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ORIGINAL RESEARCH |
From the *Departments of Obstetrics and Gynaecology and Immunology, University of Liverpool, Liverpool; and the Department of Biochemistry and Immunology, St George's Hospital Medical School, London, United Kingdom.
Address reprint requests to: Siobhan Quenby, First Floor, Department of Obstetrics and Gynaecology, Liverpool Women's Hospital, Crown Street, Liverpool, L87SS, UK; e-mail: squenby{at}liv.ac.uk.
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ABSTRACT |
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 ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: Chorion from term placentas was digested and extravillous trophoblast isolated. Extravillous trophoblast formed giant multinuclear cells that were counted after 24, 36, and 48 hours of culture. This model was then used to investigate the effect of unfractionated, low-molecular-weight heparin and aspirin on in vitro extravillous trophoblast differentiation at both therapeutic and supratherapeutic doses. In addition, the effect of unfractionated and low-molecular-weight heparin on hepatocyte growth factor–stimulated SGHPL-4 cell (extravillous trophoblast cell line) motility was determined by time-lapse microscopy.
RESULTS: At therapeutic doses unfractionated heparin promoted extravillous trophoblast differentiation. However, low-molecular-weight heparin inhibited giant multinuclear cells formation. At supratherapeutic doses, both low-molecular-weight and unfractionated heparin promoted extravillous trophoblast differentiation. Low-dose aspirin had minimal effects on the extravillous trophoblast differentiation. Both unfractionated and low-molecular-weight heparin inhibited hepatocyte growth factor–stimulated extravillous trophoblast motility at supratherapeutic doses. At a therapeutic dose of 0.25 IU/mL, only unfractionated heparin inhibited hepatocyte growth factor–stimulated motility, whereas low-molecular-weight heparin had no effect.
CONCLUSION: Our data suggest that unfractionated and low-molecular-weight heparin have differing effects on trophoblast differentiation and motility at therapeutic doses. This finding may be one of many factors that contribute to the clinical scenario.
LEVEL OF EVIDENCE: II-2
The use of heparin and aspirin to prevent pregnancy loss was based on the premise that some pregnancy losses were caused by a placental thrombosis and infarction and that thromboprophylaxis with heparin could prevent this process. However, more recent data have challenged these findings.
Initial retrospective reports suggested that there was a significant association between thrombophilia and pregnancy loss.4–6 However, more recent meta-analysis of all published literature found that some, but not all, thrombophilias were associated with fetal loss.7 There has been a failure to associate other thrombophilias with placental thrombosis.8 Hence, there is little evidence that a maternal predisposition to maternal thrombosis necessarily leads to placental thrombosis.
The most extensively investigated thrombophilia, antiphospholipid syndrome, is characterized by the presence of anticardiolipin antibodies. However, examination of placentas and first-trimester decidua from antiphospholipid syndrome complicated pregnancies has found little evidence of specific thrombotic placental pathology.9–12 There is evidence that antiphospholipid syndrome may be associated with direct inhibition of trophoblast invasion, and this, rather than placental thrombosis, may be the reason for pregnancy loss. Defective decidual endovascular trophoblast invasion, rather than excessive intervillous thrombosis, was the most frequent histological abnormality in antiphospholipid syndrome associated early pregnancy loss.11 Antiphospholipid antibodies have a direct effect on in vitro trophoblast cell function when using choriocarcinoma cell lines or primary villous trophoblast cultures (Quenby S, Mountfield S, Cartwright JE, Whitley GS, Chamley L, Vince G, submitted for publication).13–19 However, poor invasion of extravillous trophoblast is thought to be the cause of uteroplacental failure associated with antiphospholipid syndrome. We have isolated extravillous trophoblasts and found that antiphospholipid antibodies inhibit their differentiation (Quenby S, Mountfield S, Cartwright JE, Whitley GS, Chamley L, Vince G, submitted for publication), suggesting that a failure of uteroplacental development may be an underlying pathology in antiphospholipid syndrome–associated pregnancy loss.
Clinically, trials using heparin to prevent fetal loss have produced conflicting results. A combination of unfractionated heparin and aspirin was significantly more successful in the prevention of fetal loss in antiphospholipid syndrome than aspirin alone in 2 controlled trials.1,2 In these trials, the majority of the excess fetal deaths in the control groups occurred in the first trimester of pregnancy. Thus, in patients with antiphospholipid syndrome, heparin appeared to facilitate early pregnancy implantation and trophoblast invasion. However, our randomized controlled trial used low-molecular-weight heparin and aspirin for the prevention of antiphospholipid syndrome–associated miscarriage with no improvement in the live birth rate over aspirin alone.20 There have been no prospective randomized controlled trials comparing low-molecular-weight heparin and unfractionated heparin.
Low-dose aspirin frequently is prescribed to prevent miscarriage. However, a meta-analysis of randomized controlled trials into low-dose aspirin in antiphospholipid syndrome–associated miscarriage21 and a large observational study into its use in recurrent miscarriage22 have shown no improvement in pregnancy outcome. In contrast, a meta-analysis into the use of low-dose aspirin for the prevention of still birth and preeclampsia has suggested a benefit in improving pregnancy outcome in women with a past history of preeclampsia,23,24 suggesting that low-dose aspirin may have a beneficial effect on placentation.
The aim of this study was to investigate the effect of low-molecular-weight heparin, unfractionated heparin, and aspirin on primary extravillous trophoblast differentiation using an in vitro model (Quenby S, Mountfield S, Cartwright JE, Whitley GS, Chamley L, Vince G, submitted for publication). Because extravillous trophoblast need to perform multiple functions for successful invasion, the effect of low-molecular-weight heparin and unfractionated heparin on a second in vitro model, that of extra-villous trophoblast motility, also was investigated.
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MATERIALS AND METHODS |
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The protocol for isolation of term trophoblast was based on the method developed by Shorter et al.25 The placental membranes were cut from the term placenta and the villous placenta itself discarded. The amnion was then peeled away from the chorion and decidua, and the chorion was placed decidual side upwards on a paper towel to allow the decidua to be scraped off using a razor blade. The chorion was washed in phosphate-buffered saline (PBS), pH 7.4, and placed in a Petri dish containing 50 mL of 1 mg/mL Protease Type XIV (Sigma, Poole, UK) in RPMI 1640 (Sigma, St. Louis, MO) for 1 hour. All incubations were conducted at 37°C in 5% CO2. The tissue was then washed in sterile PBS and resuspended in 50 mL of RPMI 1640 containing 2 mg/mL hyaluronidase (Sigma), 0.5 mg/mL collagenase type IV (Sigma), and 0.05 mg/mL DNAse type IV (Sigma) with 10% fetal calf serum (Sigma) for 1 hour. The tissues were then removed and disrupted using a plastic pipette and returned to the incubator for a further 15 minutes. The digest was filtered through 100-µm mesh, and the cells were washed twice in PBS at 400g for 10 minutes. The resulting pellets were resuspended in 20 mL of 25% (v/v) Percoll (Sigma), and 12 mL of 60% Percoll was layered underneath. The gradients were centrifuged at 500g for 30 minutes at 12°C. The cells at the 25%/60% interface were collected, washed twice in PBS as above, and counted.
The isolated primary extravillous trophoblast cells were plated out at 1 x 106 cells/mL in RPMI 1640 containing 10% (v/v) fetal calf serum in 24-well plates (Nalge Nunc International, Rochester, NY) containing tissue culture–treated coverslips (Thermanox, Nalge Nunc International). At 24, 36, and 48 hours, coverslips were removed and left to air dry before staining. All cultures were incubated at 37°C in 5% CO2.
SGHPL-4 cells are a well-characterized cell line derived from primary first-trimester human extravillous trophoblasts. They retain many features of normal extravillous trophoblasts, including expression of human leukocyte antigen (HLA)-G, cytokeratin-7, CD9, human placental lactogen (hPL), and human chorionic gonadotropin (hCG), and have been used extensively as a model of extravillous trophoblast.26 SGHPL-4 cells were cultured as previously described.27
Extravillous trophoblasts were cultured with low-molecular-weight heparin (Daltiperin; Pharmacia and Upjohn, Milton Keynes, Buckinghamshire, UK) and unfractionated heparin (heparin sodium; CP Pharmaceuticals, Wrexham, UK) at a range of concentrations, from that found in the serum of women receiving low-dose subcutaneous unfractionated heparin/low-molecular-weight heparin (0.025–0.25 IU/mL)28 to supratherapeutic doses (2.5–250 IU/mL). Aspirin (acetyl salicylic acid; Sigma-Aldrich, Dorset, UK) was used at a concentration equivalent to that in the serum of women receiving low-dose aspirin (75–150 mg a day; 10–5–10–3 mol/L).29 Previous experiments found some interpatient variability in the rate of formation of giant multinuclear cells (Quenby S, Mountfield S, Cartwright JE, Whitley GS, Chamley L, Vince G, submitted for publication). To account for this variability, each experiment included control wells containing extravillous trophoblasts derived from the same chorion in RPMI 1640 and 10% fetal calf serum without additions to establish the "baseline" rate of giant multinuclear cell formation for each trophoblast culture. Experiments were repeated with extravillous trophoblasts isolated from 5 different placental membranes.
Coverslips were mounted on ordinary glass slides using Aquamount (BDH, Poole, UK) and left to dry overnight. The cells were fixed in ice-cold ethanol for 5 minutes and washed twice in Tris-buffered saline pH 7.6. The slides were incubated with primary antibody (cytokeratin, CK7; Dako, Ely, UK) diluted in Tris-buffered saline containing 0.05% (wt/vol) bovine serum albumin for 30 minutes. Slides were then washed twice in Tris-buffered saline and incubated for 30 minutes with rabbit anti-mouse immunoglobulin (Ig)G (DAKO) and diluted (1:25) in Tris-buffered saline and 5% (v/v) normal human serum. Slides were then washed in Tris-buffered saline and incubated for 30 minutes with alkaline phosphatase antialkaline phosphatase complex (Serotec, Oxford, UK) diluted 1:50 in Tris-buffered saline. Slides were then washed again with Tris-buffered saline and incubated for 20 minutes with alkaline phosphatase substrate containing Fast Red TR salt, levamisole, and phenyl-glycine-glycine. Slides were washed in Tris-buffered saline followed by deionized water. Finally, slides were counterstained in Hemalum (BDH) washed in water, and mounted using Aquamount. The purity of the original trophoblast cultures was 92.5 ± 2% (mean ± standard deviation of 10 isolations; Quenby et al, submitted).
Immunohistochemistry with cytokeratin 7 was used to ensure that only trophoblasts were counted. A giant multinuclear cell was defined as a cell containing a minimum of 5 nuclei but with no evidence of any internal cell membranes and with a constant outer membrane. Ten high-power fields (x 400) were selected at random, and the number of giant multinuclear cells observed was recorded. This process was repeated on 3 separate coverslips for each time period. Results are expressed as number of giant multinuclear cells per 30 high-power fields for each time point and then converted to a percentage of those in the "baseline" control wells. Two independent observers counted a random sample of 50 coverslips, and the interobserver error was found to be less than 1%.
SGHPL-4 cells (2–13 x 103 cells per 35-mm well) were allowed to adhere overnight in Hams F10 containing 10% (v/v) fetal calf serum, and the medium was then replaced with Hams F10 containing 0.5% (v/v) fetal calf serum for a further 24 hours. At the end of this period, cell motility was determined in the presence or absence of hepatocyte growth factor (HGF) at 10 ng/mL at 37°C in an atmosphere of 5% CO2 in air. Where appropriate, cells were incubated with unfractionated heparin or low-molecular-weight heparin at 0.025–250 IU/mL. Images were captured every 15 minutes over a 6-hour period using a Hamamatsu CCD camera (Hamamtsu City, Japan) connected to an Olympus IX 70 phase contrast microscope (Tokyo, Japan). At least 20 cells in a field of view were chosen at random, and the distance moved was quantified using Image Pro-Plus software (Media Cybernetics, San Diego, CA).
The number of giant multinuclear cells in 30 high-power fields was expressed as a percentage of the control or "baseline" cultures. Then a mean percentage of giant multinuclear cell formation compared with the control cultures was calculated for data from 5 different normal women's placentas. Next, an analysis of variance for repeated observations was used to analyze the differences in giant multinuclear cell formation between low-molecular-weight heparin and unfractionated heparin in the therapeutic and nontherapeutic ranges (Arcus; Research Solutions, Cambridge, UK). Cell motility results were expressed as mean ± standard error of the mean and were analyzed using the nonparametric Mann-Whitney U test. Statistical significance was assumed at the .05 level. Experimenters who were blinded to the treatment that was used conducted all analyses of trophoblast cell motility.
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RESULTS |
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At all time points studied, the therapeutic doses of low-molecular-weight heparin inhibited giant multinuclear cells formation (Fig. 2a–c). In contrast, unfractionated heparin had little effect on giant multinuclear cell formation after 24 hours of culture (Fig. 2a) but promoted giant multinuclear cell formation after 36 and 48 hours of culture (Fig. 2b and 2c). There were significantly more giant multinuclear cells formed with therapeutic doses (0.025–0.25 IU/mL) of unfractionated heparin compared to low-molecular-weight heparin (P < .001; Fig. 2a–c). However, there was a similar amount of giant multinuclear cell formation with supratherapeutic doses (2.5–250 IU/mL) of unfractionated heparin compared with low-molecular-weight heparin (P = .96; Fig. 2a–c).
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After 48 hours of culture with aspirin 10–4 mol/L (Fig. 1f), there was a similar appearance to the control (Fig. 1a). Aspirin had minimal effect on giant multinuclear cell formation at all time points and doses studied (Fig. 3).
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As we have previously described, HGF significantly stimulated SGHPL-4 cell motility.27,30 Low-molecular- weight heparin at the therapeutic doses of 0.025 and 0.25 IU/mL had no effect on HGF-stimulated motility; however, doses greater than 2.5 IU/mL significantly inhibited HGF-stimulated motility (Fig. 4a). In contrast to low-molecular-weight heparin at 0.25 IU/mL, unfractionated heparin at 0.25 IU/mL significantly inhibited HGF-stimulated motility (Fig. 4b). Higher doses of unfractionated heparin also inhibited motility. Neither unfractionated heparin nor low-molecular-weight heparin had any effect on basal motility.
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DISCUSSION |
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Heparin exerts its anticoagulant function by binding to and potentiating antithrombin and by mobilizing tissue factor pathway inhibitor into the circulation. There is growing evidence of the superior efficacy and safety of low-molecular-weight heparin over unfractionated heparin for the treatment of both venous and arterial thromboembolism.31 There are significant differences between unfractionated heparin and low-molecular-weight heparin. Unfractionated heparin has a higher molecular weight (3–30 kDa, average 15 kDa) than low-molecular-weight heparin (4–6 kDa); unfractionated heparin has a different effect on antithrombin and tissue factor pathway inhibitor than low-molecular-weight heparin in vivo31,32; low-molecular-weight heparin has a decreased clearance time and is longer acting33; low-molecular-weight heparin has increased bioavailability partly because of lower affinity for plasma matrix proteins33; low-molecular-weight heparin does not bind to endothelial cells33; and low-molecular-weight heparin is less capable of activating resting platelets.33 In addition, unfractionated heparin is monitored using prothrombin-based assays, whereas low-molecular-weight heparin is monitored using a factor Xa–based assay. However, the known differences between unfractionated heparin and low-molecular-weight heparin do not explain the different effects on extravillous trophoblast differentiation in vitro. In the present study, we tested only one type of low-molecular-weight heparin, Daltiperin (Pharmacia and Upjohn, Milton Keynes); thus, the data may not apply to all types of low-molecular-weight heparin.
In addition to its anticoagulant function, heparin also binds to growth factors that are considered to be important in trophoblast invasion and placental development. For example heparin-binding epidermal growth factor has been found to be important in early implantation34 and deficient heparin-binding epidermal growth factor signaling during placental development could impair trophoblast survival, differentiation, and invasion, leading to poor placental perfusion and hypertension in preeclampsia.35 Vascular endothelial growth factor, placental growth factor, and HGF are thought to be important regulators of extravillous trophoblast invasion.27,36 Heparin was found to be a critical component for regulating vascular endothelial growth factor–165 and placental growth factor–2 interactions with its receptor, NRP1, by physically interacting with both receptor and ligands.37 Therefore, any difference in binding of low-molecular-weight heparin and unfractionated heparin to these growth factors may explain the differing effects of unfractionated heparin and low-molecular-weight heparin on extravillous trophoblasts in vitro.
Low-dose aspirin inhibits cyclo-oxygenase and, thereby, thromboxane synthesis. Low-dose aspirin is thought to exert its beneficial effect on women with a history of preeclampsia by having an effect on platelet aggregation.23 Thus, the lack of effect of aspirin on the in vitro model of extravillous trophoblast differentiation presented here is in agreement with current theories on the pathogenesis and treatment of preeclampsia. Furthermore, the efficacy of aspirin in preventing pregnancy loss in vivo is in doubt.21,22
There is conflicting clinical data regarding the effect of unfractionated heparin compared with low-molecular-weight heparin on placentation.1,2,20 The in vitro data presented here cannot be directly translated to the clinical scenario because there are multiple influences on both placentation and obstetric outcome. However, the direct effects of low-molecular-weight heparin and unfractionated heparin on extravillous trophoblast function may be one of many important factors that affect placentation.
It is interesting that both unfractionated heparin and low-molecular-weight heparin had an inhibitory effect on HGF-stimulated motility at the supratherapeutic concentrations, but only unfractionated heparin had any significant effects at the therapeutic concentrations used. Because cell motility is an important component of the invasive process, it may be that normal trophoblast function is, in fact, inhibited by unfractionated heparin, which is contrary to its proposed therapeutic benefits. In contrast, the lack of effect of low-molecular-weight heparin is more reassuring because it does not harm this aspect of invasion in vitro. Similarly, unfractionated heparin significantly stimulates differentiation, which could be regarded as inhibiting invasion. There is, therefore, an urgent need to further investigate the effects of heparin on trophoblast function, using different models and in different centers.
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Footnotes |
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This study was funded by The Arthritis Research Council and The Newborn Appeal of the Liverpool Women's Hospital.
10.1097/01.AOG.0000128902.84876.d4
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REFERENCES |
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14. Rote NS, Vogt E, DeVere G, Obringer AR, Ng AK. The role of placental trophoblast in the pathophysiology of the antiphospholipid syndrome [review]. Am J Reprod Immunol 1998;39:125–6
15. Katsuragawa H, Kanzaki H, Inoue T, Hirano T, Mori T, Rote NS. Monoclonal antibody against phosphatidylserine inhibits in vitro human trophoblastic hormone production and invasion. Biol Reprod 1997;56:50–8.[Abstract]
16. Chamley LW, Duncalf AM, Mitchell MD, Johnson PM. Action of anticardiolipin and antibodies to beta2-glycoprotein-1 on trophoblast proliferation as a mechanism for fetal death. Lancet 1998;352:1037–8.[Medline]
17. Di Simone N, Ferrazzani S, Castellani R, De Carolis S, Mancuso S, Caruso A. Heparin and low-dose aspirin restore placental human chorionic gonadotrophin secretion abolished by antiphospholipid antibody-containing sera. Hum Reprod 1997;12:2061–5.
18. Di Simone N, Caliandro D, Castellani R, Ferrazzani S, De Carolis S, Caruso A. Low-molecular weight heparin restore in-vitro trophoblast invasiveness and differentiation in presences of immunoglobulin G fractions obtained from patients with antiphospholipid syndrome. Hum Reprod 1999;14:489–95.
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