Molecular Human Reproduction, Vol. 7, No. 4, 357-363,
April 2001
© 2001 European Society of Human Reproduction and Embryology
Expression of 11ß-hydroxysteroid dehydrogenase isozymes and corticosteroid hormone receptors in primary cultures of human trophoblast and placental bed biopsies
Divisions of Medical Sciences and Reproductive and Child Health, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK
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Interconversion of active and inactive glucocorticoids, e.g. cortisol (F) and cortisone (E) is catalysed by 11ß-hydroxysteroid dehydrogenase (11ß-HSD) which exists as two isoforms. We have used human placental bed biopsies and an in-vitro cytotrophoblast cell culture system to examine the expression and activity of the 11ß-HSD isoforms along with that of the glucocorticoid and mineralocorticoid receptors (GR and MR). Immunohistochemistry localized 11ß-HSD1 to decidualized stromal cells and 11ß-HSD2 to villous cytotrophoblast, syncytiotrophoblasts and trophoblast cells invading the placental bed and maternal vasculature. In primary cultures of human cytotrophoblast, 11ß-HSD2, GR and MR mRNA were expressed. Low levels of 11ß-HSD1 mRNA were noted in these cultured cells, but could be explained on the basis of contaminating, vimentin-positive decidual stromal cells (
5%). Enzyme activity studies confirmed the presence of a high-affinity, NAD-dependent dehydrogenase activity (Km 137 nmol/l and Vmax 128 pmol E/h/mg protein), indicative of the 11ß-HSD2 isoform. No reductase activity was observed. The presence of functional MR and GR was determined using Scatchard analyses of dexamethasone and aldosterone binding (MR Kd 1.4 nmol/l Bmax 3.0; GR Kd 6.6 nmol/l Bmax 16.2 fmol/ng protein). The expression of 11ß-HSD1 in maternal decidua and 11ß-HSD2 in adjacent trophoblast suggests an important role for glucocorticoids in determining trophoblast invasion. The presence of the MR within trophoblast indicates that some of the effects of cortisol could be MRrather than GR-mediated. 11ß-hydroxysteroid dehydrogenase/glucocorticoid receptor/mineralocorticoid receptor/placenta/trophoblast
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Ligand-dependent transactivation via the mineralocorticoid (MR) and glucocorticoid (GR) receptors is dependent on a variety of tissue-specific mechanisms, including the relative concentrations of the receptors. Previous studies have also highlighted `pre-receptor' regulation as an important mechanism regulating corticosteroid hormone action. This intracrine system is modulated by the enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD), which catalyses the interconversion of active glucocorticoids [e.g. cortisol (F)] and inactive glucocorticoids [e.g. cortisone (E)] (Stewart and Krozowski, 1999
). The type 1 isozyme (11ß-HSD1), found predominantly in GR-rich tissues such as liver and adipose tissue, shows predominantly E to F conversion and as such appears to be involved in the local stimulation of GR-mediated responses. In contrast, expression of the type 2 isozyme (11ß-HSD2) has been closely associated with tissues expressing MR. 11ß-HSD2 inactivates F to E in MR-rich tissues such as the kidney and distal colon, thereby preventing the illicit occupation of the MR by F, and enabling aldosterone to bind to the receptor in vivo (Edwards et al., 1988; Funder et al., 1988).
Recent studies by our group have highlighted novel patterns of expression of both isozymes. In particular, 11ß-HSD2 was shown to be widely expressed in fetal tissues including the placenta, where it co-localized with GR rather than MR (Condon et al., 1998). Functionally, the enzyme has been proposed to protect the developing fetus from the deleterious effects of relative maternal hypercortisolaemia and may therefore have an important role in regulating fetal growth and differentiation. Indeed, reduced placental 11ß-HSD2 expression has been documented in pregnancies complicated with intrauterine growth restriction (Shams et al., 1998). However, within the maternal/placental unit there remains debate surrounding the cellular localization of 11ß-HSD isozymes, and the relationship between the enzyme and MR/GR expression. In the baboon, both 11ß-HSD isoforms are expressed in placental syncytiotrophoblasts, with expression of both enzymes increasing as gestation advances (Pepe et al., 1996). Detailed microdissection of human placenta and maternal membranes including decidua indicate expression of only 11ß-HSD2 in the placental trophoblast and 11ß-HSD1 in decidua-stromal cells of the basal placentae and chorion (Stewart et al., 1995; Ricketts et al., 1998). These data are in keeping with the observation of significant conversion of cortisol to cortisone by placental homogenates at all gestational ages, increasing at term (Giannopoulos et al., 1982; Shams et al., 1998). However, immunocytochemical studies have also described the presence of immunoreactive 11ß-HSD1 in the intermediate trophoblasts and the endothelium of the fetal vasculature (Sun et al., 1997). This raises the possibility of a coordinated interaction between these two enzymes within the placenta itself. To address this issue, we have used human placental bed samples and, in addition, have developed and utilized an in-vitro human cytotrophoblast primary cell culture system for detailed analysis of the expression of 11ß-HSD isozymes, MR and GR in these cells.
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Materials and methods |
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Subjects
Placentae and placental bed samples (comprising myometrial biopsies at the site of placental implantation) were obtained from women (22–34 years of age) undergoing elective Caesarean section at term (38–41 weeks) at the Birmingham Women's Hospital, according to local ethical committee approval.
Immunohistochemistry
Together with placental bed biopsies from six individuals, multiple biopsies were taken from both the centre and periphery of corresponding placentae (trophoblast free from chorionic tissue). These tissues were washed in isotonic saline to remove any adherent blood clot prior to 10% formalin fixation and embedding in paraffin wax. Sections 3 µm thick were cut and placed on charged glass slides (Superfrost plus; BDH, Leicester, UK). The immunoperoxidase method employed was modified from a published technique (Ricketts et al., 1998). Following de-waxing, sections were subjected to microwave pressure cooking in 0.01 mol/l citrate buffer, pH 6, for 5 min. Endogenous peroxidase was blocked using 0.3% hydrogen peroxide in methanol for 30 min. Sections were incubated, for 1 h at room temperature, with primary antiserum diluted in 0.05 mol/l Tris-buffered saline, pH 7.6, containing 1% normal swine serum (NSS/TBS) (11ß-HSD1 1:300, 11ß-HSD2 1:80, cytokeratin 1:50, vimentin 1:25). After rinsing with NSS/TBS, a 1:50 dilution of donkey anti-sheep or rabbit anti-mouse horseradish peroxidase conjugate (Binding Site, Birmingham, UK) was applied for 30 min and developed using a 1 mg/ml 3,3'-diaminobenzidine tetrahydrochloride solution, with 0.003% hydrogen peroxide. Staining was intensified with a solution containing 4.2 g CuSO4 and 7.2 g NaCl per litre. The characterization of the sheep anti-human 11ß-HSD1 and 11ß-HSD2 antibodies has been reported (Shimojo et al., 1997; Ricketts et al., 1998). Mouse anti-human cytokeratin (MNF116) and vimentin (VIM3B4) antibodies were supplied by Dako, Ely, UK. Positive control tissues of adult human liver and kidney were employed for 11ß-HSD1 and -2 respectively. In addition, parallel staining was carried out in the presence of the appropriate immunizing peptide (100-fold excess), to ensure that all primary antibody binding was specific.
Dual fluorescence immunocytochemistry was also conducted on primary cell cultures. This was achieved by air drying cells spun onto glass slides, fixing in 4% formaldehyde at 4°C for 30 min and applying the appropriate primary antibodies (11ß-HSD2 and cytokeratin, 11ß-HSD1 and vimentin) as described above. The secondary antibodies used were a 1:400 dilution of a donkey anti-sheep-fluoroscein isothiocyanate conjugate (Binding Site) and rabbit anti-mouse rhodamine conjugate. The nuclear staining was obtained using Hoechst 33342 (Sigma).
Placental cytotrophoblast cell culture
Placental cytotrophoblast cells were isolated from term human placentae using previously described methods (Kliman et al., 1986; Greenwood et al., 1993). Cytotrophoblast cells were plated at a density of 4x106 cells/well in 6-well plates and cultured at 37°C and 5% CO2 in medium consisting of Dulbecco's modified Eagle's medium with 25 mmol/l HEPES (Life Technologies Ltd, Paisley, UK), 44% Ham's F-12 nutrient mix (Gibco, Paisley, UK), 10% fetal bovine serum (FBS; Life Technologies), 1% PSG (0.6 g benzylpenicillin, 1 g streptomycin sulphate, 2.92g L-glutamine, Sigma, Poole, UK, in 100 ml ultra-pure water); 0.1% gentamicin (Sigma). Cytotrophoblast cells were cultured for a minimum of 72 h and media changed to charcoal-stripped FBS for 24 h immediately prior to experimentation.
11 ß -HSD activity and kinetics in cultured human placenta cytotrophoblasts
Interconversion of F and E was assessed by enzyme activity studies using tritiated steroids as substrates as previously reported by our group (Stewart et al., 1994). Stock solutions of unlabelled cortisone and cortisol (Sigma), were prepared in absolute ethanol, such that ethanol concentrations were <0.1% in subsequent assays. Activity assays were performed in triplicate on intact cells from 72 h cultures of cytotrophoblasts. In 6-well plates, cells were incubated for 1 h in serum-free media, at 37°C with 50 nmol/l and 500 nmol/l F or E and appropriate tritiated tracer (50 000 cpm). Following extraction of steroids from media using dichloromethane and separation of F and E using thin-layer chromatography, the fractional conversion of F to E (or E to F) was determined and enzyme activity expressed as pmol F or E produced/mg protein per h. In all cases protein concentrations were determined using a microplate protein assay (Bio-rad, Hemel Hempstead, UK). The cofactor requirement for 11ß-HSD activity was determined using cell lysates. Cytotrophoblast cultures were sonicated (three 5 s pulses) in phosphate-buffered saline. Aliquots of the lysate containing 50 µg protein were incubated in phosphate buffer (0.1 mol/l, pH 7.6) containing 200 µmol/l NAD, 200 µmol/l NADP, or no cofactor, in the presence of 50 nmol/l F and radiolabelled tracer, at 37°C in a shaking water bath for 1 h. The conversion of F to E was determined as described above. The apparent Km and Vmax values for 11ß-HSD in intact cytotrophoblast cultures were determined by repetition of the whole-cell activity assay outlined above using a range of F concentrations (5–500 nmol/l). Km and Vmax were calculated from the mean of four experiments using Lineweaver-Burk plots of 1/[V] (rate of reaction) versus 1/[S] (substrate concentration). For all activity assays, blank control reactions were performed involving incubations without cells/lysate, and any background conversion was subtracted from activity assays.
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of 11 ß -HSD isozyme, MR and GR expression
Total RNA was extracted from 72 h primary cytotrophoblast cultures, adult human liver and kidney biopsies (11ß-HSD type 1 and 2 positive tissues, respectively) using a published method (Chomczynski and Sacchi, 1987) (RNAzol B; ams Biotechnology, Oxford, UK). cDNA was synthesized from 1 µg total RNA, using avian myeloblastoma viral (AMV) RT-driven primer extension from random hexamer oligonucleotides (Promega, Southampton, UK). 10% of each RT reaction was used as template for PCR amplification using oligonucleotide primers designed specifically for each 11ß-HSD isozyme, MR and GR. The primers for 11ß-HSD1 (5'-CAGGTGTGACCCATGACTTG-3' and 5'-CAAGTTTGCTTTGG ATGGGT-3') amplified a region of 451 bp (exons 1–4). Primers for 11ß-HSD2 (5'-GGATTCTTTAGGCCAGGGTC-3' and 5'-TGGAGGTGAATTTCTTTGGC-3') amplified a region of 773 bp (exons 3–5). Primers employed for the specific amplification of GR and MR (regions of 450 and 693 bp respectively) were those previously reported by our group (Bland et al., 1999). For all primers an initial denaturing step (95°C for 6 min) was followed by 32 cycles of annealing (11ß-HSDs 60°C; MR/GR 55°C for 1 min), extension (72°C for 1 min) and denaturing (95°C for 1 min), and a single extension termination step (72°C for 10 min). Negative controls employed RT products from reactions without AMV or template RNA. Positive/negative controls of human liver and kidney RNA were also used. PCR products were visualized using 2% agarose gel electrophoresis.
MR and GR binding studies
Cytotrophoblast cultures (72 h), in 24-well plates, were washed twice with serum-free media and then incubated in media containing 0.1% BSA and increasing concentrations (0.625–20 nmol/l) of [3H]aldosterone (specific activity 64 Ci/mmol) or [3H]dexamethasone (80 Ci/mmol) (Amersham, Little Chalfont, UK), with or without a 200-fold excess of unlabelled ligand. Aldosterone binding studies were also carried out in the presence or absence of a 200-fold excess of the GR antagonist RU38486 (Roussel Uclaf, France). Cells were incubated with radiolabelled steroids for 1 h at 37°C in a 5% CO2 incubator, and then washed three times to remove unincorporated steroids. 500 µl distilled H2O was added to each well and then subjected to two rounds of freeze-thawing. An aliqout (450 µl) of the resulting lysate was removed and radioactivity counted. The remaining 50 µl was used to determine protein concentration as described above. Scatchard analysis was used to determine Kd and Bmax. In each case, binding studies were conducted in triplicate on three separate occasions.
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11 ß -HSD expression in placental bed
In placental and placental bed tissue sections, 11ß-HSD2 immunoreactivity was consistently detectable in cells which were also cytokeratin positive, namely fused syncytiotrophoblast and invasive extravillous trophoblast, including those lining the maternal spiral arteries. 11ß-HSD1 immunoreactivity was consistently absent from trophoblast cells; positive staining was confined exclusively to vimentin-positive decidual stromal cells (Figure 1
).
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Dual-fluorescence immunocytochemistry confirmed that primary trophoblast cultures consisted of a population of cells that were both 11ß-HSD2 and cytokeratin positive (>95%) (Figure 2
). Only rarely was contamination with a vimentin-positive decidual cell observed.
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11 ß -HSD activity and kinetics in cultured human placenta cytotrophoblasts
11ß-HSD assays conducted on primary cytotrophoblast cultures indicated high affinity dehydrogenase activity. Thus in the presence of 50 nmol/l F, specific activity was 50 ± 10 (mean ± SE of triplicate assays, n = 12) pmol E/h/mg protein; with 500 nmol/l F conversion the specific activity was 355 ± 63 (n = 10) pmol E/h/mg protein (Figure 3A
). Kinetic studies on 72 h cytotrophoblast cultures indicated the presence of 11ß-HSD with an apparent Km for F of 137 nmol/l and Vmax of 128 (n = 4) pmol E/H/mg protein (Figure 4). In addition, cell lysates incubated with 200 µmol/l NAD showed significantly more activity when compared to those incubated with NADP or with no cofactor (Figure 3B). These data are consistent with expression of the type 2 11ß-HSD isozyme. Reductase activity (i.e. conversion of E to F) was absent in every primary cell preparation.
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Analysis of 11 ß -HSD isozyme and MR/GR mRNA expression
RT–PCR analyses of mRNA isolated from primary cultures of cytotrophoblasts demonstrated the presence of transcripts for both 11ß-HSD1 and 11ß-HSD2, with products corresponding to the predicted sizes of 451 and 773 kb respectively (Figure 5A,B
). Further analyses showed expression of both GR and MR, with RT-PCR products at 693 and 450 bp respectively (Figure 5C,D).
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MR and GR binding analysis
Studies were also carried out to confirm the presence of functional GR and MR. Whole cell assays demonstrated specific binding for [3H]aldosterone and [3H]dexamethasone, suggesting that cytotrophoblast cell cultures possess both MR and GR respectively. Scatchard analysis revealed specific binding of [3H]dexamethasone to the GR with a maximal binding capacity (Bmax) of 16.2 fmol/ng protein and a dissociation constant (Kd) of 6.6 nmol/l. Analysis of [3H]aldosterone binding kinetics indicated a Bmax of 3.0 fmol/ng protein and a Kd of 1.4 nmol/l (Figure 6
).
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Discussion |
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We have documented the expression of mRNA and protein for both type 1 and type 2 11ß-HSD in the human placenta and placental bed biopsies at term. Immunolocalization of the two isoforms of 11ß-HSD appeared in distinct morphological areas of the placental bed. Consistent with our own and previous reports, 11ß-HSD2 expression appears to be most intense in the fused syncytiotrophoblast (Albiston et al., 1994
; Brown et al., 1996; Shams et al., 1998; Hirasawa et al., 2000), with the inner cytotrophoblast layer also expressing this isoform. The role of 11ß-HSD2 in the placenta remains unclear. It has been suggested that it may protect the fetus from relatively high concentrations of maternally derived glucocorticoids which may result in deleterious effects on fetal development and growth (Reinisch et al., 1978). The most intense localization of 11ß-HSD2 to syncytiotrophoblasts in contact with maternal blood supports this proposed role. Alterations in placental 11ß-HSD2 activity have been postulated to explain the strong epidemiological links emerging between fetal environment, reduced fetal growth and adult cardiovascular disease (Benediktsson et al., 1993). Conversely in the fetal circulation, placental 11ß-HSD2 may ensure rapid and efficient metabolism of endogenously derived cortisol and a high adrenocorticotrophic hormone drive to the fetal adrenal gland, a mechanism which appears to enhance the ontogeny of development of the fetal hypothalamic-pituitary-adrenal axis in both primates and humans (Stewart et al., 1995; Pepe et al., 1996, 1999).
11ß-HSD2 is also expressed in many other fetal tissues (Condon et al., 1998), where its ontogeny and localization more closely relates to the GR rather than the MR. This suggests that despite the established role of 11ß-HSD2 in protecting the MR from glucocorticoid in adult mineralocorticoid-target tissues such as kidney and colon, in fetal tissues including the placenta, this enzyme may be regulating cortisol exposure to the GR. However, using RT-PCR, we have demonstrated the presence of mRNA transcripts for both GR and MR in primary cytotrophoblast cultures. Furthermore, Scatchard analyses using these cultured cells revealed the presence of functional MR due to specific binding of [3H]aldosterone in the presence of the GR antagonist RU38486. The dissociation constant (Kd) for aldosterone binding was in the order of 1 nmol/l, consistent with previous studies (Krozowski and Funder, 1983; Arriza et al., 1987). Dexamethasone is also known to bind to the MR with a similar affinity (0.7 nmol/l) (Hellal-Levy et al., 1999). However, data from our study indicated a Kd value for dexamethasone binding of ~6.6 nmol/l. This suggests that dexamethasone was binding predominantly to GR in trophoblast cells. Thus, both GR and MR appear to be functional in human cytotrophoblast cells.
Recent studies have also noted MR immunoreactivity in human placental syncytiotrophoblast and cytotrophoblast cells (Hirasawa et al., 2000). There are no data on the expression and mineralocorticoid regulation of gene products mediating epithelial sodium transport within the placenta (e.g. epithelial sodium channels, serum and glucocorticoid-induced kinase). However, cortisol has been shown to inhibit placental 15-hydroxyprostaglandin dehydrogenase, and the fact that this occurs at low nanomolar concentrations and is enhanced following incubation with the 11ß-HSD inhibitor, carbenoxolone, suggests that it may be a MR-mediated effect (Patel et al., 1999). This aside, recombinant mice lacking the MR have no obvious fetal/placental phenotype (Berger et al., 2000), and placental MR expression is very low compared to reported levels in the kidney (Brown et al., 1996b). Nevertheless with such an abundant expression of 11ß-HSD2, inactivating F to E, even low levels of MR expression may be of relevance. Further studies are now required to define the biological role of the MR in human placenta.
We have shown that cultured cytotrophoblasts express active 11ß-HSD2, consistent with immunohistochemical data. By contrast, 11ß-HSD1 was localized only to maternal decidual stromal cells, with no expression being observed in trophoblast tissue. Using RT-PCR on total RNA extracted from cultured cytotrophoblast, lower levels of 11ß-HSD1 mRNA were detected, but we attribute this to minor (<5%) decidual contamination of our culture preparations. Thus >95% of the primary cytotrophoblast cultured cells stained positive for cytokeratin, but very rarely an isolated, vimentin-positive cell was observed. 11ß-HSD1 immunoreactivity in some extravillous cytotrophoblasts and the vascular endothelium of the villous placenta has been reported (Sun et al., 1997), a finding which has been endorsed in the baboon placenta, but not in this study. The explanation for this discrepancy is unclear; we have utilized validated antisera against human 11ß-HSD isozymes and in every case have contrasted cellular expression with established markers of trophoblast epithelial cells and decidual stromal cells. Nevertheless, the close apposition of 11ß-HSD2 in the cytokeratin-expressing cells lining the modified spiral arteries and 11ß-HSD1 in adjacent decidual cells in the implanting placenta seems likely to be important in modulating cortisol concentrations in an autocrine/paracrine fashion which may in turn be pivotal in the placentation process. It remains unclear why, in normal physiology, the decidua needs to generate cortisol whilst an adjacent fetal trophoblast cell apparently needs to inactivate it. Haemochorial placentation in human pregnancy is a complex procedure in which the fetal and maternal circulations become juxtaposed (Pijnenborg et al., 1980). From the point at which the trophectoderm implants on the decidualized endometrium, cytotrophoblast stem cells begin to differentiate along two discrete pathways (Pijnenborg et al., 1983; Cross et al., 1994). The villous cytotrophoblasts fuse to form a multinucleated syncytiotrophoblast layer, which is in direct contact with maternal blood, whilst those from the anchoring villi form mononucleated cell columns to attach the placenta to the uterine wall and progress endovascular invasion (Cross et al., 1994). The `invasion' of cytotrophoblast along spiral arteries has been thought to be controlled, at least in part, by extracellular matrix (ECM)-degrading enzyme (MMP-9) (Librach et al., 1991) and the expression of MMP-9 in cytotrophoblasts is down-regulated by corticosteroids (Librach et al., 1994). Thus one possibility is that the inactivation of cortisol by trophoblast 11ß-HSD2 would promote invasion, whilst the generation of cortisol at the maternal interface may inhibit this process.
In summary, we have documented expression of 11ß-HSD2 within the placenta including invasive extravillous trophoblast. The expression of 11ß-HSD1, in contrast, appears to be confined to adjacent, vimentin-positive, decidual stromal cells. These results suggest that the local control of glucocorticoid concentrations may play a crucial role in regulating placentation and invasion. The expression of 11ß-HSD2 may be a crucial factor in determining trophoblast invasion, and our in-vitro model comprising of primary trophoblast cultures represents an excellent model to further evaluate this. Finally, the presence of both MR and GR within trophoblast indicates that some of the effects of cortisol could be MRrather than GR-mediated.
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We wish to thank Colin Sibley and Susan Greenwood for advice and technical assistance regarding the primary cell culture technique, and Dipah Gill for help with immunohistochemistry. This work was supported by the MRC and Action Research. P.M.S. is an MRC Senior Clinical Fellow.
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1 To whom correspondence should be addressed. E-mail: m.d.kilby{at}bham.ac.uk
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Submitted on September 19, 2000; accepted on January 31, 2000.
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K. J. Barber, J. A. Franklyn, C. J. McCabe, F. L. Khanim, J. N. Bulmer, G. S. J. Whitley, and M. D. Kilby The in Vitro Effects of Triiodothyronine on Epidermal Growth Factor-Induced Trophoblast Function J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1655 - 1661. [Abstract] [Full Text] [PDF]
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J. W. Tomlinson, E. A. Walker, I. J. Bujalska, N. Draper, G. G. Lavery, M. S. Cooper, M. Hewison, and P. M. Stewart 11{beta}-Hydroxysteroid Dehydrogenase Type 1: A Tissue-Specific Regulator of Glucocorticoid Response Endocr. Rev., October 1, 2004; 25(5): 831 - 866. [Abstract] [Full Text] [PDF]
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C. C. W. Chan, T. T. Lao, P. C. Ho, E. O. P. Sung, and A. N. Y. Cheung The Effect of Mifepristone on the Expression of Steroid Hormone Receptors in Human Decidua and Placenta: A Randomized Placebo-Controlled Double-Blind Study J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 5846 - 5850. [Abstract] [Full Text] [PDF]
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S. Chan, S. Kachilele, E. Hobbs, J. N. Bulmer, K. Boelaert, C. J. McCabe, P. M. Driver, A. R. Bradwell, M. Kester, T. J. Visser, et al. Placental Iodothyronine Deiodinase Expression in Normal and Growth-Restricted Human Pregnancies J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4488 - 4495. [Abstract] [Full Text] [PDF]
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D. B. Hardy and K. Yang The Expression of 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Is Induced during Trophoblast Differentiation: Effects of Hypoxia J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3696 - 3701. [Abstract] [Full Text] [PDF]
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V. E. Murphy, T. Zakar, R. Smith, W. B. Giles, P. G. Gibson, and V. L. Clifton Reduced 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Activity Is Associated with Decreased Birth Weight Centile in Pregnancies Complicated by Asthma J. Clin. Endocrinol. Metab., April 1, 2002; 87(4): 1660 - 1668. [Abstract] [Full Text] [PDF]
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C. L. McTernan, N. Draper, H. Nicholson, S. M. Chalder, P. Driver, M. Hewison, M. D. Kilby, and P. M. Stewart Reduced Placental 11{beta}-Hydroxysteroid Dehydrogenase Type 2 mRNA Levels in Human Pregnancies Complicated by Intrauterine Growth Restriction: An Analysis of Possible Mechanisms J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4979 - 4983. [Abstract] [Full Text] [PDF]
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