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Departments of Obstetrics and Gynecology (R.J.G., V.K.M.H.), Physiology (R.J.G., M.G.), and Pediatrics (D.M.M., K.N., V.K.M.H.), Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada N6A 4V2
Address all correspondence and requests for reprints to: Dr. R. J. Gratton, Department of Obstetrics and Gynecology, Lawson Health Research Institute, St. Joseph’s Health Center, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. E-mail: rgratton{at}uwo.ca.
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Abstract |
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Introduction |
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In pathological pregnancies, alterations in ADM concentrations in the maternal plasma and in fetal placental tissues may mediate compensatory vascular responses in the placental or fetal circulation (5, 6, 7, 8). Preeclampsia is a serious pathological condition that affects 7–10% of all pregnancies and is a leading cause of maternal and fetal morbidity and mortality (9). Failure of the endovascular component of trophoblast invasion (10, 11) and reduced placental perfusion result in placental ischemia/reperfusion and the release of reactive oxygen species. Maternal predisposing factors (obesity, diabetes, and hypertension) contribute to the oxidative stress and result in systemic endothelial dysfunction (12). Clinically, preeclampsia presents as hypertension and proteinuria, and in the most severe cases, vasospasm and widespread microvascular occlusive thrombi cause thrombocytopenia, hepatic and renal dysfunction, and seizures. The development of uteroplacental insufficiency may result in intrauterine growth restriction and intrauterine fetal demise.
The roles of ADM in the pathogenesis of preeclampsia and in the regulation of fetal/placental perfusion are unknown. Circulating ADM levels in women with preeclampsia have been reported to be increased (13), decreased (14), or unchanged (6, 15). Preeclamptic patients have been reported to have higher concentrations of ADM in the amniotic fluid and in umbilical venous plasma (6). Immunoreactive ADM has been identified in the fetal membranes (16, 17) and placentae (5, 15, 16, 17, 18, 19), suggesting that fetal placental tissues may contribute to the maternal and fetal plasma, and amniotic fluid levels of ADM in pregnancy. The two previous studies of ADM mRNA expression in the placenta of women with hypertension in pregnancy reported conflicting changes in ADM expression (17, 20). These studies included very different groups of women and are limited by the placental sampling methods. The purpose of this study was to further our understanding of the potential role of ADM in the placenta of normal and preeclamptic women by determining the regional localization and cellular expression of ADM mRNA in the placentae of normal and preeclamptic women.
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Materials and Methods |
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Thirty-seven pregnant women were recruited for this study in the following four groups: pregnancies complicated by severe preeclampsia and delivered before 28 completed wk of gestation (n = 7) or after 28 completed wk of gestation (n = 15), and control normotensive pregnancies matched for gestational age and route of delivery before 28 completed wk of gestation (n = 6) or after 28 completed wk of gestation (n = 13). The mean gestational age, birth, and placental weights for the early and late gestational age groups are presented in Table 1
. Patients were excluded from the study if any of the following were present: evidence of intrapartum infection, other pregnancy complications [multiple gestation, diabetes, premature rupture of membranes, lupus anticoagulant, fetal anomalies, fetal growth restriction (birth weight <10th percentile for gestational age), or chromosomal abnormalities], prepregnancy medical conditions (chronic hypertension, renal disease, or collagen vascular disease), and maternal smoking. The diagnosis of severe preeclampsia was based on a blood pressure of at least 160/110 mm Hg (on two occasions over 6 h), proteinuria (>3+ on dipstick or >3 g/24 h), presence of headaches, visual disturbances, epigastric pain, oliguria (<30 ml/h), thrombocytopenia (<100 x 106/liter), or abnormal liver function tests (>2 times normal) (9). Placentae were collected immediately after delivery. Using a template for consistent collection, a total of eight samples were taken from central cotyledons, and four samples were taken from peripheral cotyledons and fetal membranes. Peri-infant samples were taken after visual inspection of the preeclampsia placentae. For analysis, we selected 1) chorionic villi tissue from normal appearing central cotyledons at three sites, 2) chorionic villi tissue from peri-infarct areas (two sites) in preeclampsia, 3) basal plate decidua from the corresponding regions, and 4) full thickness membranes (four sites) (amnion, chorion, and decidua parietalis). Samples were frozen in liquid nitrogen immediately and stored at -70 C or fixed in neutral buffered formalin and embedded in paraffin, and 0.5-µm tissue sections were prepared and mounted on SuperFrost Plus slides (VWR, Mississauga, Canada) for in situ hybridization.
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Total RNAs were extracted from the placental samples using the single step method of RNA isolation by guanidinium-thiocyanate-phenol-chloroform extraction (21), and Northern blotting was performed as previously described (22). Briefly, total RNA (20 µg/lane) was denatured and subjected to electrophoresis in a 1% (wt/vol) agarose gel containing 2.2 M formaldehyde. The RNA was transferred to a zeta-probe nylon membrane (Bio-Rad Laboratories, Hercules, CA) by capillary transfer technique. After transfer, the blots were hybridized with 32P-labeled human ADM cDNA probes (2 x 106 cpm/ml buffer containing 5x SSPE (0.75 M NaCl, 44 mM NaH2PO4·2H2O, and 5 mM EDTA, pH 7.4), 7% sodium dodecyl sulfate, and 5 µg/ml denatured salmon sperm DNA. The 576-bp RTP cDNA probe was generated from total RNA of a chorionic villous region of a control placenta by RT-PCR using the following primers designed based on published sequence (22): 5' sense, 5'-ATGAAGCTGGTTTCCGTCGCCCTGATGTA-3' (position 157–185); and 3' antisense, 5'-ACCATGGGCGCCTAAATCCTAAAGAAAGTG-3' (position 732–703).
The cDNA inserts were labeled with [32P]deoxy-CTP (Pharmacia Canada, Inc., St. Laurent, Canada) to specific activities of 1–2 x 109 cpm/µg by the random priming technique using an oligo labeling kit (Pharmacia Canada, Inc.). Blots were washed twice for 30 min each time in 1x SSC (0.15 M NaCl and 15 mM sodium citrate, pH 7.0)/0.1% sodium dodecyl sulfate at 42 C and twice for 30 min each time in 0.1x SSC/0.1% sodium dodecyl sulfate at 42 C, air-dried, and subjected to autoradiography at -70 C. Consistency in loading and transfer of total RNA in each lane was checked by probing the blots for 18S ribosomal RNA. Autoradiograms were quantified using phosphorimaging (Phosphorimager S.I., Molecular Dynamics, Sunnyvale, CA).
In situ hybridization and immunohistochemistry
In situ hybridization was performed as described previously (23). Briefly, 5-µm tissue sections were deparaffinized, rehydrated, and hybridized with 35S-labeled antisense cRNA probes overnight at 55 C and washed at maximum stringency of 0.1x SSC at 65 C for 30 min. The 35S-radiolabeled antisense and sense RNA probes were designed in our laboratory from the published sequence as described above (22).
A combined in situ hybridization for ADM mRNA and immunohistochemistry for cytokeratin (to identify epithelial cells and trophoblasts) was performed as described previously (23). Briefly, in situ hybridization with a 35S-labeled cRNA probe was performed to the stage of completion of washing with 0.1x SSC solution at 65 C, after which the tissue sections were incubated with a rabbit antiserum against cytokeratin (1:2500; Dako, Glostrup, Denmark) at 4 C for 24 h. Immunostaining was identified by the avidin-biotin-peroxidase technique using a Vectastain kit (Vector Laboratories, Inc., Burlingame, CA) with diaminobenzidine as the chromagen. After dehydration, tissue sections were washed, dehydrated, coated with NTB-3 nuclear track emulsion (Eastman Kodak Co., Rochester, NY), and exposed at 4 C for 3–14 d. The photoemulsion was developed with a D19 Developer (Kodak), fixed, stained with Harris hematoxylin and eosin, and mounted with Permount (Fisher Scientific, Fairlawn, NJ). The specificity of the in situ hybridization was demonstrated by the absence of hybridization signal when adjacent tissue sections were subjected to an identical in situ hybridization procedure with radiolabeled sense cRNA probes.
Chorionic villous explant studies
In a separate group of placentae, chorionic villous tissue was dissected from normal term placentae (n = 6) delivered by cesarean section, and explant cultures were established and maintained in standard culture and reduced oxygen tension conditions. Three cotyledons were extracted from each placentae and rinsed with sterile saline. Explants were prepared by removing the decidua and placental vessels from a 1-cm3 sample of chorionic villi under a dissecting microscope. The explants were cultured in six-well NUNC plates containing 3 ml/well RPMI media (Life Technologies, Inc., Burlington, Canada), 50 U/ml penicillin (Life Technologies, Inc.), 50 µg/ml streptomycin (Life Technologies, Inc.), and 10% fetal calf serum. All explants were incubated in a 100% humidified atmosphere at 37 C in 5% CO2 for 1, 3, and 6 h. Explants were incubated in 37 C anaerobic chambers (Billuns-Rothenburg, Del Mar, CA) that were used to maintain explants at 1% or 0% oxygen, and room air was used to maintain cultures at 21% oxygen. Explants collected from three- to six-well plates were pooled, and total RNA was extracted (20) after 12, 24, and 48 h of culture, and the mRNA abundance was determined by Northern blot analysis.
Statistical analysis
ADM mRNA abundance in control and preeclamptic placenta was compared using an unpaired t test. To combine data from more than one Northern blots, a z-score was calculated. After the mRNA abundance for each sample was normalized for 18S, the mean ADM mRNA abundance/18S was determined for each blot. The z-score was calculated by the [sample ADM mRNA/18S ratio - mean ADM mRNA/18S ratio]/SD for the blot. A z-score of 0 is equivalent to the mean abundance in a specific placental region, whereas a negative score represents less and a positive score represents more expression than the mean for the region. The mean and SE of the z-scores from placental regions of central and preeclamptic placenta are presented. One-way ANOVA was used to compare mRNA abundance in chorionic explant studies. Significance was considered at P < 0.05.
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Results |
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). Only weak expression was identified in the isolated amnion membranes. There was no difference in expression in central compared with peripheral cotyledons or in placental regions from early gestation compared with late gestation control women.
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). Although there was a trend toward increased ADM mRNA abundance in chorionic villi and basal plate decidua regions in preeclampsia, this was not significantly significant. There was no statistical difference in ADM mRNA abundance in the chorionic villi, basal plate, or choriodecidua in placenta of women with early onset (<28 wk) preeclampsia compared with gestational age-matched normotensive controls (Fig. 3); however, as in later gestation there appeared to be an increase in choriodecidua regions. In situ hybridization studies showed that ADM mRNA was expressed in the cytotrophoblasts in the basal plate decidua regions similarly in both control and preeclampsia (Fig. 4, A, B, and G). In the chorionic villi regions ADM mRNA was expressed in the syncytiotrophoblasts of control (Fig. 4, A, B, and E) placentae. In the chorionic villi from preeclampsia placentae, increased hybridization was seen in the syncytial knots (Fig. 4, C, D, and F) in early and late gestation preeclampsia, which was consistent with the Northern blots where there was a trend to increased abundance in the chorionic villi region. There was also high ADM mRNA expression in the cytotrophoblasts in the regions surrounding placental infarcts in placenta of women with preeclampsia (Fig. 5, A–C). In the fetal membranes, there was weak expression in the amnion. ADM mRNA expression was high in the trophoblast cells of the chorion leave in control placentae (Fig. 5D) and was increased in the trophoblast cells of the chorion in late-onset preeclampsia, consistent with the Northern blot results (Fig. 5, E and F).
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), and this was maximally increased at 24 h. In situ hybridization studies demonstrated that ADM mRNA was expressed in the syncytiotrophoblasts and chorionic mesoderm in the chorionic villi regions (Fig. 5
, G and H).
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Discussion |
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Moriyama et al. (19) recently identified ADM mRNA expression in placental trophoblastic tissue from each trimester and in JAR cells by RT-PCR. We have furthered these observations by studying multiple placental regions in normal pregnancy. ADM mRNA was expressed in the chorionic villi and at the maternal-fetal interface in the basal plate decidua region. In situ hybridization studies demonstrated ADM mRNA expression in syncytiotrophoblasts in chorionic villi regions and in cytotrophoblasts of basal plate decidua regions. Apodaca et al. (5) identified ADM mRNA expression in amnion and chorion by RT-PCR. We have shown that ADM mRNA was maximally expressed in trophoblast cells of chorion leave and was weakly expressed in amnion in control placentae. Previous immunohistochemical studies have identified ADM expression in syncytiotrophoblasts (18, 20) and cytotrophoblasts in chorionic villi (19), extravillous trophoblasts (18, 20), and amnion and trophoblasts of the chorion (18, 20). The regional and cellular localizations of ADM mRNA and peptide in villous and extravillous trophoblasts and in fetal membranes suggest that fetal/placental tissues contribute to the maternal and fetal plasma and amniotic fluid levels of ADM and support a role for ADM in the vascular adaptation in pregnancy and in the regulation of placental vascular tone.
In pathological pregnancies, alterations in ADM concentrations in maternal plasma and in fetal placental tissues may mediate compensatory vascular responses in the placental or fetal circulation (5, 6, 7, 8). The ADM concentration in the fetal placental circulation is increased in intrauterine fetal growth restriction with abnormal umbilical artery Doppler wave forms (7), and amniotic fluid ADM levels are increased in diabetic pregnancy (8). ADM mRNA abundance was increased 5-fold in placentae of women with pregnancies complicated by oligohydramnios (5). Jerat et al. (3) demonstrated that ADM significantly relaxed stem villous arteries from both normal and preeclamptic patients. In preeclampsia, trophoblast invasion into the maternal decidua and spiral arteries at the maternal-fetal interface is impaired (10, 11). The resulting shallow placental invasion and reduced perfusion may reduce oxygen tension in the placenta and result in fetal growth restriction and the release of vasoactive factors that contribute to the pathophysiology of preeclampsia (24, 25, 26). The role of ADM in the pathogenesis and in the regulation of fetal/placental perfusion in pregnancies complicated by preeclampsia is unknown.
Maternal concentrations of ADM in preeclampsia have been recently reported. Hata et al. (14) reported that women with preeclampsia have decreased circulating ADM levels. Conversely, Lauria et al. (13) reported that women with preeclampsia have elevated ADM levels compared with normotensive pregnant women. Other studies have shown that ADM concentrations do not change in women with pregnancies complicated by preeclampsia (3, 6, 15) or gestational hypertension (3). Women with preeclampsia have higher concentrations of ADM in the amniotic fluid and umbilical vein plasma, and Di Iorio et al. (6) suggested that increased ADM concentrations may be necessary to maintain placental vascular resistance and/or the fetal circulation at a physiological level.
The two studies of ADM in the placenta of women with hypertension in pregnancy have reported conflicting results. In immunohistochemical studies, Kanenishi et al. (20) found that ADM expression in the placentae obtained from women with preeclampsia was decreased compared with that in normotensive controls. They found was no difference in the expression in the amnion or chorion. Makino et al. (17) reported that immunoreactive ADM was significantly increased in the fetal membranes of patients with pregnancy-induced hypertension. In the placenta, there was no difference in ADM mRNA or protein expression. The differences in these studies are explained in part by the differences in the study groups, as Kanenishi et al. (20) studied women with term preeclampsia and significant fetal growth restriction, while Makino et al. (17) studied women with term gestational hypertension, but not preeclampsia. In addition, the placenta is a heterogeneous tissue, and the specific site of sampling is not clear.
We have studied women with severe preeclampsia of early onset (<28 wk) and late onset (>28 wk) in gestation with normal birth weight fetuses and have sampled multiple specific regions of the placenta. We chose to study ADM mRNA abundance, instead of peptide levels, because it reflects the synthetic capacity of the placenta. In the placentae of women with severe preeclampsia, ADM mRNA was expressed in the syncytiotrophoblasts of the chorionic villi regions and in the cytotrophoblasts of the basal plate decidua regions similar to control placentae. However, increased hybridization was seen in the syncytial knots of the chorionic villi regions in the early and late gestation preeclampsic groups. Syncytial knots are more common in preeclampsia, particularly early-onset preeclampsia, and they are an indication of utero-placental ischemia. There was also increased ADM mRNA expression in cytotrophoblasts in the regions surrounding placental infarcts in preeclampsia. In fetal membranes, ADM mRNA expression was significantly increased in the choriodecidua in pregnancies complicated by severe preeclampsia (>28 wk) consistent with the previous report of increased immunoreactive ADM in fetal membranes (17). The choriodecidua represents the maternal-fetal interface over the entire uterine surface area. Therefore, our findings suggest that ADM is expressed in placental regions that are undergoing hypoxic stress. The increased expression in the choriodecidua, syncytial knots, and perinfarct areas in women with severe preeclampsia may reflect reduced oxygen tension within the placenta in the second half of pregnancy as a consequence of reduced uterine blood flow.
One method to study in vitro the effects of hypoxia on the placenta involves short-term cultures of placental explants. In the villous explant studies we demonstrated that ADM mRNA expression in the placenta is induced by hypoxia. Hypoxia has been shown to induce ADM expression in other tissues (27, 28), and ADM appears to be under hypoxia-inducible factor-1 transcriptional regulation (28). This is the first demonstration of hypoxia-mediated increased ADM expression in the placenta. Increased ADM expression in the trophoblast from the peri-infarct areas and in the syncytial knot is consistent with the expression patterns of other hypoxia-inducible genes in preeclamptic placentae (29). Di Iorio et al. (6) and others (17, 20) suggested that ADM may act in a paracrine fashion and mediate fetal/placental adaptive responses through interaction with other vasoactive agents, such as nitric oxide. We speculate that the increase in ADM mRNA in the villous placenta, in the peri-infarct regions, and at the maternal-fetal interface in the choriodecidua represents a local physiological adaptive response to the reduced perfusion and vasospasm in the placenta of women with preeclampsia.
In conclusion, ADM expressed in syncytiotrophoblasts of the chorionic villi and extravillous trophoblasts in the basal plate and choriodecidua may contribute to the maternal and fetal plasma and amniotic fluid levels in pregnancy, and this supports a role for ADM in the regulation of placental vascular tone. In preeclampsia, regional increases in ADM mRNA abundance observed in syncytial knots, trophoblasts in the peri-infarct regions, and chorion levae may reflect lower oxygen tension in the placenta. Increased ADM expression may mediate compensatory vascular responses to maintain fetal/placental perfusion in preeclampsia.
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Footnotes |
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Received February 25, 2003.
Accepted August 26, 2003.
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