homogenized, and then total RNA was extracted using the commercially available product Trizol (Invitrogen Life technologies Ltd, UK) according to the manufacturers instructions. Following extraction, total RNA was resuspended in 50 µl of RNA storage buffer (Ambion, USA) and stored at 80°C.
Samples of total RNA to be used for quantification of AR mRNA required pre-treatment with RNase-free DNase (Invitrogen) as this primer probe preparation did not span an intron. Samples of RNA to be used for quantification of 17HSD2 mRNA did not require pre-treatment with RNase-free DNase, as this primer probe preparation spanned an intron.
The RTPCR reaction was performed in a 10 µl volume of reaction solution containing 1xTaqman RT buffer, MgCl2 (5.5 mmol/l), deoxyNTP, random hexamers (2.5 µmol/l), multiscribe reverse transcriptase (1.25 IU/µl), RNase inhibitor (0.40 IU/µl), and nuclease-free water (all reagents from Applied Biosystems, UK). The mix was divided into aliquots in individual tubes (8 µl/tube) and template RNA 200 ng (100 µg/l) was added. The samples were over-layered with mineral oil and RT reaction was conducted at 25°C for 60 min, 48°C for 45 min, and 95°C for 5 min for 1 cycle. Thereafter samples were stored at 20°C.
Real time quantitative PCR was performed in an ABI 7700 Sequence Detection System (PerkinElmer, USA). Oligonucleotide forward and reverse primers and oligonucleotide Taqman probes for AR and 17HSD2 were designed, with the use of Primer Express version 1.0 (PerkinElmer Applied Biosystems, UK), from sequences entered in the GenBank database.
The sequences for the AR and 17HSD2 primers and probe are shown in Table I. Quantitative PCR reaction mixtures were made containing Sure Start Taq DNA polymerase (0.025 IU/µl; Stratagene, UK), dNTP (all at 200 µmol/l) and specific forward and reverse primers (300 nmol/l) and probe (200 nmol/l) for either AR or 17
HSD2. Primers and VIC-labelled probe (all at 50 nmol/l; Applied Biosystems, UK) for ribosomal 18s RNA were also added to the same reaction mixture.
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Copy numbers were not used as we performed relative quantification as described above and as outlined in the PerkinElmer guidelines. No DNA or RNA standard was used.
The sizes of the amplicons that are generated by the primer probe set for AR and 17HSD2 are 65 and 72 bp respectively (see Table I).
The accuracy of the quantitative PCR was tested by serial dilution (to x32 dilution) of a pool of AR cDNA and a pool of 17HSD2 cDNA. The slope of the line plotting the cycle number at which the curve crossed a threshold (Ct) against dilution had a gradient of <0.14 and 0.13
Ct units/x32 dilution respectively. All menstrual cycle data and LNG-IUS-treated endometrium data were obtained from separate single PCR runs and related to standard AR cDNA and 17
HSD2 cDNA preparations using the formula 2
Ct that relates the ratio of 18S and specific amplicon in the sample cDNA with that of the standard preparation. Intra-assay variability for AR was calculated to 1% and for 17
HSD2 to 3.3%.
Statistical analysis
Numeric data from the real time quantitative RTPCR were analysed by one-way analysis of variance, followed by Fishers protected least significant difference test.
Antibody specificity
The properties of the antibodies for the immunolocalization of the AR (F39.4; Slayden et al., 2001) and 17
HSD2 (Mab C2-12; Moghrabi et al., 1997
) have been previously described and validated.
Immunohistochemistry
Paraffin sections (5 µm) were dewaxed and rehydrated through a series of alcohols, then washed in distilled water and 0.01 mol/l phosphate-buffered saline (PBS; SigmaAldrich Ltd, UK). The slides were then subjected to antigen retrieval by pressure-cooking in 0.01 mol/l sodium citrate at pH 6 for 5 min. After cooling for 20 min, the slides were washed in PBS. Endogenous peroxidase activity was quenched with immersion in 3% hydrogen peroxide (Merck, UK) in distilled water for 10 min at room temperature. Non-specific binding of the antibodies was blocked by incubating the sections for 2030 min at room temperature in non-immune horse serum (Vectastain; Vector Laboratories, Inc., UK).
For localization of the AR, sections were incubated with the mouse monoclonal antibody F39.4 (IgG1; Biogenex Laboratories, Inc., USA) overnight at 4°C at a 1:480 dilution in PBS/bovine serum albumin gelatin or similarly with a control mouse IgG1 antibody at a matched IgG1 concentration to the F39.4 antibody (1:600 dilution). For localization of 17HSD2, sections were incubated with the mouse monoclonal antibody C2-12 (gift from Dr Stefan Andersson) overnight at 4°C at a 1:20 dilution in normal horse serum or similarly with a control mouse IgG antibody at a matched IgG concentration to the C2-12 antibody (1:400 dilution). Following a wash in PBS with Tween 20, the slides were incubated in biotinylated horse anti-mouse secondary antibody (Vectastain) in normal horse serum for 60 min at room temperature, reacted with the avidinbiotin peroxidase complex (Vectastain Elite) for 60 min at room temperature and visualized with the substrate and chromagen 3,3'-diaminobenzidine (Dako Liquid, Dako Corp., USA).
Scoring and analysis of immunoreactivity
The immunostaining intensity of epitopes in all tissue sections was assessed in a semi-quantitative manner on a 4-point scale: 0 = no immunostaining, 1 = mild immunostaining, 2 = moderate immunostaining and 3 = intense immunostaining. All tissue sections were scored blind by two observers. We had previously validated this scoring system in a subset of tissue sections in which immunoreactivity was measured with a computerized image analysis system, and a strong correlation between quantitative data derived from image analysis and subjective scores by a trained observer was obtained (Wang et al., 1998). Semi-quantitative scoring results were analysed by a non-parametric method, the KruskallWallis test, followed by Dunns post-hoc multiple comparison test.
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Results |
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Significantly higher levels of AR mRNA were observed in the early and mid proliferative phase endometrial tissue samples compared with the remainder of the menstrual cycle stages (Figure 1).
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Comparison revealed a significantly greater production of AR mRNA in the proliferative phase when compared with any duration of treatment with the LNG-IUS (P < 0.01) (Figure 1). No significant differences were noted between the secretory phase and the LNG-IUS-treated endometrium.
Temporal expression of 17HSD2 messenger RNA in endometrium through the menstrual cycle and following treatment with LNG-IUS
The same samples analysed for AR were used for the 17HSD2 assays. The highest levels of 17
HSD2 mRNA expression was noted in the early proliferative phase, with the levels being significantly greater than the other stages of the menstrual cycle apart from the late secretory phase (P < 0.008) (Figure 2).
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AR immunostaining in levonorgestrel-treated endometrium
Presence of LNG-IUS resulted in a typically pseudo-decidualized stroma with atrophic glands and these histological features were observed in all post-insertion endometrial biopsies, as previously shown (Silverberg et al., 1986). Prior to insertion, all proliferative phase endometrial tissue samples displayed strongly positive nuclear immunoreactivity for AR in the endometrial stroma with minimal glandular nuclear immunoreactivity. As we previously noted (Slayden et al., 2001
), AR immunoreactivity in secretory phase biopsies was also localized to stromal nuclei and was clearly decreased (Figure 3).
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Discussion |
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Further, the present data demonstrate that endometrium exposed to high-dose intrauterine delivery of levonorgestrel exhibits a significant decrease in stromal AR protein immunoreactivity when compared with control proliferative endometrium. This significant down-regulation is in keeping with levels exhibited in the secretory phase and is maintained for 12 months following insertion of the LNG-IUS. In addition, there is a down-regulation of AR messenger RNA in LNG-IUS-exposed endometrium, which, in this case, is closely reflected by the pattern of expression of the AR protein.
Levonorgestrel binds to both the AR and PR (Kloosterboer et al., 1988; Lemus et al., 1992
). The role of androgens in the regulation of the AR protein and mRNA is not yet known, with some studies suggesting an up-regulation in response to androgens and others suggesting a down-regulation (Chada et al., 1994
; Fujimoto et al., 1994
; Iwai et al., 1995
; Apparao et al., 2002
). In-situ hybridization studies in the primate uterus suggested that testosterone can synergize with estradiol to up-regulate AR (Adesanya-Famuyiwa et al., 1999
). Levonorgestrel has dual progestogenic and androgenic activity shown by its relative binding affinity for the PR when compared with AR (Kloosterboer et al., 1988
). It is therefore unclear as to the mechanism of AR down-regulation in levonorgestrel-exposed endometrium, but it could be a consequence of both progestogenic and androgenic action.
In human endometrium, steroidal regulation of receptor action is dependent upon ligand availability. The 17HSD enzyme family comprises eight members, of which six human isoforms have been characterized. Its primary role is the inactivation of estradiol to estrone as well as testosterone to androstenedione, but in addition it converts the inactive 20
-dihydroprogesterone to active progesterone (Peltoketo et al., 1999
; Labrie et al., 2000
). Previous publications also report an up-regulation of 17
HSD2 in the progesterone-dominated secretory phase (Casey et al., 1994
). With intrauterine delivery of levonorgestrel, we found an increase in the expression of 17
HSD2 protein when compared with the proliferative phase of the menstrual cycle followed by a down-regulation of the 17
HSD2 protein in glandular epithelium after 3 months of treatment that was maintained up to the 12 month time-point. A significant up-regulation of the 17
HSD2 mRNA in the initial 3 months was also noted followed by a down-regulation in patients treated for >6 months. This is consistent with the down-regulation of the PR receptor over this same time frame (Critchley et al., 1998
).
Of interest, the decline in 17HSD2 mRNA expression appears to be staggered from that of the protein with a decrease in protein expression being noted prior to mRNA decline. This may be due to enhanced stability of 17
HSD2 mRNA possibly resulting in a persistence of the mRNA for long periods without translation into protein. It has been shown that mRNA can be stable for days at a time and this may be regulated in some cases by sex steroids (Day et al., 1998
; Staton et al., 2000
). Analysis of 17
HSD2 mRNA through the menstrual cycle also shows a lack of correlation with protein expression, because the highest levels of mRNA noted in the early proliferative phase occurred when no protein expression was noted. A similar pattern of 17
HSD2 mRNA expression is seen in the data reported by Casey et al. (1994
), Mustonen et al. (1998
) and Kitawaki et al. (2000
). Northern blot analysis revealed 17
HSD2 mRNA in the early proliferative phase (Casey et al., 1994
) and Kitawaki et al. found comparable levels of 17
HSD2 mRNA in proliferative and secretory phase tissue specimens (Kitawaki et al., 2000
). Quantitative analysis of 17
HSD2 mRNA in human endometrium reported by Mustonen et al. (1998
) also revealed a lack of correlation with protein levels, higher levels of mRNA expression occurring during the late secretory phase after the peaks in both serum progesterone and 17
HSD2 protein expression.
Another consistent feature in the endometrium of long-term progestin-only contraceptive users is the abnormalities reported in the structure of endometrial microvessels (Hickey et al., 2000). There is an associated increase in blood vessel fragility that may contribute to the pathogenesis of endometrial BTB. In the endometrium, progesterone receptors are absent in the endothelium but prominent in the perivascular cells (Koji et al., 1994
; Critchley et al., 2001
). Levonorgestrel is reported to induce changes in vessel fragility that may be mediated through perivascular cells rather than the endothelium (Roberts et al., 1992
).
In summary, exposure of the endometrium to intrauterine LNG results in decidualization and atrophy. The BTB problems reported by a proportion of users with this method of long-term progestin delivery are maximal during the first few months following insertion of an LNG-IUS. During this period, the 17HSD2 enzyme is elevated and steroid receptors, including AR, are suppressed. As a consequence, intracellular levels of the estrone are raised while those of estradiol, the more potent estrogen, are lowered. Problematic BTB usually improves after 36 months of LNG-IUS usage. At that time, levels of 17
HSD2 protein are low and thus intracellular concentrations of estradiol would be raised and estrone lowered. The levels of both AR mRNA and protein remain suppressed. We suggest that these gradual changes in endocrine relationships during treatment with the LNG-IUS mitigate the tendency for vascular fragility in the LNG-IUS-treated endometrium. However, it is likely that the mechanisms underlying the BTB associated with the use of LNG-IUS, and indeed other progestin-only contraceptive methods, are complex and involve multiple factors. Hopefully, data such as those presented here will contribute further to our understanding of local progestin action and associated tendency for BTB.
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Acknowledgements |
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Submitted on March 11, 2003; resubmitted on July 4, 2003; accepted on September 4, 2003.