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Research Article |
Schools of Surgical and Reproductive Sciences,3 Clinical & Laboratory Sciences,4 and Medical Education Development,5 University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE2 4HH, United Kingdom
ABSTRACT
Extravillous trophoblast cell (EVT) invasion in early pregnancy occurs in a relatively low-oxygen environment. The role of oxygen in regulation of EVT invasion remains controversial. We hypothesized that 1) culture in 3% oxygen inhibits EVT invasion compared with culture at 8% or 20% oxygen and 2) inhibition of invasion is due to alterations in levels of components of the urokinase plasminogen activator (PLAU, uPA) system rather than through increased apoptosis and/or decreased proliferation. Placental samples (8–10, 12–14, and 16–20 wk gestation) were obtained from women undergoing elective surgical termination of pregnancy or after cesarean section delivery (term) at the Royal Victoria Infirmary, Newcastle upon Tyne, U.K. EVT invasion from placental explants cultured at 3%, 8%, or 20% oxygen was assessed using Matrigel invasion assays. Invasion was assessed on Day 6, explants were harvested for analysis of apoptosis and proliferation, and medium was stored for analysis of PLAU system components by ELISA and casein zymography. Culture at 3% oxygen inhibited EVT invasion. PLAU receptor and plasminogen activator inhibitor-2 protein levels were increased and PLAU activity decreased in these cultures. There was no difference in the proliferation in explants cultured at the three different oxygen concentrations. Apoptosis, assessed by M30 immunostaining, was increased in EVT at both 3% and 8% oxygen. The reduction in the invasive capacity of EVT cultured at 3% oxygen appears to be mediated both by a general inhibition of the PLAU system and a decrease in the number of cells available to invade.
apoptosis, early development, explants, implantation, invasion, oxygen, proteases, trophoblast
INTRODUCTION
Extravillous trophoblast (EVT) cell invasion of the uterine decidua and inner myometrium is a critical step in the establishment of successful pregnancy [1]. Invasion occurs by an endovascular route, whereby EVTs migrate up the lumen of the uterine spiral arteries, and an interstitial route, involving attachment of cytotrophoblast cell columns to decidua, proteolytic degradation of the extracellular matrix (ECM), and movement of EVT between decidual and subsequently myometrial cells [2–4]. Invasion of maternal tissues by EVT is a tightly controlled process, known to be regulated by various autocrine and paracrine growth factors and cytokines that are proposed to act via altered cell adhesion, modulation of protease activity, and/or induction of apoptosis [5, 6]. Local oxygen and glucose concentrations, as well as tissue pH, are also likely to regulate cellular phenotype [7].
It is well recognized that many tissues are exposed to very low PO2 values under normal conditions, including the pericentral cells of the liver, retinal epithelium, thymus [8], and the first-trimester placenta [9, 10]. Yedwab et al. [11] demonstrated that PO2 levels within the lumen of the uterus during the time of blastocyst implantation are as low as 10–15 mm Hg (1–2%). Further, before the 10th week of gestation, when trophoblast invasion is maximal, placental oxygen levels are much lower than those in the surrounding endometrium; PO2 values increase from approximately 18 mm Hg (2–3%) at 8–10 wk gestation to 60 mm Hg (7–8%) at 12–13 wk gestation [9, 10]. Oxygen concentrations within the placental bed during these critical periods of placentation have been reported to be physiologically normal (approximately 7–8%) [10].
The role of oxygen in determining the invasive capacity of trophoblasts has been widely debated [12, 13]. Genbacev et al. [14] demonstrated a decrease in the invasive capacity of primary isolates of first-trimester trophoblast cells when cultured in 2% oxygen for 72 h. In contrast, Graham et al. [15] demonstrated increased invasion of a first-trimester EVT cell line, HTR-8/SVneo, in a Matrigel invasion assay on exposure to 1% oxygen for 24 h.
There are several mechanisms by which changes in oxygen concentrations may regulate trophoblast invasion. Low oxygen concentrations have been shown to increase proliferation of cells in cytotrophoblast columns [16–18]. While the role of low oxygen concentrations in apoptosis of villous trophoblast cells has been widely studied [18–20], the results differ depending on whether explants or primary trophoblast cell isolates were used. Furthermore, apoptosis of EVT in response to differing oxygen concentrations has not been studied. Trophoblast cells use two main proteolytic pathways to facilitate their invasion through decidua, namely the matrix metalloproteinases (MMPs) [21] and the urokinase plasminogen activator (PLAU, uPA) system [22]. Previous studies have demonstrated that levels of several components of the PLAU system, but not the MMPs, are regulated by oxygen [15, 23, 24].
We hypothesized that EVT invasion would be inhibited by culture in 3% oxygen when compared with cultures maintained at 8% or 20% oxygen. We also sought to examine the potential mechanism by which oxygen may elicit these effects and therefore investigated apoptosis and proliferation in explants cultured at the different oxygen concentrations as well as secreted levels of the different components of the PLAU system, namely the serine proteinase PLAU, the PLAU receptor (PLAUR, uPAR), and the plasminogen activator inhibitors 1 and 2 (SERPINE1 and 2, PAI-1 and –2) [25].
MATERIALS AND METHODS
Placental Explants
Placental samples were obtained from women undergoing elective surgical termination of pregnancy or normal term delivery at the Royal Victoria Infirmary, Newcastle upon Tyne, U.K. The study was approved by the Joint Ethics Committee of Newcastle and North Tyneside Health Authority and the University of Newcastle and all women gave informed written consent. Samples were obtained from four gestational age periods; 8–10 wk, 12–14 wk, 16–20 wk (as determined by ultrasound measurement of crown-rump length or biparietal diameter) and 37–40 wk. Following collection, placental tissue was immediately suspended in sterile saline and transported to the laboratory, where it was washed 2–3 times in sterile PBS to remove excess blood. Chorionic villous tips were dissected, minced to approximately 0.5 mm3, and resuspended in culture medium (DMEM:F12 containing 10% fetal bovine serum [FBS], penicillin/streptomycin, and amphoteracin B [all from Sigma, Poole, U.K.]) such that 15 µl of the suspension constituted approximately 10 mg of tissue [6].
Invasion Assay
To assess the effects of oxygen on EVT invasion, Matrigel invasion assays were performed as previously described [6]. Matched plates were incubated for 6 days in standard tissue culture conditions (20% O2), at an oxygen concentration considered to be more physiological in the developing placenta and placental bed (8% O2) or at a reduced oxygen concentration (3% O2). Culture at 20% oxygen was performed in a standard 37°C 5% CO2 in air incubator (Heraeus). Culture in 8% or 3% oxygen was in a Sanyo Multigas incubator, which was continually flushed with 5% CO2/balance nitrogen to maintain the desired oxygen concentration. At the end of the incubation period, the Matrigel and explant were removed and the upper side of the membrane cleaned with a cotton wool bud. For assessment of the number of invaded cells, the filters were stained with hematoxylin and eosin, mounted on glass microscope slides with SuperMount (BioGenex, San Ramon, CA), and coverslipped with DPX synthetic resin (Raymond Lamb, London, U.K.). Each slide was blinded and the total number of invaded cells on the underside of the filter was counted. We have previously demonstrated that the invaded cells have an EVT phenotype [6].
Depending on the experiment, data are expressed either as the total number of cells invaded (effect of gestational age) or as an invasion index, where the level of invasion was normalized to the 20% oxygen control within each experiment.
Explant Culture
To assess the potential mechanism by which oxygen regulates EVT invasion, explants were grown in 24-well tissue culture plates coated with 10 µl Matrigel (diluted 1:5 with media containing 2% FBS). Explants were cultured under standard conditions for 2–4 h to allow adherence to the Matrigel. The wells were then flooded with 750 µl media and matched plates were then incubated in 20%, 8%, or 3% oxygen for 6 days. The explants were then harvested, fixed in 10% neutral buffered formalin for 24 h, and processed into paraffin wax for immunohistochemistry. Conditioned medium was stored at –80°C until required for analysis. Each experiment was repeated using five different placentas for each gestational age group (8–10 and 12–14 wk).
Immunohistochemistry
Immunohistochemistry was performed using an avidin-biotin peroxidase method (Vectastain Elite mouse kit, Vector Laboratories, Peterborough, U.K.) using the primary monoclonal antibodies described below. The reaction was developed with Fast diaminobenzidine tablets. Washes between each step were performed in TBS (0.15 M TRIS buffered 0.05 M saline, pH 7.6). Sections were counterstained in Mayer hematoxylin (BDH, Poole, U.K.) and mounted in DPX synthetic resin (Raymond Lamb). Omission of primary antibody or substitution with nonimmune mouse serum for the primary antibody was included as controls.
To determine the role of cell viability and numbers (apoptosis and proliferation) in the oxygen-mediated regulation of EVT invasion, sections (3 µm) from explants were immunostained for M30 (neo-epitope of cytokeratin 18 exposed after caspase-mediated cleavage; pretreatment 10 min trypsin at 37°C, 1:200; Roche Diagnostics Ltd., Lewes, U.K.) and MKI67 (proliferation marker; pretreatment pressure cooking for 1 min in citrate buffer, pH 6.0, 1:200; Novocastra Laboratories, Newcastle upon Tyne, U.K.). Sections were also stained for HLA-G (pretreatment pressure cooking for 1 min in citrate buffer, pH 6.0, 1:200; Serotec, Oxford, U.K.) and cytokeratin 7 (pretreatment pressure cooking for 1 min in citrate buffer, pH 6.0, 1:20; Novocastra Laboratories) to aid distinction of extravillous (HLA-G positive) from villous trophoblast. The sections were scored for the approximate percentage of immunopositive cells (1 = <10% immunopositive cells, 2 = 11–24% immunopositive cells, 3 = 25–74% immunopositive cells, 4 = >75% immunopositive cells) by two independent investigators (J.N.B. and B.A.I.), who were blinded to the identity of the sample.
Analysis of the PLAU Protease System
PLAU (American Diagnostica, Greenwich, CT), PLAUR (R&D Systems), SERPINE1 (American Diagnostica), and SERPINE2 (American Diagnostica) were assayed in conditioned culture media using commercially available ELISA kits according to the manufacturers' instructions. Preliminary experiments demonstrated that there was no difference between the levels of PLAUR detected in the culture media and those in protein homogenates of tissue samples (data not shown). Therefore, experiments were performed on conditioned media.
To determine levels of active PLAU, casein zymographic analysis was performed on conditioned media as described previously [6]. Briefly, 20 µg total protein (Bio-Rad protein assay; Bio-Rad, Hemel Hempstead, Herts., U.K.) was resolved in a 12% SDS-PAGE containing 2 mg/ml casein and 0.025 units/ml plasminogen (American Diagnostica). The gels were then washed in 2.5% Triton X-100 to remove the SDS and incubated overnight at 37°C in a solution of 50 mM Tris and 5 mM CaCl2 to allow the enzymes to digest the substrate. After incubation, the gels were stained with Coomassie Brilliant Blue R250, destained, preserved, and dried. The dried gels were then scanned and densitometry performed (UnScan-It, Silk Scientific Co., Orem, UT). To remove intersubject variability, all results are normalized to their respective controls.
Statistical Analysis
Data are presented as means ± SEM. Statistical calculations were performed using the StatView statistical software package (Abacus Concepts Inc., Berkley, CA). Statistical significance was determined by use of one-way ANOVA followed by Fisher post hoc analysis unless otherwise stated. A two-way ANOVA was used to assess the impact of gestational age on the inhibition of invasion by culture in 3% oxygen. Student t-test was used when only two sets of data were compared. All statistical tests were two sided and differences were considered statistically significant at P < 0.05.
RESULTS
Effect of Gestational Age on the Invasive Capacity of EVT Cells
There was a significantly (P < 0.03) higher number of invaded EVT cells in samples at 8–10 wk gestational age (n = 5) compared with the other gestational age groups (12–14 wk [n = 5], 16–20 wk [n = 5], and term [n = 5]) (Fig. 1). There was no difference in the number of invaded EVT cells between the other three gestational age groups. Therefore, for further experiments, only two gestational age groups (8–10 and 12–14 wk) were tested.
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Effect of Oxygen on the Invasive Capacity of EVT Cells
To test the effect of reduced oxygen concentrations on the invasiveness of EVT cells, placental explants from 8–10 and 12–14 wk gestation were cultured in 20%, 8%, or 3% oxygen for 6 days. There was no difference in EVT cell invasion when the assays were performed at 8% O2 compared with 20% O2 (Fig. 2). However, for both the gestational age groups tested, there was a significant decrease in the number of invaded EVT cells when explants were cultured at 3% O2 compared with 20% or 8% oxygen (8–10 wk, P < 0.0004; 12–14 wk, P < 0.01; Fig. 2). The gestational age of the explants did not affect the level of inhibition of invasion by culture in 3% oxygen (P = 0.1, two-way ANOVA).
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Role of Cellular Apoptosis/Proliferation in Oxygen-Regulated Trophoblast Invasion
The role of reduced oxygen concentrations in EVT cell death (apoptosis) and proliferation in placental explants was investigated by culturing explants at 20%, 8%, and 3% oxygen for 6 days (Fig. 3; Table 1). There was no difference in the proportion of M30-positive villous cytotrophoblast cells at either gestational age at any of the oxygen concentrations tested. There was also no difference in the proportion of M30-positive EVT cells derived from the earlier gestational age group at any of the oxygen concentrations tested. However, in the later gestational age group, the proportion of M30-positive EVT cells was increased in explants cultured at 8% and 3% oxygen compared with those cultured at 20% oxygen (12–14 wk: 20% O2 1.0 ± 0.2, 8% O2 1.8 ± 0.2, P < 0.02, 3% O2 2.0 ± 0.4, P < 0.009). MKI67-positive trophoblast cells were only observed in the villous trophoblast of the explants and the most proximal parts of the cytotrophoblast columns, HLA-G-positive EVT cells being universally MKI67 negative. There was no difference in the proportion of MKI67-positive cells at either gestational age in any of the experimental conditions tested.
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Oxygen Regulation of the PLAU Protease System in Placental Explants
The role of four components of the PLAU protease system (PLAU, PLAUR, SERPINE1, and SERPINE2) in the oxygen-mediated regulation of EVT invasion was tested in the two gestational age groups (8–10 and 12–14 wk), after 6 days of culture at three different oxygen concentrations (20%, 8%, and 3%) using sandwich-based ELISAs and casein zymography.
There was no difference in total secreted levels of PLAU (as determined by ELISA) between samples cultured at 3% O2 and 20% O2 (Fig. 4A), but there was a significant reduction in total secreted PLAU levels in explants cultured at 8% O2 compared with those cultured at 20% and 3% O2 (Fig. 4A) in both gestational age groups tested (8–10 wk, P < 0.007; 12–14 wk, P < 0.05). There was a significant decrease in the levels of active PLAU (as determined by casein zymography) secreted from explants cultured at 3% and 8% O2 compared with those cultured at 20% O2 for both gestational age periods studied (8–10 wk, 3% P < 0.009, 8% P < 0.004; 12–14 wk, 3% P < 0.0001, 8% P < 0.005; Fig. 4B). There was no difference in the levels of PLAU with respect to the gestational age of the explants at any oxygen concentration tested.
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There was a significant increase (approximately 5-fold) in the level of PLAUR in the conditioned media of explants cultured at 3% oxygen compared with those cultured at 20% or 8% oxygen (8–10 wk, P < 0.006; 12–14 wk, P < 0.04; Fig. 5). There was no difference in PLAUR levels with respect to the gestational age of the explants at any oxygen concentration tested. Culture at 8% oxygen did not alter the level of PLAUR compared with culture at 20% oxygen (Fig. 5).
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While SERPINE1 levels were not altered at any of the oxygen concentrations or gestational ages tested (Fig. 6A), there was an increase in secreted SERPINE2 levels into the culture media of explants cultured at 3% oxygen compared with those cultured at 20% or 8% oxygen (8–10 wk, P < 0.04; 12–14 wk, P < 0.003; Fig. 6B). Levels of SERPINE2 did not differ between explants cultured at 20% or 8% oxygen (Fig. 6B). There was no difference in levels of SERPINE1 or SERPINE2 with respect to the gestational age of the explants at any oxygen concentration tested.
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DISCUSSION
Oxygen is thought to be a key regulator of trophoblast function at the feto-maternal interface. In the present study, we have demonstrated that culture of placental explants at 3% oxygen inhibited invasion of EVT cells in a Matrigel invasion assay compared with culture at 8% or 20% oxygen. This inhibition in invasion at 3% oxygen was associated with an increase in PLAUR and SERPINE2 levels. In addition, there was an increase in EVT apoptosis but only at the later gestational age studied. However, there was no evidence of altered trophoblast proliferation at either gestational age.
In agreement with the current study, both Genbacev et al. [14] and Crocker et al. [26] have demonstrated an inhibition in trophoblast invasion by low oxygen concentrations using different models. However, this contrasts with the findings of Graham et al. [15], who demonstrated an increase in invasiveness of the first-trimester trophoblast cell line, HTR-8/SVneo, when cultured at 1% oxygen compared with 20% oxygen. The discrepancies in these studies are likely due to the cell type and methodology used. In the current study, explants were chosen, as they were considered to be a more physiologically relevant source of EVT than enzyme-digested primary isolates of cytotrophoblast or a transformed cell line. In addition, Newby et al. [27] demonstrated no difference in the level of outgrowth from first-trimester explants cultured on Matrigel in 2% oxygen compared with 20% oxygen. The difference in these results and those of the current study are likely due to differences in the technique used. In the current study, the level of invasion was quantified by the number of cells that had invaded through Matrigel and a porous membrane, whereas Newby et al. [27] semiquantitatively assessed outgrowth of villous tips on the surface of a thick layer of Matrigel rather than invasion. Interestingly, the findings of Newby et al. [27] also contrast with those of Caniggia et al. [17], who demonstrated increased outgrowth from explants cultured at 3% oxygen, although these authors did not assess migration or invasion. Interestingly, there was no difference in the invasiveness of the explants cultured at 8% oxygen compared with those cultured at 20% oxygen, which, although standard for tissue culture, is considered hyperoxic for trophoblast cells [12].
There was an intrinsic difference in the invasiveness of EVT derived from explants of different gestational ages. Explants collected from 8–10 wk gestation were approximately twice as invasive as those from later gestational age groups (12–14 wk, 16–20 wk, and term) but interestingly, there was no difference between these later gestational age groups. This difference was only observed when explants were cultured at 20% oxygen (data not shown for 3% oxygen). The mechanism underlying this intrinsic difference in invasiveness at 20% oxygen is not clear. Indeed, we could find no differences in levels of components of the PLAU system between the different gestational age groups at any of the oxygen concentrations tested. Genbacev et al. [14] reported similar gestational age differences when primary isolates of trophoblast cells were cultured at 20% oxygen, although they made no comment on the physiological relevance of this observation.
In the present study, the proportion of MKI67 immunopositive trophoblast cells did not differ in any of the experimental conditions for either gestational age group. Several previous studies have reported an increase in the proliferation (determined by MKI67 staining) of cytotrophoblast cells in the proximal column in explants cultured at 2–3% oxygen compared with higher oxygen concentrations [17, 18]. However, neither of these groups quantified the proportion of MKI67-positive cells in explants cultured at different oxygen concentrations. In addition, Genbacev and colleagues [14, 16] reported an increase in [3H] thymidine incorporation by primary isolates of cytotrophoblast cells (10–12 wk gestation [14]) and explants (6–8 wk gestation [16]) cultured at 2% oxygen compared with those cultured at 20% oxygen. Incorporation of [3H] thymidine is more sensitive than MKI67 immunostaining as used in the current study and, hence, the results obtained by Genbacev et al. may reflect the role of low oxygen in trophoblast proliferation than those reported here. Therefore, a further study of thymidine incorporation in villous explants under different oxygen conditions would be useful.
The effect of low-oxygen concentrations on trophoblast apoptosis has been widely studied [18–20]. However, these studies have focused on cytotrophoblast apoptosis, and its relationship to formation of syncytia rather than to EVT cell apoptosis, and have shown different results depending on methodology used. In the present study, we observed no difference in the proportion of M30-immunopositive villous trophoblast cells in explants cultured at any of the oxygen concentrations tested. In contrast, there was an increase in the proportion of M30-immunopositive EVT in explants (12–14 wk gestation only) cultured at 3% or 8% oxygen compared with those exposed to 20% oxygen. This result may, in part, provide a mechanism for the observed decrease in invasion observed at 3% oxygen; cells that are committed to an apoptotic pathway lose their invasive phenotype. Indeed, we have not detected any M30-immunopositive cells among the invading cells on the underside of the filters at the end of the culture period (data not shown). However, while there was an increase in EVT cell apoptosis in explants cultured at 8%, there was no corresponding alteration in invasive capacity in these cells. The increase in the proportion of M30-immunopositive cells observed in EVT from 12- to 14-wk explants cultured at 8% and 3% oxygen may be a reflection of the gestational age of these explants and their ability to sense oxygen rather than an effect of oxygen on the apoptotic process per se. Indeed, oxygen concentrations in the intervillous space rise rapidly between 10 and 12 wk gestation, giving rise to severe oxidative stress in the placenta that takes several weeks to recover [10]. Therefore, while other functions of trophoblast may not be altered at this gestational age, the cells of the placenta may be at higher risk of oxidative damage leading to apoptosis.
In the present study, there was an increase in the levels of PLAUR and SERPINE2 secreted by explants cultured at 3% oxygen compared with the higher concentrations. In addition there were no differences in the levels of PLAU and SERPINE2 in cultures at 3%, compared with 20%, oxygen as determined by ELISA. However, there was an overall decrease in the activity of PLAU secreted by explants cultured at 3% oxygen compared with those cultured at 20% oxygen as determined by casein zymography. The difference in levels of total and active PLAU observed at 3% oxygen can be accounted for by considering how the components of the PLAU system interact and the increases in PLAUR and SERPINE2 observed at this lowered oxygen concentration. On binding to PLAUR, pro-PLAU is cleaved into active PLAU that can bind and cleave plasminogen into plasmin [28]. The PLAUR:PLAU complex can be bound to and inhibited by SERPINE1 or SERPINE2, whereby the whole complex is internalized, where SERPINE and PLAU are degraded and PLAUR recycled to the cell surface [28]. In addition to the action of PLAUR as a cell surface receptor for PLAU, it can bind several different integrins and vitronectin and, as such, is important in cell-ECM attachment [29]. This attachment can be inhibited by SERPINE1, although it is not known whether SERPINE2 plays a similar role [29]. Taken together, the observed changes in the components of the PLAU system in explants cultured at 3% oxygen (compared with 20% oxygen) would lead to a decrease in PLAU activity and an increase in cell-ECM attachment, giving rise to an overall decrease in invasiveness, as observed.
The effect of hypoxia on various components of the PLAU system has been studied in a wide range of cell types with varying results, suggesting that there are cell type-specific modulators of oxygen sensing [30, 31] even though hypoxia response elements have been identified in the promoter region of both PLAUR and SERPINE1 genes [32, 33]. However, the only other studies to investigate components of the PLAU system under different oxygen concentrations in trophoblasts have used the transformed cell line, HTR-8/Svneo, which, in contrast with the explants used in the present study, shows increased invasion at 1% oxygen. These studies demonstrated an increase in PLAUR, a decrease in PLAU activity, and an increase in SERPINE1 levels in cells cultured at 1% oxygen compared with 20% oxygen [15, 23]. The HTR-8/SVneo cell line shows a decrease in adhesion to vitronectin and fibronectin when cultured at 1% oxygen due to the competitive inhibition of PLAUR binding by the increased levels of SERPINE1 [23, 34], contributing to the increased invasiveness observed in these cells at 1% oxygen.
The role of oxygen in altering levels of SERPINE2 has not been widely studied and this is the first study to demonstrate an increase in secreted SERPINE2 levels in response to hypoxia. Unlike SERPINE1, SERPINE2 is generally found as a major intracellular source in a nonglycosylated form [35]. Secreted SERPINE2 is heavily glycosylated, but the mechanisms and triggers underlying its secretion remain unclear [35]. It is possible that low-oxygen concentrations may trigger secretion of SERPINE2 into the extracellular environment. In addition, SERPINE2 expression can be regulated by several different factors, including cytokines and glucocorticoids [28, 35], some of which are in turn hypoxically upregulated, suggesting there may be an indirect mechanism by which hypoxia stimulates SERPINE2 expression.
In the current study, we also observed lower levels of PLAU assayed by ELISA and casein zymography from samples cultured at 8% oxygen compared with those at 20% oxygen. However, there was no difference in the invasive capacity of EVT derived from explants cultured at 8% oxygen compared with those cultured in 20% oxygen. This suggests that the increase in PLAUR and SERPINE2 observed at 3% oxygen may be more important in dictating the invasive phenotype of the cell than the active PLAU levels.
On balance, therefore, at 3% oxygen, the excess of PLAUR and SERPINE2 would facilitate inhibition of active PLAU and potentially increase binding of trophoblasts to several components of the extracellular matrix. As oxygen levels increase, levels of PLAUR and SERPINE2 decrease, leading to a more invasive phenotype. Jauniaux et al. [10] have proposed that the oxygen levels in the intervillous space at less than 10 wk of gestation are significantly lower than those in the uterus and that this oxygen differential decreases as gestation increases. Therefore, we propose that, in very early gestation, when the cytotrophoblast columns are forming in low-oxygen conditions, the predominant effect of the low oxygen is to promote proliferation of the trophoblast cells in the cytotrophoblast columns, which then acquire a much more invasive phenotype as they reach the higher oxygen concentrations of the uterus and invade into decidua. This represents a possible mechanism for initiation of the invasive process that is further controlled and regulated by many other uterine and trophoblast-derived factors, including, among others, vascular endothelial growth factor-A [36]; transforming growth factor-beta1, 2, and 3 [6]; tumor necrosis factor-alpha; and interferon-gamma (H.A. Otun, personal communication).
ACKNOWLEDGMENTS
The authors wish to acknowledge the staff at the Royal Victoria Infirmary, Newcastle upon Tyne, for their assistance in sample collection.
FOOTNOTES
1 Supported by funding from BBSRC (S19967). 
2 Correspondence: Gendie E. Lash, School of Surgical and Reproductive Sciences, 3rd Floor, William Leech Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, U.K. FAX: 44 191 222 5066; g.e.lash{at}ncl.ac.uk
Received: 8 September 2005.
First decision: 26 September 2005.
Accepted: 26 October 2005.
REFERENCES
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  S.K.M. Seeho, J.H. Park, J. Rowe, J.M. Morris, and E.D.M. Gallery Villous explant culture using early gestation tissue from ongoing pregnancies with known normal outcomes: the effect of oxygen on trophoblast outgrowth and migration Hum. Reprod., May 1, 2008; 23(5): 1170 - 1179. [Abstract] [Full Text] [PDF]
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