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Role of aspartyl-(asparaginyl) β-hydroxylase in placental implantation: relevance to early pregnancy loss
Human Pathology (2007) 38, 50 – 59
www.elsevier.com/locate/humpath
Original contribution
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation: relevance to early pregnancy loss Fusun Gundogan MDa,*, Gwen Elwoodd, David Greco BS a, Lewis P. Rubin MDb, Halit Pinar MDa, Rolf I. Carlson BS d, Jack R. Wands MDd, Suzanne M. de la Monte MD, MPHc,d a
Department of Pathology, Women and Infants Hospital, Brown Medical School, Providence, RI 02905, USA Department of Pediatrics, Women and Infants Hospital, Brown Medical School, Providence, RI 02905, USA c Department of Pathology, Liver Research Center, Rhode Island Hospital, Brown Medical School, Providence, RI 02905, USA d Department of Medicine, Liver Research Center, Rhode Island Hospital, Brown Medical School, Providence, RI 02905, USA b
Received 2 May 2006; accepted 6 June 2006
Keywords: Aspartyl-(asparaginyl); b-hydroxylase; Placenta; Implantation; Trophoblast; Spontaneous abortion
Summary Aspartyl-(asparaginyl) b-hydroxylase (AAH) is a type 2 transmembrane protein with catalytic activity that hydroxylates epidermal growth factor–like domains of proteins that have a functional role in cell motility and invasion. Extravillous cytotrophoblasts (CTB) are motile and invasive unpolarized epithelial cells that mediate early implantation through interaction with the endometrium. This study characterizes the potential role of AAH in CTB implantation using human placentas from (1) terminated pregnancies (n = 11), (2) normal term deliveries (n = 21), (3) spontaneous abortuses (n = 21), and (4) small-for-gestational-age (SGA) term deliveries (n = 21). The SGA cases all had established clinical histories of intrauterine growth restriction or preeclampsia. Formalin-fixed, paraffin-embedded sections of placenta were immunostained using the 15C7 monoclonal antibody generated to recombinant AAH. In addition, snap-frozen or RNAlater-preserved specimens (Ambion, Austin, TX) were used for RNA analysis of AAH expression by real-time quantitative reverse transcriptase–polymerase chain reaction and protein analysis by Western blotting. The immunohistochemical staining studies demonstrated AAH expression in amniocytes, villous CTB, syncytiotrophoblast, extravillous CTB, decidua, and endometrial glands at all gestational ages and in all 4 groups. Higher levels of AAH immunoreactivity were observed in extravillous CTB compared with villous CTB. Immunohistochemical staining and RNA analysis demonstrated abundant AAH expression in placental trophoblastic cells as well as in decidua and endometrial glands, with reduced expression in spontaneous abortion and SGA, suggesting that AAH may serve as a biomarker of impaired implantation. The high levels of AAH in decidua and endometrial glands suggest a role for this molecule in breceptivityQ of endometrium. D 2007 Elsevier Inc. All rights reserved.
1. Introduction * Corresponding author. E-mail address: [email protected] (F. Gundogan). 0046-8177/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2006.06.005
Normal growth and development of fetus requires a well-functioning placenta, including all of its subsets of trophoblastic cells, which are derived from outer blastomeres
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation of the blastocyst. Optimum implantation of the blastocyst is dependent upon the expression of growth factors, hormones, enzymes, and cytokines that are derived from both embryonal and maternal tissues [1]. However, for implantation to occur, the endometrium must be receptive, and this bwindow of receptivityQ is restricted to a very limited interval during the menstrual cycle. A much-needed biomarker for endometrial receptivity would likely improve the success rates of in vitro fertilization and also aid in the detection of suboptimum implantation that could lead to spontaneous abortion or small-for-gestational-age (SGA) infants. Villous cytotrophoblasts (CTBs) are immotile, polarized epithelial stem cells that terminally differentiate into syncytia along the fusion pathway. Extravillous CTBs are derived from anchoring villi that are in direct contact with maternal endometrium at the floor of the intervillous space. Extravillous CTBs differentiate into motile and invasive epithelial cells along the invasive pathway, and they mediate early implantation. There are 2 subsets of extravillous CTBs: interstitial CTBs, which invade the perivillous fibrin deposits, decidualized endometrium and the proximal one third of myometrium; and endovascular CTBs, which invade the uterine spiral arteries [2]. After invading the decidual stroma, interstitial CTBs reach the superficial myometrium by the eighth week of gestation [3]. Interstitial CTBs then continue to invade through the myometrium and eventually fuse to form multinuclear giant cells. Impaired function of extravillous CTB could adversely affect implantation and result in spontaneous abortion (SAB), preeclampsia, or intrauterine growth restriction (IUGR) [2]. The 80 to 100 spiral arteries that perforate the basal plate provide maternal blood supply to the intervillous space. Endovascular CTBs invade the lumens of spiral arteries, beginning at 4 to 6 weeks of gestation, and then progressively extend into myometrial vessels [4]. This process, termed physiologic change, transforms the normally small muscular arteries into distended flaccid vessels. Without physiologic conversion of myometrial vessels, blood flow into the intervillous space is reduced and compromised. Impaired uteroplacental blood flow has been linked to IUGR [5]. Therefore, proper functioning of trophoblastic cells is critical for maintaining normal pregnancy, and the availability of a biomarker that reflects trophoblastic cell function could aid in the early identification of high-risk pregnancies. Aspartyl-(asparaginyl) b-hydroxylase (AAH) is a a-ketoglutarate –dependent dioxygenase that catalyzes posttranslational hydroxylation of b carbons of aspartyl and asparaginyl residues present in epidermal growth factor– like domains of certain proteins. The initial finding that trophoblastic cells, which have motile and invasive properties, have high levels of AAH [6] led to the hypothesis that AAH has a functional role in cell migration and invasiveness. In addition, the finding that AAH is frequently overexpressed in malignant epithelial neoplastic cells and at the infiltrating margins of tumors or in metastatic lesions
51
[6-9] suggests that AAH has a functional role in the infiltrative and metastatic growth of malignant neoplasms. Further experimental work demonstrated that overexpression of AAH results in increased motility, whereas antisense inhibition of AAH expression results in decreased cell motility [10]. Normal CTB cells have motile and invasive properties that are critical for placental implantation. Therefore, understanding the role of AAH expression in relation to CTB function could help in the early detection and treatment of disease states that are caused by suboptimal placental implantation or impaired placental function. This study characterizes AAH expression in human placentas from normal term deliveries, terminated pregnancies (TOP), SAB, and in SGA term deliveries associated with IUGR or preeclampsia. These investigations enabled us to compare the spatiotemporal pattern of AAH expression in normal pregnancy and gestations associated with impaired implantation.
2. Materials and methods 2.1. Case selection Cases used for immunohistochemical staining to characterize AAH expression were identified in the surgical pathology archives of the Rhode Island Women and Infant’s Hospital. All placentas used in this study were harvested between January and April 2005. The cases included in this study were: (1) placentas from normal term deliveries with gestational ages of 37 weeks or longer (n = 21), (2) termination of pregnancy cases with gestational ages of 6 to 16 weeks (n = 11), (3) spontaneous abortuses with gestational ages of 5 to 12 weeks (n = 23), and (4) SGA placentas with gestational ages of 37 weeks or longer (n = 21). The SGA placentas included in this study had clinically established diagnosis of IUGR with or without preeclampsia. Cases in which the gestational ages were unknown, the site of implantation could not be determined, the placental tissue was markedly degenerated and necrotic, or additional major medical conditions such as diabetes mellitus or chronic hypertension existed were excluded from the study.
2.2. Immunohistochemistry Placental tissue samples were fixed in 10% buffered formalin and embedded in paraffin. The hematoxylin and eosin–stained sections were reviewed to select blocks that contained both chorionic villi and the implantation site. Sections 5 lm thick were deparaffinized in xylenes and rehydrated in graded alcohol solutions. Endogenous peroxidase activity was quenched by treating the sections with 0.6% hydrogen peroxide in methanol for 30 minutes. After rinsing in Tris-buffered saline (TBS; 50 mmol/L Tris, 150 mmol/L NaCl, pH 7.4), the slides were incubated for 30 minutes at room temperature with a 1:3000 dilution of
52 the 15C7 mouse monoclonal antibody to AAH. The 15C7 antibody was raised against the C-terminal region of recombinant human AAH protein, which contains the catalytic domain (Wands et al, unpublished data, 2005). Antibody binding was detected with a biotin-free system in which the antimouse IgG was conjugated to a horseradish peroxidase–labeled polymer (Envision+ Dual Link polymer, DakoCytomation, Carpenteria, CA), and diaminobenzidine tetrahydrochloride was used as the chromogen. The sections were counterstained lightly with hematoxylin. Adjacent sections were immunostained with antibodies to CK7 (OVTL 12/30, DakoCytomation) to distinguish the extravillous CTB from decidual cells at the implantation site. In addition, adjacent sections were immunostained with antibodies to vimentin (V9, DakoCytomation) as a positive control and with the primary antibody omitted as a negative control. All immunohistochemical staining reactions were
F. Gundogan et al. performed using the Dako Autostainer (DakoCytomation) according to the manufacturer’s protocol. The levels of AAH immunoreactivity detected in villous CTB, syncytiotrophoblast, extravillous CTB, decidua, and endometrial glands were independently assigned semiquantitative grades designated as 0 (absent), 1+ (low level), 2+ (moderate), or 3+ (intense), as illustrated in Fig. 1. The assigned grades reflect the levels of AAH immunoreactivity observed in at least 50% of the cells.
2.3. Western blot analysis AAH protein expression in human placental tissue was confirmed by Western blot analysis. Fresh, snapfrozen samples of normal placental tissue (n = 2) stored in liquid nitrogen were homogenized in 5 volumes of radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl, pH 7.5, 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl,
Fig. 1 Grading scheme of AAH immunoreactivity. The levels of AAH immunoreactivity detected in villous CTB, extravillous CTB, decidua, and endometrial glands were independently assigned semiquantitative grades designated as 0 (absent), 1+ (low level), 2+ (moderate), or 3+ (intense). The designated grade reflected the levels of AAH immunoreactivity observed in at least 50% of the cells. All the cases had at least 1+ immunoreactivity in all cell types examined. Adjacent sections immunostained with CK7 were examined to differentiate the extravillous trophoblastic cells from decidual stromal cells. Villous CTB (A-C), from 1+ to 3+; extravillous CTB/decidua (D-F), from 1+ to 3+; endometrial glands (G-I), from 1+ to 3+ (original magnification 400).
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation Table 1
53
Primer pair sequences for real-time quantitative RT-PCR
Primer
Sequence (5V Y 3V)
Position (mRNA)
GAPDH
ForTCA TCA GCA ATG CCT CCT GC RevTCC TTC CAC GAT ACC AAA GTT GTC ForGGG AGA TTT TAT TTC CAC CTG GG RevCCT TTG GCT TTA TCC ATC ACT GC
461 543 1650 1906
AAH
1 mmol/L EDTA, 2 mmol/L EGTA) containing protease (1 mmol/L phenylmethylsulfonyl fluoride, 0.1 mmol/L tosyl phenylalanyl chloromethylketone, 1lg/mL pepstatin A, 0.5 lg/mL leupeptin, 1 mmol/L NaF, 1 mmol/L Na4P2 7) and phosphatase (2 mmol/L Na3VO4) inhibitors. The supernatant fractions obtained after centrifuging the samples at 12 000g for 15 minutes at 48C were used for Western blotting. Protein concentrations were determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Protein samples (40 lg) were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis [7]. After transferring the proteins to Immobilon-P membranes (Millipore Corp, Bedford, MA), nonspecific binding sites were adsorbed with SuperBlock-TBS (Pierce). The membranes were incubated overnight at 48C with the monoclonal antibody to AAH diluted in TBS containing 1% bovine serum albumin and 0.05% Tween-20 (tris-buffered saline tween-20 [TBST]–bovine serum albumin). After thorough rinsing in TBST, the blots were incubated for 1 hour at room temperature with horseradish peroxidase-conjugated antimouse IgG (1:30 000) diluted in TBST + 0.5% casein. Immunoreactivity was revealed using enhanced chemiluminescence reagents (Pierce Chemical Company, Rockford, IL) and digital imaging with the Kodak Digital Science Imaging Station (NEN Life Sciences, Boston, MA).
Amplicon size (bp) 83 257
England). PCR amplifications were performed in 25-lL reactions containing cDNA generated from 2.5 ng of original RNA template, 300 nmol/L each of gene-specific forward and reverse primer (Table 1), and 12.5 lL of 2 QuantiTect SYBR Green PCR Mix (Qiagen Inc, Valencia, CA). The amplified signals were detected continuously with the BIO-RAD iCycler iQ Multi-Color RealTime PCR Detection System (Bio-Rad, Hercules, CA). The amplification protocol used was as follows: initial 15-minute denaturation and enzyme activation at 958C and 45 cycles of 958C 15 seconds, 608C 30 seconds, and 728C
2.4. Analysis of AAH messenger RNA expression Real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis was used to demonstrate AAH messenger RNA (mRNA) expression in placental tissue. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Samples containing 2 lg of RNA were reverse-transcribed with the AMV First Strand complementary DNA (cDNA) synthesis kit (Roche, Basel, Switzerland) and random oligodeoxynucleotide primers. Polymerase chain reaction (PCR) primers for AAH were designed using Mac Vector 7.0 software (Accelrys Inc, Oxford Molecular Ltd, Oxford, Table 2
Clinical data
TOP SAB Normal term SGA term
Case no. (n)
Gestational age (wk)
Maternal age (y)
11 23 21 21
6-16 5-12 37-40 37-40
26.4 30.8 29.4 24.9
F F F F
6.2 6.1 6.7 6.0
Fig. 2 Down-regulation of AAH gene in clinical conditions associated with impaired placental implantation. Real-time quantitative RT-PCR was performed on the limited numbers (n) of available samples that had been preserved in RNAlater. AAH and GAPDH mRNA transcripts were detected in cases of normal term pregnancy (n = 6), TOP (n = 2), SAB (n = 2), and SGA term placentas (n = 6) with clinical history of IUGR. The median relative levels of AAH (normalized to GAPDH levels in same samples) were similar in the normal term placentas compared with the early gestation placentas (TOP). The median relative levels of AAH mRNA were consistently reduced in the SGA and SAB relative to normal term and TOP placentas, respectively (A and B). Western blot analysis was used to verify AAH protein expression in placental tissue. Two fresh, snap-frozen samples of normal term delivery placentas were available for study. Western blot analysis was done using a monoclonal antibody, which binds to the N-terminal region of AAH. We detected the expected 86-kd fulllength AAH in both samples (C).
54 30 seconds. Annealing temperatures were optimized using the temperature gradient program provided with the iCycler software (Bio-Rad Laboratories, Hercules, CA). Experiments were performed in triplicate. Primer-target specificity was checked using National Center for Biotechnology Information BLASTn (Basic Local Alignment Search Tool–nucleotide) searches. In addition, SYBR Green–labeled PCR products were evaluated by agarose gel electrophoresis, and the authenticity of each amplicon was verified by nucleic acid sequencing. The cDNAs were cloned into the PCRII vector (Invitrogen). Serial dilutions of known quantities of recombinant plasmid DNA containing the specific target sequences were used as standards in the PCR reactions, and the regression lines generated from the Ct values of the standards were used to calculate mRNA abundance [11,12]. Relative mRNA abun-
F. Gundogan et al. dance was determined from the ng ratios of specific mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) measured in the same samples. Results were normalized to GAPDH because standard housekeeping genes usually suffice as internal control genes. Control studies included realtime quantitative PCR analysis of (1) template-free reactions; (2) RNA that had not been reverse-transcribed; (3) RNA samples that were pretreated with DNAse I; (4) samples treated with RNAse A before RT-PCR; and (5) genomic DNA.
2.5. Statistical analysis Intergroup statistical comparisons were made by v 2 analysis or analysis of variance and the post hoc TukeyKramer significance test using the Number Cruncher Statistical System (Dr Jerry L. Hintze, Kaysville, UT) or
Fig. 3 AAH immunoreactivity in terminated pregnancy (TOP) cases. AAH highlighted the cytoplasm of villous CTB and syncytiotrophoblast with 1+ intensity in most cases (A). Polarized extravillous CTB/cell columns (arrow) away from the implantation site showed no immunoreactivity (A), whereas the cell columns (arrow) of anchoring villi at the implantation site showed 2+ to 3+ immunoreactivity (B). The extravillous CTBs that invade the decidua expressed 3+ immunoreactivity in most cases (B). The endovascular CTB and extravillous trophoblast that invade and transform the maternal vessels, had 2+ to 3+ AAH immunoreactivity (C). In addition to trophoblastic cells, endometrial glands and decidual cells expressed 3+ cytoplasmic AAH immunoreactivity in more than 78% of cases (D) (original magnification 200).
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation GraphPad Prism 4 software (GraphPad Software, Inc, San Diego, CA). P b .05 was considered statistically significant.
3. Results 3.1. Sample profiles The placentas used in this study were obtained from deliveries that occurred between January 2005 and April 2005 (Table 2). The 21 normal and 21 SGA placentas had gestational ages of 37 to 40 weeks. The 11 TOP cases had gestational ages of 6 to 16 weeks, and the 23 SAB cases had gestational ages ranging from 5 to 12 weeks. The cases of SGA had clinical histories of IUGR or preeclampsia. The maternal ages were similar for the cases of normal term delivery, TOP, and SGA, whereas the mean maternal age of the SAB group was significantly higher than the SGA and TOP groups ( P b .05).
55
was also graded as 2+ (10 cases [48%]) or 3+ (11 cases [52%]). Because the term placenta basal plates are devoid of endometrial glands, AAH immunoreactivity could not be evaluated in these structures in late gestation. Within the TOP group and irrespective of gestational age, the intensity of AAH immunoreactivity in villous CTB was 1+ in 7 (64%) of 11 cases (Fig. 3A) and 2+ in 4 (36%) of 11 cases, although 3+ AAH immunoreactivity was focally present in 3 of the cases. Syncytiotrophoblast exhibited similar levels and patterns of AAH immunoreactivity, as observed for the villous CTB. In extravillous interstitial CTB, 3 (27%) of 11 cases had 2+ AAH expression, and in 8 of 11, AAH immunoreactivity was 3+ (Fig. 3B). Endovascular CTB, that is, extravillous CTB that transforms the decidual vessels and phenocopy the endothelial cells, also exhibited 2+ or 3+ levels of AAH immunoreactivity (Fig. 3C), as observed in interstitial CTB. Cell columns that give rise to extravillous trophoblasts and form connections
3.2. AAH expression demonstrated by RT-PCR and Western blot analysis Real-time quantitative RT-PCR was performed on the limited numbers of available samples that had been preserved in RNAlater. In total, 6 cases of each normal term pregnancy and SGA with established IUGR and 2 cases each of TOP and SAB were analyzed. AAH mRNA and GAPDH mRNA transcripts were detected in all samples. The relative levels of AAH mRNA (normalized to GAPDH levels in the same samples) were similar in the normal term placentas compared with the TOP cases. However, the relative levels of AAH mRNA were consistently reduced in the SGA and SAB relative to the normal term placentas and TOP, respectively (Fig. 2A and B). Western blot analysis was used to verify AAH protein expression in placental tissue. Two fresh, snap-frozen samples of normal term delivery placentas were available for study. Western blot analysis using monoclonal antibody, which binds to the N-terminal region of AAH, detected the expected approximately 86-kd full-length AAH (Fig. 2C).
3.3. AAH immunoreactivity in normal gestation Terminated pregnancy cases were used to examine AAH expression during early gestation, and term delivery cases were used to examine AAH expression during late gestation. AAH cytoplasmic staining was detected in villous CTB, syncytiotrophoblast, extravillous (both interstitial and endovascular) CTB, decidual cells, and endometrial glands in all cases. In the normal term delivery placentas, cytoplasmic AAH immunoreactivity was detected in villous CTB, extravillous CTB, and decidualized stromal cells. Within villous CTB, AAH immunoreactivity was 1+ in 15 (71%) of the 21 cases and 2+ in 6 cases (29%). In contrast, AAH immunoreactivity in extravillous CTB was 2+ in 11 of 21 cases (52%), and 3+ in 10 (48%) of 21 cases. Within decidualized stromal cells, AAH immunoreactivity
Fig. 4 Differentiation of decidual cells from extravillous CTB with the aid of CK7 immunohistochemistry. The diffuse cytoplasmic AAH expression at implantation site and decidua (A) required CK7 immunostaining to differentiate the trophoblastic cells from decidual cells. CK7, a trophoblastic marker, highlighted the extravillous CTB and endometrial glands (B) revealing the extent of AAH expression in decidual cells (original magnification 200).
56 between anchoring villi and the basal plate also had 2+ or 3+ levels of AAH immunoreactivity at the implantation site (Fig. 3B), although cell columns away from the implantation site had no detectable AAH immunoreactivity (Fig. 3A). In decidua, AAH immunoreactivity was 2+ in 2 (18%) and 3+ in 9 (82%) of the 11 cases (Fig. 3D). Endometrial glands were present in 9 of the 11 cases. Two (22%) had 2+ and 7 (78%) had 3+ levels of AAH immunoreactivity (Fig. 3D). In these studies, CK7 immunohistochemical staining of adjacent sections was used to distinguish decidual cells from trophoblastic cells (Fig. 4) and to map the distribution and grade AAH immunoreactivity in decidual cells.
3.4. Altered AAH expression with impaired implantation Because preeclampsia and IUGR have been associated with shallow or improper placental implantation, to evaluate the potential role of AAH in relation to implantation,
F. Gundogan et al. we compared the AAH expression profiles in placentas from normal term and SGA cases. In the SGA cases with clinically established IUGR, the intensity profiles of AAH immunoreactivity in villous CTB, extravillous CTB, and decidua were similar to those of the normal term placenta (Fig. 5A-C). AAH immunoreactivity in SGA extravillous CTB was 2+ in 12 (57%) and 3+ in 9 (43%) of the 21 cases. In addition, AAH immunoreactivity in SGA villous CTB was 1+ in 15 (71%), 2+ in 6 (29%), and 3+ in 0 of the 21 cases. Decidualized stromal cells in the SGA cases had 2+ AAH immunoreactivity in 14 cases (67%), and 3+ in 7 of the 21 cases (33%). As observed in the normal term placentas, the SGA basal plates were devoid of endometrial glands. To further evaluate the potential role of AAH in relation to implantation, we compared the AAH expression profiles in placentas from TOP and SAB. Although the cellular distribution of AAH immunoreactivity was similar in the SAB and TOP cases, the AAH expression levels were more
Fig. 5 Comparison of extravillous and villous cytotrophoblastic AAH expression in late and early gestation placentas with/without clinical implication of impaired implantation. The graphs depict the distribution profile of AAH immunoreactivity in a percentage of cases. Extravillous CTB from late gestational, normal term, and SGA placentas with clinically established diagnosis of IUGR did not reveal significant difference in AAH expression (A). However, AAH immunoreactivity was significantly reduced ( P = .046) in extravillous CTB of SAB cases compared with TOP placentas (D). The intensity of villous cytotrophoblastic AAH expression was comparable in late- and earlygestation placentas and did not differ in SGA and SAB cases relative to normal term and TOP placentas, respectively (B and E). Decidual expression of AAH in late gestation was comparable in normal term and SGA placentas (C). SAB cases showed significantly reduced AAH expression in decidual cells ( P = .04) (F) and endometrial glands ( P = .005) relative to TOP cases.
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation variable and, overall, conspicuously lower in the extravillous CTB ( P = .046), decidua ( P = .04), and endometrial glands ( P = .005, data not shown) in the SAB relative to the TOP cases (Fig. 5D-F). In this regard, the intensity of AAH immunoreactivity in extravillous CTB was judged to be 1+ in 5 (22%) of 23 cases (Fig. 6C), 2+ in 12 (52%), and 3+ in 6 (26%) (Fig. 6A). In the SAB cases, AAH immunoreactivity in villous CTB and syncytiotrophoblast was similar to TOP group (Fig. 5E). In the SAB group, the distribution profile corresponding to the levels of AAH immunoreactivity in decidua was similar to that observed in extravillous trophoblasts, that is, 1+ immunoreactivity was observed in 7 (30%), 2+ in 7 (30%), and 3+ in 9 (40%) of the 23 cases (Fig. 5F). Immunohistochemical staining of adjacent tissue sections with anti-CK7 was used to differentiate decidualized endometrial stromal cells from interstitial CTB. Endometrial glands were present in 21 of the 23 SAB specimens. AAH immunoreactivity in endometrial glands
57
was 1+ in 7 (33%) (Fig. 6D), 2+ in 9 (43%), and 3+ in 5 (24%) of 21 cases (Fig. 6B). As a positive control, adjacent sections were immunostained with antibodies to vimentin. Those studies demonstrated similarly robust levels of vimentin immunoreactivity in the TOP and SAB cases.
4. Discussion This study characterizes the spatiotemporal expression pattern and the potential role of AAH in placental implantation for the first time. We showed the presence of AAH in different subsets of trophoblast, decidua, and endometrial glands throughout the gestation by immunohistochemistry and verified the AAH protein expression by Western blot analysis. The implantation process is currently considered the most limiting factor for establishment of successful pregnancy in which trophoblastic cells play a
Fig. 6 AAH expression in spontaneous abortuses. Villous CTB expressed 1+ or 2+ (A) AAH immunoreactivity in all the cases. Extravillous CTB, decidua (A), and endometrial glands (B) had 2+ to 3+ AAH expression in most cases. However, 5 of 23 cases revealed 0 to 1+ AAH immunoreactivity in extravillous CTB, and 7 of 23 cases had 1+ immunoreactivity in decidual cells (C). Same cases also revealed 1+ AAH expression in endometrial glands (D). Vimentin immunostaining showed strong immunoreactivity in all the cases with decreased AAH expression (original magnification 200).
58 crucial role. This study will aid in the understanding of the molecular interactions at the embryomaternal interface during implantation.
4.1. Overexpression of AAH in extravillous CTB Cytoplasmic AAH expression was detected in villous CTB, extravillous CTB, amniocytes, decidua, and endometrial glands in early- and late-gestation placentas. Regardless of the gestational age, extravillous CTB and both interstitial and endovascular CTB demonstrated stronger AAH immunoreactivity in comparison with villous CTB. Considering that extravillous CTBs are the motile and invasive subgroup of trophoblast, AAH overexpression in this group, in comparison with immotile villous CTB [2], further confirms the role of AAH in cell migration and invasiveness [6,9,10]. Although the cell columns away from the implantation site revealed no AAH immunoreactivity, the cell columns of anchoring villi, where the trophoblastic attachment and infiltration into endometrium takes place, expressed intense AAH immunoreactivity. It has been shown that from the proximal to the distal end of the cell column, extravillous CTBs gradually change their phenotype from a proliferative epithelial cell to a nondividing and invasive mesenchymallike cell [2]. AAH immunostaining demonstrated this phenotypic change strikingly.
4.2. AAH expression in decidua and endometrial glands One of the most interesting findings of this study was demonstration of AAH expression in decidua and endometrial glands. It has been known that decidua is one of the key players in receptivity of endometrium and, thus, in implantation [13-16]. Decidual AAH expression was diffuse at the implantation site and also in hormonally active fragments revealing 2+ to 3+ immunoreactivity. Endometrial glandular AAH expression was also diffuse, and the glands with Arias Stella features showed the most intense immunoreactivity. To find the specificity of this staining pattern for pregnancy, we randomly selected secretory phase endometrial biopsy cases from different periods: 16, 22 to 23, and 24 days of cycle. AAH immunohistochemistry revealed no reactivity in early secretory phase, multifocal 2+ to 3+ glandular immunoreactivity on days 22 to 23, and diffuse 3+ glandular immunoreactivity on 24th day of cycle. bDecidualizedQ stroma in none of these cases revealed AAH immunoreactivity (unpublished data). This staining pattern suggests that the decidual AAH expression is specific for the implantation period.
4.3. Altered AAH expression with impaired implantation After demonstrating the expression pattern of AAH in early and late gestation placenta cases, we evaluated AAH expression patterns in clinical conditions associated with
F. Gundogan et al. impaired implantation. Although it is a well-established fact that 50% to 60%, approaching to 90% as it occurs earlier, of early spontaneous abortuses are associated with chromosomal abnormality, the underlying mechanism is still not completely understood [17] and molecular and hormonal factors involved in trophoblastic invasion are under investigation [13,15,16,18]. Anatomical evidence of defective placentation defined as shallow implantation, thin and fragmented trophoblastic shell, and deficient physiologic conversion of maternal vessels have been reported in two thirds of early pregnancy failures [17]. Except for deficient physiologic conversion of maternal vessels in 2 cases, we did not identify other features by hematoxylin and eosin examination in our SAB study group. This observation further supported the need for a biomarker that reflects trophoblastic cell function. Comparison of AAH expression profiles in placentas from SAB and TOP cases revealed a striking shift toward lower intensity of AAH immunoreactivity in extravillous CTB, decidua, and endometrial glands of SAB cases. The AAH expression pattern of villous CTB was similar in TOP and SAB cases. The altered AAH expression in extravillous CTB has 2 clinical consequences: (1) decreased trophoblast motility and invasiveness at the maternal-placental interface result in shallow implantation and (2) defective invasion of endovascular trophoblast cause deficient or absent physiologic conversion of maternal vessels. In addition to lower-intensity expression of AAH in extravillous CTB, we also demonstrated that AAH expression is shifted toward lower intensity in decidua and endometrial glands in SAB cases. Decreased trophoblast invasion may be a consequence of altered AAH expression in the extravillous CTB or in the decidua that the trophoblasts are attempting to invade, or both. We also demonstrated that altered AAH expression in SAB cases is at the transcriptional level by RT-PCR. In addition to SAB cases, we also evaluated SGA placentas with established diagnosis of IUGR with or without preeclampsia. Decreased or absent trophoblastic invasion into the maternal spiral arteries has been documented in cases of preeclampsia [19]. However, this finding can only be reliably documented in placental bed biopsies. Basal plates of term placentas were devoid of maternal vessels and could not be evaluated in late gestation group. The cellular distribution and intensity of AAH immunoreactivity was similar in normal term and SGA term placentas. However, the relative levels of AAH mRNA were reduced in the SGA placentas relative to the normal term placentas. In conclusion, we demonstrated abundant AAH expression in placental trophoblastic cells, as well as in decidua and endometrial glands irrespective of gestational age. Reduced expression at the transcriptional level in SAB and IUGR with/without preeclampsia cases suggest that AAH may serve as a biomarker of impaired implantation. In addition, the high levels of AAH in decidua
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation and endometrial glands suggest a role for this molecule in breceptivityQ of endometrium.
Acknowledgments
[9]
[10]
The authors thank Terry Pasquariello, BS, for her assistance in immunohistochemical staining of the cases.
[11]
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clonal antibodies: potential biomarkers for pancreatic carcinoma. Pancreas 2002;25:39 - 44. Maeda T, Taguchi K, Aishima S, et al. Clinicopathological correlates of aspartyl (asparaginyl) beta-hydroxylase over-expression in cholangiocarcinoma. Cancer Detect Prev 2004;28:313 - 8. Sepe PS, Lahousse SA, Gemelli B, et al. Role of aspartyl (asparaginyl) beta-hydroxylase gene in neuroblastoma cell motility. Lab Invest 2002;82:881 - 91. Steen E, Terry BM, Rivera EJ, et al. Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer’s disease: is this type 3 diabetes? J Alzheimers Dis 2005;7:63 - 80. de la Monte SM, Xu XJ, Wands JR. Ethanol inhibits insulin expression and actions in the developing brain. Cell Mol Life Sci 2005;62:1131 - 45. Dunn CL, Kelly RW, Critchley HO. Decidualization of the human endometrial stromal cell: an enigmatic transformation. Reprod Biomed Online 2003;7:151 - 61. Norwitz ER, Schust DJ, Fisher SJ. Implantation and survival of early pregnancy. N Engl J Med 2001;345:1400 - 8. Pollheimer J, Knofler M. Signaling pathways regulating the invasive differentiation of human trophoblasts: a review. Placenta 2005; 26(Suppl A):S21-S30. Fazleabas AT, Kim JJ, Strakova Z. Implantation: embryonic signals and the modulation of the uterine environment: a review. Placenta 2004;25(Suppl A):S26-S31. Jauniaux E, Burton GJ. Pathophysiology of histological changes in early pregnancy loss. Placenta 2005;26:114 - 23. Bischof P, Meisser A, Campana A. Involvement of trophoblast in embryo implantation: regulation by paracrine factors. J Reprod Immunol 1998;39:167 - 77. Pijnenborg R, Luyten C, Vercruysse L, Van Assche FA. Attachment and differentiation in vitro of trophoblast from normal and preeclamptic human placentas. Am J Obstet Gynecol 1996;175:30 - 6.
www.elsevier.com/locate/humpath
Original contribution
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation: relevance to early pregnancy loss Fusun Gundogan MDa,*, Gwen Elwoodd, David Greco BS a, Lewis P. Rubin MDb, Halit Pinar MDa, Rolf I. Carlson BS d, Jack R. Wands MDd, Suzanne M. de la Monte MD, MPHc,d a
Department of Pathology, Women and Infants Hospital, Brown Medical School, Providence, RI 02905, USA Department of Pediatrics, Women and Infants Hospital, Brown Medical School, Providence, RI 02905, USA c Department of Pathology, Liver Research Center, Rhode Island Hospital, Brown Medical School, Providence, RI 02905, USA d Department of Medicine, Liver Research Center, Rhode Island Hospital, Brown Medical School, Providence, RI 02905, USA b
Received 2 May 2006; accepted 6 June 2006
Keywords: Aspartyl-(asparaginyl); b-hydroxylase; Placenta; Implantation; Trophoblast; Spontaneous abortion
Summary Aspartyl-(asparaginyl) b-hydroxylase (AAH) is a type 2 transmembrane protein with catalytic activity that hydroxylates epidermal growth factor–like domains of proteins that have a functional role in cell motility and invasion. Extravillous cytotrophoblasts (CTB) are motile and invasive unpolarized epithelial cells that mediate early implantation through interaction with the endometrium. This study characterizes the potential role of AAH in CTB implantation using human placentas from (1) terminated pregnancies (n = 11), (2) normal term deliveries (n = 21), (3) spontaneous abortuses (n = 21), and (4) small-for-gestational-age (SGA) term deliveries (n = 21). The SGA cases all had established clinical histories of intrauterine growth restriction or preeclampsia. Formalin-fixed, paraffin-embedded sections of placenta were immunostained using the 15C7 monoclonal antibody generated to recombinant AAH. In addition, snap-frozen or RNAlater-preserved specimens (Ambion, Austin, TX) were used for RNA analysis of AAH expression by real-time quantitative reverse transcriptase–polymerase chain reaction and protein analysis by Western blotting. The immunohistochemical staining studies demonstrated AAH expression in amniocytes, villous CTB, syncytiotrophoblast, extravillous CTB, decidua, and endometrial glands at all gestational ages and in all 4 groups. Higher levels of AAH immunoreactivity were observed in extravillous CTB compared with villous CTB. Immunohistochemical staining and RNA analysis demonstrated abundant AAH expression in placental trophoblastic cells as well as in decidua and endometrial glands, with reduced expression in spontaneous abortion and SGA, suggesting that AAH may serve as a biomarker of impaired implantation. The high levels of AAH in decidua and endometrial glands suggest a role for this molecule in breceptivityQ of endometrium. D 2007 Elsevier Inc. All rights reserved.
1. Introduction * Corresponding author. E-mail address: [email protected] (F. Gundogan). 0046-8177/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2006.06.005
Normal growth and development of fetus requires a well-functioning placenta, including all of its subsets of trophoblastic cells, which are derived from outer blastomeres
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation of the blastocyst. Optimum implantation of the blastocyst is dependent upon the expression of growth factors, hormones, enzymes, and cytokines that are derived from both embryonal and maternal tissues [1]. However, for implantation to occur, the endometrium must be receptive, and this bwindow of receptivityQ is restricted to a very limited interval during the menstrual cycle. A much-needed biomarker for endometrial receptivity would likely improve the success rates of in vitro fertilization and also aid in the detection of suboptimum implantation that could lead to spontaneous abortion or small-for-gestational-age (SGA) infants. Villous cytotrophoblasts (CTBs) are immotile, polarized epithelial stem cells that terminally differentiate into syncytia along the fusion pathway. Extravillous CTBs are derived from anchoring villi that are in direct contact with maternal endometrium at the floor of the intervillous space. Extravillous CTBs differentiate into motile and invasive epithelial cells along the invasive pathway, and they mediate early implantation. There are 2 subsets of extravillous CTBs: interstitial CTBs, which invade the perivillous fibrin deposits, decidualized endometrium and the proximal one third of myometrium; and endovascular CTBs, which invade the uterine spiral arteries [2]. After invading the decidual stroma, interstitial CTBs reach the superficial myometrium by the eighth week of gestation [3]. Interstitial CTBs then continue to invade through the myometrium and eventually fuse to form multinuclear giant cells. Impaired function of extravillous CTB could adversely affect implantation and result in spontaneous abortion (SAB), preeclampsia, or intrauterine growth restriction (IUGR) [2]. The 80 to 100 spiral arteries that perforate the basal plate provide maternal blood supply to the intervillous space. Endovascular CTBs invade the lumens of spiral arteries, beginning at 4 to 6 weeks of gestation, and then progressively extend into myometrial vessels [4]. This process, termed physiologic change, transforms the normally small muscular arteries into distended flaccid vessels. Without physiologic conversion of myometrial vessels, blood flow into the intervillous space is reduced and compromised. Impaired uteroplacental blood flow has been linked to IUGR [5]. Therefore, proper functioning of trophoblastic cells is critical for maintaining normal pregnancy, and the availability of a biomarker that reflects trophoblastic cell function could aid in the early identification of high-risk pregnancies. Aspartyl-(asparaginyl) b-hydroxylase (AAH) is a a-ketoglutarate –dependent dioxygenase that catalyzes posttranslational hydroxylation of b carbons of aspartyl and asparaginyl residues present in epidermal growth factor– like domains of certain proteins. The initial finding that trophoblastic cells, which have motile and invasive properties, have high levels of AAH [6] led to the hypothesis that AAH has a functional role in cell migration and invasiveness. In addition, the finding that AAH is frequently overexpressed in malignant epithelial neoplastic cells and at the infiltrating margins of tumors or in metastatic lesions
51
[6-9] suggests that AAH has a functional role in the infiltrative and metastatic growth of malignant neoplasms. Further experimental work demonstrated that overexpression of AAH results in increased motility, whereas antisense inhibition of AAH expression results in decreased cell motility [10]. Normal CTB cells have motile and invasive properties that are critical for placental implantation. Therefore, understanding the role of AAH expression in relation to CTB function could help in the early detection and treatment of disease states that are caused by suboptimal placental implantation or impaired placental function. This study characterizes AAH expression in human placentas from normal term deliveries, terminated pregnancies (TOP), SAB, and in SGA term deliveries associated with IUGR or preeclampsia. These investigations enabled us to compare the spatiotemporal pattern of AAH expression in normal pregnancy and gestations associated with impaired implantation.
2. Materials and methods 2.1. Case selection Cases used for immunohistochemical staining to characterize AAH expression were identified in the surgical pathology archives of the Rhode Island Women and Infant’s Hospital. All placentas used in this study were harvested between January and April 2005. The cases included in this study were: (1) placentas from normal term deliveries with gestational ages of 37 weeks or longer (n = 21), (2) termination of pregnancy cases with gestational ages of 6 to 16 weeks (n = 11), (3) spontaneous abortuses with gestational ages of 5 to 12 weeks (n = 23), and (4) SGA placentas with gestational ages of 37 weeks or longer (n = 21). The SGA placentas included in this study had clinically established diagnosis of IUGR with or without preeclampsia. Cases in which the gestational ages were unknown, the site of implantation could not be determined, the placental tissue was markedly degenerated and necrotic, or additional major medical conditions such as diabetes mellitus or chronic hypertension existed were excluded from the study.
2.2. Immunohistochemistry Placental tissue samples were fixed in 10% buffered formalin and embedded in paraffin. The hematoxylin and eosin–stained sections were reviewed to select blocks that contained both chorionic villi and the implantation site. Sections 5 lm thick were deparaffinized in xylenes and rehydrated in graded alcohol solutions. Endogenous peroxidase activity was quenched by treating the sections with 0.6% hydrogen peroxide in methanol for 30 minutes. After rinsing in Tris-buffered saline (TBS; 50 mmol/L Tris, 150 mmol/L NaCl, pH 7.4), the slides were incubated for 30 minutes at room temperature with a 1:3000 dilution of
52 the 15C7 mouse monoclonal antibody to AAH. The 15C7 antibody was raised against the C-terminal region of recombinant human AAH protein, which contains the catalytic domain (Wands et al, unpublished data, 2005). Antibody binding was detected with a biotin-free system in which the antimouse IgG was conjugated to a horseradish peroxidase–labeled polymer (Envision+ Dual Link polymer, DakoCytomation, Carpenteria, CA), and diaminobenzidine tetrahydrochloride was used as the chromogen. The sections were counterstained lightly with hematoxylin. Adjacent sections were immunostained with antibodies to CK7 (OVTL 12/30, DakoCytomation) to distinguish the extravillous CTB from decidual cells at the implantation site. In addition, adjacent sections were immunostained with antibodies to vimentin (V9, DakoCytomation) as a positive control and with the primary antibody omitted as a negative control. All immunohistochemical staining reactions were
F. Gundogan et al. performed using the Dako Autostainer (DakoCytomation) according to the manufacturer’s protocol. The levels of AAH immunoreactivity detected in villous CTB, syncytiotrophoblast, extravillous CTB, decidua, and endometrial glands were independently assigned semiquantitative grades designated as 0 (absent), 1+ (low level), 2+ (moderate), or 3+ (intense), as illustrated in Fig. 1. The assigned grades reflect the levels of AAH immunoreactivity observed in at least 50% of the cells.
2.3. Western blot analysis AAH protein expression in human placental tissue was confirmed by Western blot analysis. Fresh, snapfrozen samples of normal placental tissue (n = 2) stored in liquid nitrogen were homogenized in 5 volumes of radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl, pH 7.5, 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl,
Fig. 1 Grading scheme of AAH immunoreactivity. The levels of AAH immunoreactivity detected in villous CTB, extravillous CTB, decidua, and endometrial glands were independently assigned semiquantitative grades designated as 0 (absent), 1+ (low level), 2+ (moderate), or 3+ (intense). The designated grade reflected the levels of AAH immunoreactivity observed in at least 50% of the cells. All the cases had at least 1+ immunoreactivity in all cell types examined. Adjacent sections immunostained with CK7 were examined to differentiate the extravillous trophoblastic cells from decidual stromal cells. Villous CTB (A-C), from 1+ to 3+; extravillous CTB/decidua (D-F), from 1+ to 3+; endometrial glands (G-I), from 1+ to 3+ (original magnification 400).
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation Table 1
53
Primer pair sequences for real-time quantitative RT-PCR
Primer
Sequence (5V Y 3V)
Position (mRNA)
GAPDH
ForTCA TCA GCA ATG CCT CCT GC RevTCC TTC CAC GAT ACC AAA GTT GTC ForGGG AGA TTT TAT TTC CAC CTG GG RevCCT TTG GCT TTA TCC ATC ACT GC
461 543 1650 1906
AAH
1 mmol/L EDTA, 2 mmol/L EGTA) containing protease (1 mmol/L phenylmethylsulfonyl fluoride, 0.1 mmol/L tosyl phenylalanyl chloromethylketone, 1lg/mL pepstatin A, 0.5 lg/mL leupeptin, 1 mmol/L NaF, 1 mmol/L Na4P2 7) and phosphatase (2 mmol/L Na3VO4) inhibitors. The supernatant fractions obtained after centrifuging the samples at 12 000g for 15 minutes at 48C were used for Western blotting. Protein concentrations were determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Protein samples (40 lg) were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis [7]. After transferring the proteins to Immobilon-P membranes (Millipore Corp, Bedford, MA), nonspecific binding sites were adsorbed with SuperBlock-TBS (Pierce). The membranes were incubated overnight at 48C with the monoclonal antibody to AAH diluted in TBS containing 1% bovine serum albumin and 0.05% Tween-20 (tris-buffered saline tween-20 [TBST]–bovine serum albumin). After thorough rinsing in TBST, the blots were incubated for 1 hour at room temperature with horseradish peroxidase-conjugated antimouse IgG (1:30 000) diluted in TBST + 0.5% casein. Immunoreactivity was revealed using enhanced chemiluminescence reagents (Pierce Chemical Company, Rockford, IL) and digital imaging with the Kodak Digital Science Imaging Station (NEN Life Sciences, Boston, MA).
Amplicon size (bp) 83 257
England). PCR amplifications were performed in 25-lL reactions containing cDNA generated from 2.5 ng of original RNA template, 300 nmol/L each of gene-specific forward and reverse primer (Table 1), and 12.5 lL of 2 QuantiTect SYBR Green PCR Mix (Qiagen Inc, Valencia, CA). The amplified signals were detected continuously with the BIO-RAD iCycler iQ Multi-Color RealTime PCR Detection System (Bio-Rad, Hercules, CA). The amplification protocol used was as follows: initial 15-minute denaturation and enzyme activation at 958C and 45 cycles of 958C 15 seconds, 608C 30 seconds, and 728C
2.4. Analysis of AAH messenger RNA expression Real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis was used to demonstrate AAH messenger RNA (mRNA) expression in placental tissue. Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Samples containing 2 lg of RNA were reverse-transcribed with the AMV First Strand complementary DNA (cDNA) synthesis kit (Roche, Basel, Switzerland) and random oligodeoxynucleotide primers. Polymerase chain reaction (PCR) primers for AAH were designed using Mac Vector 7.0 software (Accelrys Inc, Oxford Molecular Ltd, Oxford, Table 2
Clinical data
TOP SAB Normal term SGA term
Case no. (n)
Gestational age (wk)
Maternal age (y)
11 23 21 21
6-16 5-12 37-40 37-40
26.4 30.8 29.4 24.9
F F F F
6.2 6.1 6.7 6.0
Fig. 2 Down-regulation of AAH gene in clinical conditions associated with impaired placental implantation. Real-time quantitative RT-PCR was performed on the limited numbers (n) of available samples that had been preserved in RNAlater. AAH and GAPDH mRNA transcripts were detected in cases of normal term pregnancy (n = 6), TOP (n = 2), SAB (n = 2), and SGA term placentas (n = 6) with clinical history of IUGR. The median relative levels of AAH (normalized to GAPDH levels in same samples) were similar in the normal term placentas compared with the early gestation placentas (TOP). The median relative levels of AAH mRNA were consistently reduced in the SGA and SAB relative to normal term and TOP placentas, respectively (A and B). Western blot analysis was used to verify AAH protein expression in placental tissue. Two fresh, snap-frozen samples of normal term delivery placentas were available for study. Western blot analysis was done using a monoclonal antibody, which binds to the N-terminal region of AAH. We detected the expected 86-kd fulllength AAH in both samples (C).
54 30 seconds. Annealing temperatures were optimized using the temperature gradient program provided with the iCycler software (Bio-Rad Laboratories, Hercules, CA). Experiments were performed in triplicate. Primer-target specificity was checked using National Center for Biotechnology Information BLASTn (Basic Local Alignment Search Tool–nucleotide) searches. In addition, SYBR Green–labeled PCR products were evaluated by agarose gel electrophoresis, and the authenticity of each amplicon was verified by nucleic acid sequencing. The cDNAs were cloned into the PCRII vector (Invitrogen). Serial dilutions of known quantities of recombinant plasmid DNA containing the specific target sequences were used as standards in the PCR reactions, and the regression lines generated from the Ct values of the standards were used to calculate mRNA abundance [11,12]. Relative mRNA abun-
F. Gundogan et al. dance was determined from the ng ratios of specific mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) measured in the same samples. Results were normalized to GAPDH because standard housekeeping genes usually suffice as internal control genes. Control studies included realtime quantitative PCR analysis of (1) template-free reactions; (2) RNA that had not been reverse-transcribed; (3) RNA samples that were pretreated with DNAse I; (4) samples treated with RNAse A before RT-PCR; and (5) genomic DNA.
2.5. Statistical analysis Intergroup statistical comparisons were made by v 2 analysis or analysis of variance and the post hoc TukeyKramer significance test using the Number Cruncher Statistical System (Dr Jerry L. Hintze, Kaysville, UT) or
Fig. 3 AAH immunoreactivity in terminated pregnancy (TOP) cases. AAH highlighted the cytoplasm of villous CTB and syncytiotrophoblast with 1+ intensity in most cases (A). Polarized extravillous CTB/cell columns (arrow) away from the implantation site showed no immunoreactivity (A), whereas the cell columns (arrow) of anchoring villi at the implantation site showed 2+ to 3+ immunoreactivity (B). The extravillous CTBs that invade the decidua expressed 3+ immunoreactivity in most cases (B). The endovascular CTB and extravillous trophoblast that invade and transform the maternal vessels, had 2+ to 3+ AAH immunoreactivity (C). In addition to trophoblastic cells, endometrial glands and decidual cells expressed 3+ cytoplasmic AAH immunoreactivity in more than 78% of cases (D) (original magnification 200).
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation GraphPad Prism 4 software (GraphPad Software, Inc, San Diego, CA). P b .05 was considered statistically significant.
3. Results 3.1. Sample profiles The placentas used in this study were obtained from deliveries that occurred between January 2005 and April 2005 (Table 2). The 21 normal and 21 SGA placentas had gestational ages of 37 to 40 weeks. The 11 TOP cases had gestational ages of 6 to 16 weeks, and the 23 SAB cases had gestational ages ranging from 5 to 12 weeks. The cases of SGA had clinical histories of IUGR or preeclampsia. The maternal ages were similar for the cases of normal term delivery, TOP, and SGA, whereas the mean maternal age of the SAB group was significantly higher than the SGA and TOP groups ( P b .05).
55
was also graded as 2+ (10 cases [48%]) or 3+ (11 cases [52%]). Because the term placenta basal plates are devoid of endometrial glands, AAH immunoreactivity could not be evaluated in these structures in late gestation. Within the TOP group and irrespective of gestational age, the intensity of AAH immunoreactivity in villous CTB was 1+ in 7 (64%) of 11 cases (Fig. 3A) and 2+ in 4 (36%) of 11 cases, although 3+ AAH immunoreactivity was focally present in 3 of the cases. Syncytiotrophoblast exhibited similar levels and patterns of AAH immunoreactivity, as observed for the villous CTB. In extravillous interstitial CTB, 3 (27%) of 11 cases had 2+ AAH expression, and in 8 of 11, AAH immunoreactivity was 3+ (Fig. 3B). Endovascular CTB, that is, extravillous CTB that transforms the decidual vessels and phenocopy the endothelial cells, also exhibited 2+ or 3+ levels of AAH immunoreactivity (Fig. 3C), as observed in interstitial CTB. Cell columns that give rise to extravillous trophoblasts and form connections
3.2. AAH expression demonstrated by RT-PCR and Western blot analysis Real-time quantitative RT-PCR was performed on the limited numbers of available samples that had been preserved in RNAlater. In total, 6 cases of each normal term pregnancy and SGA with established IUGR and 2 cases each of TOP and SAB were analyzed. AAH mRNA and GAPDH mRNA transcripts were detected in all samples. The relative levels of AAH mRNA (normalized to GAPDH levels in the same samples) were similar in the normal term placentas compared with the TOP cases. However, the relative levels of AAH mRNA were consistently reduced in the SGA and SAB relative to the normal term placentas and TOP, respectively (Fig. 2A and B). Western blot analysis was used to verify AAH protein expression in placental tissue. Two fresh, snap-frozen samples of normal term delivery placentas were available for study. Western blot analysis using monoclonal antibody, which binds to the N-terminal region of AAH, detected the expected approximately 86-kd full-length AAH (Fig. 2C).
3.3. AAH immunoreactivity in normal gestation Terminated pregnancy cases were used to examine AAH expression during early gestation, and term delivery cases were used to examine AAH expression during late gestation. AAH cytoplasmic staining was detected in villous CTB, syncytiotrophoblast, extravillous (both interstitial and endovascular) CTB, decidual cells, and endometrial glands in all cases. In the normal term delivery placentas, cytoplasmic AAH immunoreactivity was detected in villous CTB, extravillous CTB, and decidualized stromal cells. Within villous CTB, AAH immunoreactivity was 1+ in 15 (71%) of the 21 cases and 2+ in 6 cases (29%). In contrast, AAH immunoreactivity in extravillous CTB was 2+ in 11 of 21 cases (52%), and 3+ in 10 (48%) of 21 cases. Within decidualized stromal cells, AAH immunoreactivity
Fig. 4 Differentiation of decidual cells from extravillous CTB with the aid of CK7 immunohistochemistry. The diffuse cytoplasmic AAH expression at implantation site and decidua (A) required CK7 immunostaining to differentiate the trophoblastic cells from decidual cells. CK7, a trophoblastic marker, highlighted the extravillous CTB and endometrial glands (B) revealing the extent of AAH expression in decidual cells (original magnification 200).
56 between anchoring villi and the basal plate also had 2+ or 3+ levels of AAH immunoreactivity at the implantation site (Fig. 3B), although cell columns away from the implantation site had no detectable AAH immunoreactivity (Fig. 3A). In decidua, AAH immunoreactivity was 2+ in 2 (18%) and 3+ in 9 (82%) of the 11 cases (Fig. 3D). Endometrial glands were present in 9 of the 11 cases. Two (22%) had 2+ and 7 (78%) had 3+ levels of AAH immunoreactivity (Fig. 3D). In these studies, CK7 immunohistochemical staining of adjacent sections was used to distinguish decidual cells from trophoblastic cells (Fig. 4) and to map the distribution and grade AAH immunoreactivity in decidual cells.
3.4. Altered AAH expression with impaired implantation Because preeclampsia and IUGR have been associated with shallow or improper placental implantation, to evaluate the potential role of AAH in relation to implantation,
F. Gundogan et al. we compared the AAH expression profiles in placentas from normal term and SGA cases. In the SGA cases with clinically established IUGR, the intensity profiles of AAH immunoreactivity in villous CTB, extravillous CTB, and decidua were similar to those of the normal term placenta (Fig. 5A-C). AAH immunoreactivity in SGA extravillous CTB was 2+ in 12 (57%) and 3+ in 9 (43%) of the 21 cases. In addition, AAH immunoreactivity in SGA villous CTB was 1+ in 15 (71%), 2+ in 6 (29%), and 3+ in 0 of the 21 cases. Decidualized stromal cells in the SGA cases had 2+ AAH immunoreactivity in 14 cases (67%), and 3+ in 7 of the 21 cases (33%). As observed in the normal term placentas, the SGA basal plates were devoid of endometrial glands. To further evaluate the potential role of AAH in relation to implantation, we compared the AAH expression profiles in placentas from TOP and SAB. Although the cellular distribution of AAH immunoreactivity was similar in the SAB and TOP cases, the AAH expression levels were more
Fig. 5 Comparison of extravillous and villous cytotrophoblastic AAH expression in late and early gestation placentas with/without clinical implication of impaired implantation. The graphs depict the distribution profile of AAH immunoreactivity in a percentage of cases. Extravillous CTB from late gestational, normal term, and SGA placentas with clinically established diagnosis of IUGR did not reveal significant difference in AAH expression (A). However, AAH immunoreactivity was significantly reduced ( P = .046) in extravillous CTB of SAB cases compared with TOP placentas (D). The intensity of villous cytotrophoblastic AAH expression was comparable in late- and earlygestation placentas and did not differ in SGA and SAB cases relative to normal term and TOP placentas, respectively (B and E). Decidual expression of AAH in late gestation was comparable in normal term and SGA placentas (C). SAB cases showed significantly reduced AAH expression in decidual cells ( P = .04) (F) and endometrial glands ( P = .005) relative to TOP cases.
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation variable and, overall, conspicuously lower in the extravillous CTB ( P = .046), decidua ( P = .04), and endometrial glands ( P = .005, data not shown) in the SAB relative to the TOP cases (Fig. 5D-F). In this regard, the intensity of AAH immunoreactivity in extravillous CTB was judged to be 1+ in 5 (22%) of 23 cases (Fig. 6C), 2+ in 12 (52%), and 3+ in 6 (26%) (Fig. 6A). In the SAB cases, AAH immunoreactivity in villous CTB and syncytiotrophoblast was similar to TOP group (Fig. 5E). In the SAB group, the distribution profile corresponding to the levels of AAH immunoreactivity in decidua was similar to that observed in extravillous trophoblasts, that is, 1+ immunoreactivity was observed in 7 (30%), 2+ in 7 (30%), and 3+ in 9 (40%) of the 23 cases (Fig. 5F). Immunohistochemical staining of adjacent tissue sections with anti-CK7 was used to differentiate decidualized endometrial stromal cells from interstitial CTB. Endometrial glands were present in 21 of the 23 SAB specimens. AAH immunoreactivity in endometrial glands
57
was 1+ in 7 (33%) (Fig. 6D), 2+ in 9 (43%), and 3+ in 5 (24%) of 21 cases (Fig. 6B). As a positive control, adjacent sections were immunostained with antibodies to vimentin. Those studies demonstrated similarly robust levels of vimentin immunoreactivity in the TOP and SAB cases.
4. Discussion This study characterizes the spatiotemporal expression pattern and the potential role of AAH in placental implantation for the first time. We showed the presence of AAH in different subsets of trophoblast, decidua, and endometrial glands throughout the gestation by immunohistochemistry and verified the AAH protein expression by Western blot analysis. The implantation process is currently considered the most limiting factor for establishment of successful pregnancy in which trophoblastic cells play a
Fig. 6 AAH expression in spontaneous abortuses. Villous CTB expressed 1+ or 2+ (A) AAH immunoreactivity in all the cases. Extravillous CTB, decidua (A), and endometrial glands (B) had 2+ to 3+ AAH expression in most cases. However, 5 of 23 cases revealed 0 to 1+ AAH immunoreactivity in extravillous CTB, and 7 of 23 cases had 1+ immunoreactivity in decidual cells (C). Same cases also revealed 1+ AAH expression in endometrial glands (D). Vimentin immunostaining showed strong immunoreactivity in all the cases with decreased AAH expression (original magnification 200).
58 crucial role. This study will aid in the understanding of the molecular interactions at the embryomaternal interface during implantation.
4.1. Overexpression of AAH in extravillous CTB Cytoplasmic AAH expression was detected in villous CTB, extravillous CTB, amniocytes, decidua, and endometrial glands in early- and late-gestation placentas. Regardless of the gestational age, extravillous CTB and both interstitial and endovascular CTB demonstrated stronger AAH immunoreactivity in comparison with villous CTB. Considering that extravillous CTBs are the motile and invasive subgroup of trophoblast, AAH overexpression in this group, in comparison with immotile villous CTB [2], further confirms the role of AAH in cell migration and invasiveness [6,9,10]. Although the cell columns away from the implantation site revealed no AAH immunoreactivity, the cell columns of anchoring villi, where the trophoblastic attachment and infiltration into endometrium takes place, expressed intense AAH immunoreactivity. It has been shown that from the proximal to the distal end of the cell column, extravillous CTBs gradually change their phenotype from a proliferative epithelial cell to a nondividing and invasive mesenchymallike cell [2]. AAH immunostaining demonstrated this phenotypic change strikingly.
4.2. AAH expression in decidua and endometrial glands One of the most interesting findings of this study was demonstration of AAH expression in decidua and endometrial glands. It has been known that decidua is one of the key players in receptivity of endometrium and, thus, in implantation [13-16]. Decidual AAH expression was diffuse at the implantation site and also in hormonally active fragments revealing 2+ to 3+ immunoreactivity. Endometrial glandular AAH expression was also diffuse, and the glands with Arias Stella features showed the most intense immunoreactivity. To find the specificity of this staining pattern for pregnancy, we randomly selected secretory phase endometrial biopsy cases from different periods: 16, 22 to 23, and 24 days of cycle. AAH immunohistochemistry revealed no reactivity in early secretory phase, multifocal 2+ to 3+ glandular immunoreactivity on days 22 to 23, and diffuse 3+ glandular immunoreactivity on 24th day of cycle. bDecidualizedQ stroma in none of these cases revealed AAH immunoreactivity (unpublished data). This staining pattern suggests that the decidual AAH expression is specific for the implantation period.
4.3. Altered AAH expression with impaired implantation After demonstrating the expression pattern of AAH in early and late gestation placenta cases, we evaluated AAH expression patterns in clinical conditions associated with
F. Gundogan et al. impaired implantation. Although it is a well-established fact that 50% to 60%, approaching to 90% as it occurs earlier, of early spontaneous abortuses are associated with chromosomal abnormality, the underlying mechanism is still not completely understood [17] and molecular and hormonal factors involved in trophoblastic invasion are under investigation [13,15,16,18]. Anatomical evidence of defective placentation defined as shallow implantation, thin and fragmented trophoblastic shell, and deficient physiologic conversion of maternal vessels have been reported in two thirds of early pregnancy failures [17]. Except for deficient physiologic conversion of maternal vessels in 2 cases, we did not identify other features by hematoxylin and eosin examination in our SAB study group. This observation further supported the need for a biomarker that reflects trophoblastic cell function. Comparison of AAH expression profiles in placentas from SAB and TOP cases revealed a striking shift toward lower intensity of AAH immunoreactivity in extravillous CTB, decidua, and endometrial glands of SAB cases. The AAH expression pattern of villous CTB was similar in TOP and SAB cases. The altered AAH expression in extravillous CTB has 2 clinical consequences: (1) decreased trophoblast motility and invasiveness at the maternal-placental interface result in shallow implantation and (2) defective invasion of endovascular trophoblast cause deficient or absent physiologic conversion of maternal vessels. In addition to lower-intensity expression of AAH in extravillous CTB, we also demonstrated that AAH expression is shifted toward lower intensity in decidua and endometrial glands in SAB cases. Decreased trophoblast invasion may be a consequence of altered AAH expression in the extravillous CTB or in the decidua that the trophoblasts are attempting to invade, or both. We also demonstrated that altered AAH expression in SAB cases is at the transcriptional level by RT-PCR. In addition to SAB cases, we also evaluated SGA placentas with established diagnosis of IUGR with or without preeclampsia. Decreased or absent trophoblastic invasion into the maternal spiral arteries has been documented in cases of preeclampsia [19]. However, this finding can only be reliably documented in placental bed biopsies. Basal plates of term placentas were devoid of maternal vessels and could not be evaluated in late gestation group. The cellular distribution and intensity of AAH immunoreactivity was similar in normal term and SGA term placentas. However, the relative levels of AAH mRNA were reduced in the SGA placentas relative to the normal term placentas. In conclusion, we demonstrated abundant AAH expression in placental trophoblastic cells, as well as in decidua and endometrial glands irrespective of gestational age. Reduced expression at the transcriptional level in SAB and IUGR with/without preeclampsia cases suggest that AAH may serve as a biomarker of impaired implantation. In addition, the high levels of AAH in decidua
Role of aspartyl-(asparaginyl) b -hydroxylase in placental implantation and endometrial glands suggest a role for this molecule in breceptivityQ of endometrium.
Acknowledgments
[9]
[10]
The authors thank Terry Pasquariello, BS, for her assistance in immunohistochemical staining of the cases.
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