Characterization of human placental explants: morphological, biochemical and physiological studies using first and third trimester placenta

Suren R. Sooranna1, Eugene Oteng-Ntim2, Rafia Meah1, Timothy A. Ryder2 and Rekha Bajoria3,4

1 Department of Obstetrics and Gynaecology, Imperial College School of Medicine, Chelsea and Westminster, 2 Queen Charlotte's and Chelsea Hospital for Women, London and 3 Department of Obstetrics and Gynaecology, University of Manchester, St Mary's Hospital for Women and Children, Whitworth Park, Manchester M13 OJH, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The primary objective of this study was to characterize an in-vitro model of the human placenta using morphological, biochemical and physiological parameters. Placental villi were obtained from normal first trimester and term pregnancies. The villi were incubated with Dulbecco's modified Eagle's medium: Ham's F12 nutrient mixture in a shaking water bath at 37°C for up to 310 min. The viability was determined by the production of ß human chorionic gonadotrophin (HCG) and lactic dehydrogenase (LDH) and the incorporation of [3H]thymidine, [3H]L-leucine and L-[U14C]arginine, while ultrastructure was assessed by transmission electron microscopy. In the first and third trimester group, the release into the medium of the intracellular enzyme LDH remained unaltered throughout the experiment. By contrast, ß-HCG concentrations increased linearly and concentrations were higher in the first trimester than term villi (354.5 ± 37.8 versus 107 ± 8.1 IU/g villi protein; P < 0.001). Electron microscopy confirmed preservation of tissue viability for up to 4 h of incubation. The incorporation of thymidine (12.2 ± 2.9 versus 5.2 ± 0.5 nmol/g villi protein; P < 0.05), leucine (9.4 ± 2.1 versus 1.9 ± 0.4 nmol/g villi protein; P < 0.02) and arginine (17 ± 4.4 versus 4.2 ± 0.5 nmol/g villi protein; P < 0.05) were markedly higher in early than in term placenta. Furthermore, placental uptake of L-leucine by the first (9.4 ± 2.1 versus 17 + 4.4 mol/g villi protein; P < 0.001) and third trimester placental villi (1.9 ± 0.4 versus 4.2 + 0.5 mol/g villi protein; P < 0.001) was less than that of L-arginine. This study describes a simple technique using placental explants to determine relative rates of uptake of substrate amino acids throughout gestation.

Key words: amino acids/electron microscopy/organ culture/placenta/villi


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
With the recent awareness that an adverse intrauterine environment may predispose the fetus to development of disease in adult life, an understanding of the placental function in relation to fetal growth and development is crucial.

Although advances in imaging techniques have made the human fetal circulation accessible, in-vivo studies can only provide data on direct placental transport of a substance at almost undisturbed steady state conditions at various stages of gestation. The main disadvantage of clinical studies, however, is their inability to provide integral information on placental uptake and metabolism, as well as the mechanisms and factors influencing transport of nutrients to the fetus. Ethical considerations as well as fetal safety further restrict invasive research in human pregnancy.

Because of its easy accessibility, isolated human placental tissue has been used widely to overcome some of the restrictions placed on clinical research. Several techniques have been described, ranging from membrane vesicles (Eaton and Oakey, 1997Go), syncytiotrophoblast villous preparations (Palmer et al., 1997Go), first trimester placental villi co-cultured with decidual explants (Babawale et al., 1996Go) and placental slices (Miller and Berndt, 1974Go), to dual perfusion of either the whole organ (Panigel, 1972Go) or a single cotyledon (Schneider, 1991Go). Although experiments performed with isolated placental tissue create an artificial situation, such models can provide answers for those questions which are difficult to obtain in the complex in-vivo set-up.

Of the various systems available, perfusion of the placenta mimics in-vivo conditions closely as it retains the complexity of the intact organ and allows control of experimental variables (Schneider, 1991Go; Bajoria et al., 1996Go). Despite numerous advantages of the placental perfusion model, this experimental system cannot be used to study uptake and transfer function during first trimester pregnancies. Furthermore, it is also not technically feasible to perfuse placentae from pregnancies complicated with systemic lupus, severe pre-eclampsia and fetal growth restriction.

Alternatively, isolated cytotrophoblast cells which can be grown in primary cultures to form multinucleated cells may be used, but they are difficult to purify and have a limited lifespan (Sooranna, 1996Go). Furthermore, they are more removed from the in-vivo situation because of limited cell–cell interactions, and cells isolated from placentae obtained from abnormal pregnancies may revert to `normal' functions when in culture. These preparations do, however, have advantages over perfusion systems in that they allow comparisons of several parameters within one experiment and only small quantities of experimental substances (e.g. radiolabelled substrates) are needed (Bajoria et al., 1997Go). Another possibility is the use of choriocarcinoma cell lines, of which the BeWo line has been the most extensively characterized (Pattillo and Gey, 1968Go; van der Ende et al., 1987Go). Although these will grow very easily in culture, experiments with these should be treated with caution because they are transformed cells.

In order to obviate some of the limitations of the perfusion as well as the cell culture systems, we developed a simple in-vitro model of the human placenta using placental explants. In this paper we characterize the biochemical, morphological and physiological viability of placental villi obtained from early and term placenta.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Methyl [3H]thymidine, [3,4,5–3H(N)]-L-leucine and tissue culture reagents were obtained from Sigma Chemical Co. Ltd, Poole, Dorset, UK and L-[U14C]arginine monochloride from Amersham Life Sciences, Slough, UK. All other laboratory reagents were of the purest grade available from BDH Chemicals Ltd., Poole, Dorset, UK.

Villous preparation
First trimester human placentae between 7–12 weeks were obtained from women undergoing legal abortion. Placentae were also obtained between 37 and 41 weeks gestation following Caesarean section or vaginal deliveries from women with normal pregnancies. Gestational age was determined in all cases by ultrasound scan. From term placentae, 10–15 1 cm3 pieces of tissue were removed at random and placed in cold phosphate buffered saline (PBS) containing 100 units/ml penicillin, 100 mg/ml streptomycin and 100 units/ml nystatin. From termination of pregnancy the whole placenta was placed in PBS. The tissue was washed several times with PBS to remove as much blood as possible. The placental tissue was teased into small pieces, carefully removing any obvious blood vessels or clots. This tissue was re-washed several times with PBS. Approximately 15 ml of settled tissue was made up to 30 ml with Dulbecco's modified Eagle's medium: Ham's F12 nutrient mixture (1:1) containing 1% bovine serum albumin, 50 units/ml penicillin and 50 µg/ml streptomycin. This suspension was swirled to provide a homogeneous preparation prior to sampling for addition to incubation tubes. The incubation was carried out in sterile centrifuge tubes in DMEM: Ham's F12 medium.

Experiment protocol
Forty two experiments were undertaken using either placentae from first trimester (n = 23), or term (n = 19) pregnancies. In the first trimester group, we did six experiments each to study the production of ß human chorionic gonadotrophin (HCG) and lactic dehydrogenase (LDH), or the uptake of L-arginine and L-leucine, while five experiments were performed with thymidine. In the term group, six experiments were done to estimate ß-HCG and LDH, while five incubations were performed with thymidine only. The uptake of L-arginine and L-leucine was studied using six first trimester and five third trimester placentae.

Incubations were performed in a shaking Mickle® water bath (Fission Ltd, Dorset, UK) (70 oscillations/min) at 37°C. When radioactive substances were present the final concentrations of thymidine, leucine and arginine were 1.5 µM (175 000 d.p.m./µmol), 0.42 mM (4x106 d.p.m./µmol) and 0.7 mM (700 000 d.p.m./nmol) respectively. Incubations were carried out in duplicate for up to 310 min. At the end of the incubations, villi from each incubation tube were centrifuged for 5 min at 1420 g. The supernatant was stored for estimation of ß-HCG and LDH concentrations, and the pellet washed three times with cold PBS. After the final wash the pellet was solubilized with 2 ml of 1 M sodium hydroxide overnight at 37°C. The solubilized pellets were then diluted to 10 ml with distilled water and vortex mixed for 1 min; 2.0 ml of scintillant (Packard Ultima gold XR®; Meriden, CT, USA) was added to 2x1 ml aliquots of solubilized pellet and counted on a scintillation counter. Aliquots of the solubilized pellets were also kept for determination of protein concentration by the method of Lowry et al. (1951).

Transmission electron microscopy (TEM)
Samples of placental villi at 0, 4 and 8 h post-incubation were fixed for 1–2 h in 3% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, washed in fresh buffer and post-fixed in 1% osmium tetroxide. Samples were dehydrated through a graded series of alcohol and embedded in araldite epoxy resin. Ultrathin sections were cut perpendicular to the plane of the membrane using a diamond knife. Sections were stained with uranyl acetate and lead citrate before examination in an Hitachi HU 12A transmission electron microscope operated at 50 kV.

Analytical methods
The concentration of ß-HCG was measured by a radioimmunoassay kit purchased from ICN Biochemicals Ltd, Thame, Oxfordshire, UK. The assay had a coefficient of variation of 10–14%. The lower limit of detection was 0.1 IU/ml.

Lactic dehydrogenase activity was measured by a commercially available colorimetry assay kit from Sigma, with a coefficient of variation of 7–12%. The sensitivity of the assay was 1 U/ml.

Data analysis
Values were expressed as mean ± SEM per g of placental villi protein unless otherwise indicated. Data between two groups as a function of time were compared by two-way analysis of variance. One-way analysis of variance was used to compare blocked variables between groups. P values < 0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Concentration of ß-HCG and LDH
The biochemical viability of the placental villi during incubation as assessed by estimation of LDH and ß-HCG concentrations in the extracellular medium are shown in Figure 1Go. As the concentration of LDH in early and term placenta was comparable, data in both groups were pooled together and remained constant during incubation of up to 3.5 h of (6 ± 1.4 at 0 min to 9.3 ± 1.9 at 210 min; n = 12) % total LDH activity. By contrast, ß-HCG concentrations in first trimester placental villi increased markedly from 18.4 ± 5.11 IU/g of villi at 0 min to 354.5 ± 37.8 at 210 min (n = 6). This was markedly greater than those produced by third trimester villi (8.4 ± 2.32 U/g of villi at 0 min to 82.1 ± 10.7 at 210 min; n = 6).



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Figure 1. (A) Release of lactic dehydrogenase (LDH) by placental villi (n = 12). Data from first and third trimester placental villi were pooled together as there was no difference between two groups. (B) Release of ß human chorionic gonadotrophin (HCG) by the first trimester villi (–•–) and term villi (–{circ}–). The data in each group are shown as mean ± SEM of six experiments.

 
Ultrastructure of the tissue
Samples of placental villi were examined at 0, 4 and 8 h of incubation (Figure 2Go). The surface structure of the trophoblast epithelial cells, i.e. microvilli, were not adversely affected by the experimental procedure up until 4 h. The plasma membranes and cytomembrane system of the cells remained intact, and the mitochondria were also unaffected. However, under similar experimental conditions, formation of intracellular vacuoles was noted at 8 h incubation (Figure 2CGo).



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Figure 2. Electron micrograph of the placental villi sampled (A) at 0 h, (B) 4 h and (C) 8 h of incubation. The placental villi at time 4 h shows syncytiotrophoblast (syn) with regular cylindrical microvilli (M). The preservation of the cytoplasmic organelles is similar to that seen at 0 h. The placental villi show well preserved densely stained mitochondria and there is no evidence of intracellular oedema or vacuoles. The villi at 8 h show evidence of intracellular oedema and formation of vacuoles. Bars = 2 µm.

 
Uptake of essential amino acids
First trimester
When placental villi from first trimester were incubated with a known concentration of thymidine, the uptake increased from 2.8 ± 1.0 nmol/g of villi at 0 min to 12.2 ± 2.9 at 120 min (P < 0.001) (Figure 3BGo). L-leucine incorporation into the placental villi increased with time from 0.6 ± 0.2 nmol/g of villi at 0 min to 9.4 ± 2.1 at 180 min (Figure 3CGo). A similar increase in placental incorporation of L-arginine was also found with time (0.9 ± 0.2 nmol/g of villi at 0 min to 17.0 ± 4.4 at 180 min; Figure 3AGo). At each time point, the uptake of L-arginine was greater than L-leucine (P < 0.001).



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Figure 3. Incorporation into first trimester (–•–) and third trimester (–{circ}–) placental villi. (A) 3H-thymidine (n = 5 in each group); (B) L-leucine (n = 6 for first and n = 5 for third trimester; and (C) L-arginine (n = 6 for first trimester and n = 5 for third trimester). (D) compares uptake of leucine ({square}), arginine ({blacksquare}) at 180 min and the thymidine () at 120 min by first trimester with term placentae. The data are shown as mean ± SEM. *P < 0.001.

 
Third trimester
Similar to first trimester data, the uptake of thymidine (1.3 ± 0.2 nmol/g of villi to 5.0 ± 0.5), L-arginine (0.7 ± 0.08 nmol/g of villi to 4.2 ± 0.5), and L-leucine (0.5 ± 0.2 nmol/g of villi to 1.9 ± 0.4) by term placental villi increased with time. At each time point, uptake of L-arginine was greater than L-leucine (P < 0.001).

The uptake of thymidine (P < 0.05), L-leucine (P < 0.02), and L-arginine (P < 0.05) at each sample point by term placenta was significantly less than in the first trimester group (Figure 3DGo).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have developed a simple in-vitro model of the human placenta using short-term placental explants to study the uptake and transport of nutrients by the placenta. In this article we characterized this system using biochemical and morphological parameters. Furthermore, we used this model to study the effect of gestational age on the uptake of a neutral and a cationic amino acid.

Various investigators have used similar systems to study placental function (Ahmed and Murphy, 1988Go; Haning et al., 1988Go; Barnea et al., 1992Go; Polliotti et al., 1995Go). However, no study has systematically characterized this model to evaluate its suitability to determine placental transport of nutrients. Initially, we undertook experiments to optimize the system using various parameters. Viability of the placental tissue was maintained for up to 3.5 h as assessed by measurement of ß-HCG in the culture media. Like in-vivo data, our results also showed an eight-fold increase in ß-HCG production in early pregnancy as compared to term placenta. The documented decrease in the ratio of cytotrophoblast to syncytiotrophoblasts (Kaufmann and Castellucci, 1997Go) may explain the differences between early and late placental ß-HCG production. We also measured LDH concentration during the experimental period as an index of cellular leakage during incubation period. LDH is a cytoplasmic enzyme and minimal alteration in its concentration in the medium further substantiates that placental membrane integrity was preserved during the experiment. At the beginning of the experiment ß-HCG and LDH were present, reflecting the release of these proteins into the medium during the first wash. Similarly values for incorporation of radioactive substrates into tissues were also seen at the start of the experiment.

Cellular function in terms of the incorporation of thymidine, an index of cellular proliferation, was also assessed. [3H]Thymidine was chosen for this purpose because it is only incorporated during the S-phase of the cell cycle and therefore considered to be a reliable proliferative marker (Kaufmann and Castellucci, 1997Go). Our data showing an almost linear increase in cellular incorporation of thymidine over 2 h further confirms cellular viability. The levelling off in incorporation seen is not surprising because the DNA synthetic ability of the trophoblast is limited. The finding of greater incorporation of thymidine by the first trimester as compared to term placenta agrees with the evidence that proliferation and differentiation of placental cells decrease with advancing gestation (Kaufmann and Castellucci, 1997Go).

The viability of the villous tissue during the first 4 h of the experimental procedure was also evaluated. The ultrastructure of the syncytiotrophoblast showed no apparent changes over a 4 h incubation period. The plasma membrane remained intact with regular cylindrical microvilli. There was no evidence of intracellular oedema or damage and mitochondria, in particular, appeared to be well preserved with organized cristae. However, under similar experimental conditions, formation of intracellular vacuoles was noted at 6 and 8 h incubation. Because of these changes we studied uptake by placental villi for no longer than 3 h.

After having established the cellular viability of the system, we then studied the uptake of leucine and arginine by first and third trimester placental villi obtained from normal pregnancies. Leucine was chosen because it is a branched neutral amino acid and its uptake occurs by membrane bound sodium-dependent l transporter (Moe, 1995Go). Placental uptake of arginine was also determined because of its important role in regulation of fetal growth and pre-eclampsia. Furthermore, the uptake of arginine is mediated by a sodium-independent system y+ and y+ l (Moe, 1995Go).

Our data clearly demonstrate that the uptake of both neutral and cationic amino acids by the human placenta decreases with advancing gestational age. This observation is at odds with reports on other placental transport systems such as system A in the human (Mahendran et al., 1994Go) and the rat placenta, and system y+ and y+ l in the rat placenta, all of which increase with gestation (Malandro et al., 1994Go; Novak et al., 1997). Similarly, in relation to glucose transport by the human placenta, there is an increase in GLUT-1 from late second trimester (Jansson et al., 1993Go). However, these data agree with the observation that the concentration of free amino acid in the placental homogenate decreases with gestational age. The decrease in L-arginine uptake with gestational age parallels the changes we have found recently in nitric oxide synthase (Sooranna et al., 1995Go). Moreover, recent evidence that the concentration of L-arginine required for eNOS and iNOS activation are one to two orders of magnitude below the intracellular concentrations of L-arginine (Bogle et al., 1992Go; Myatt et al., 1993Go) suggests that nitric oxide synthesis is dependent upon the extracellular rather than intracellular concentration of L-arginine. It is possible that greater uptake of L-arginine by first trimester villi may be responsible for increased nitric oxide synthesis and hence NOS activity in early pregnancy. Increased nitric oxide synthesis in early gestation therefore may be one of the mechanisms responsible for reduced vascular resistance.

In addition, our data also indicate that for a given gestational age, the uptake of L-arginine was twice as high as that of L-leucine. This discordance in placental uptake of two essential amino acids is not altogether surprising (Rosso, 1975Go). In human pregnancies over a gestational age ranging between 20–37 weeks, placental transport of leucine is significantly more rapid than glycine (Cetin et al., 1995Go). Less is currently known about regulation of amino acid transepithelial flux. We speculate that one of the following mechanisms may be responsible for difference in uptake between leucine and arginine by placental villi.

We speculate that the difference in uptake between leucine and arginine by placental villi could be attributed to the differences in (i) intracellular concentration of the substrate, (ii) rate of protein synthesis, catabolism and production of individual amino acid within the placenta, (iii) placental permeability or affinity of the membrane-bound protein transporter. However, further studies are required to characterize the regulatory mechanism of amino acid uptake by the human placenta.

In summary, our data for the first time suggest that the uptake of leucine and arginine decreases with increasing gestational age. The current approach provides an alternative simple technique to characterize relative rates of uptake of amino acids across the human placenta throughout gestation as well as the ability to determine differences in metabolism between placentae from normal and pathophysiological conditions such as pre-eclampsia and growth restriction. This model has an added advantage that placental uptake functions can be studied in parallel to intracellular amino acid concentration. This is likely to improve our understanding of transepithelial regulatory mechanisms of amino acid flux.


    Notes
 
4 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on July 6, 1998; accepted on October 23, 1998.