This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jauniaux, E. Articles by Burton, G. J. Search for Related Content PubMed PubMed Citation Articles by Jauniaux, E. Articles by Burton, G. J.

Distribution and Transfer Pathways of Antioxidant Molecules inside the First Trimester Human Gestational Sac

Academic Department of Obstetrics and Gynaecology (E.J., J.J.), Royal Free and University College London Medical School, London WC1E 6HX; School of Health and Life Sciences (C.D., F.J.K.), King’s College, London SE1 9NN; and Department of Anatomy (T.C.-D., J.H., G.J.B.), University of Cambridge, Cambridge CB2 3DY, United Kingdom

Address all correspondence and requests for reprints to: Eric Jauniaux, Academic Department of Obstetrics and Gynaecology, University College London Medical School, 86-96 Chenies Mews, London WC1E 6HX, United Kingdom. E-mail: e.jauniaux{at}ucl.ac.uk.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The first-trimester human placenta has limited antioxidant enzyme capacity. We investigated the distribution and transfer pathways of antioxidant molecules inside the first trimester gestational sac. The coelomic fluid of the exocoelomic cavity, which borders the inside of the first-trimester placenta, contained a very low level of reduced glutathione. Glutathione disulfide was undetectable in most coelomic samples, suggesting that the role of glutathione-related detoxification system is limited in fetal fluid compartments. The coelomic fluid contained similar concentrations of ascorbic and uric acid to maternal plasma. The levels of - and -tocopherol were lower in coelomic fluid, compared with maternal plasma. The presence of these molecules inside the early gestational sac suggests that they may play an essential role in the fetal tissues’ antioxidant capacity at a time when the fetus is most vulnerable to oxidative stress. We also demonstrated by immunostaining the presence of -tocopherol transfer protein in the cytoplasm of trophoblastic cells, glandular epithelium of the decidua, and mesothelial layer of the secondary yolk sac. This finding indicates that the uterine glands and the secondary yolk sac play key roles in supplying this essential vitamin to the developing fetus before the placental circulations are established.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
FREE RADICAL SPECIES are constantly formed as a by-product of aerobic respiration and other metabolic reactions. Most of the oxygen (O₂) used during the oxidation of dietary organic molecules is converted into water, but a significant amount (1–2%) is diverted into highly reactive oxygen species (ROS), mainly the superoxide, hydroxyl, peroxyl, and hydroperoxyl anions (1). These metabolites are highly reactive, and consequently antioxidant defenses have evolved to detoxify them. Superoxide dismutase is present in all aerobic cells. It converts superoxide to hydrogen peroxide, which in turn is reduced to water by the antioxidant enzymes catalase and glutathione peroxidase. In addition to the antioxidant enzymes, other molecules such as thiols, ceruloplasmin, transferrin, and dietary vitamins, play a crucial role in tissue defense against free radicals (1). A complex homeostatic balance is thus achieved, and at physiological levels, free radicals regulate a wide variety of cell functions through their influence on the thiol-disulfide redox state (1). If generation of free radicals exceeds the cellular defenses, however, indiscriminate damage can occur to proteins, lipids, and DNA, resulting in oxidative stress.

Human placentation is more complex than that of other mammalian species, including the higher primates. Abnormalities of placentation are associated with diseases that are either unique to the human species, such as preeclampsia or hydatidiform mole, or very rare in other species, such as miscarriage. There is increasing evidence indicating that failure of placentation is associated with an imbalance of free radicals, which will further affect placental development and function and may subsequently have an influence on both the fetus and its mother. Maternal metabolic disorders (e.g. diabetes), which are associated with an increased production of free radicals species, are associated with a higher incidence of miscarriage and fetal structural defects (2). Furthermore, the teratogenicity of drugs such as thalidomide has recently been shown to involve free radical-mediated oxidative damage (3), indicating that the human fetus can be irreversibly damaged by oxidative stress.

The mammalian embryo, and subsequently the fetus, is exposed to major fluctuations in O₂ concentration during pregnancy. The O₂ tension in the oviduct and uterus of most mammalian species has been found to range between 11 and 60 mm Hg, which corresponds to approximately 1 to 9% O₂ (1). These data indicate that the earliest stages of development take place in vivo under a low O₂ concentration, compared with atmospheric conditions of 21% O₂. Ultrasound and anatomical studies have demonstrated that the intervillous circulation starts in the periphery of the placenta at around 9 wk of gestation and that it becomes continuous and diffuse only after 12 wk (4). The PO₂ measured within the human placenta in vivo is less than 20 mm Hg at 7–10 wk gestation (5). It subsequently rises to more than 50 mm Hg at 11–14 wk as the maternal intraplacental circulation becomes fully established. This inevitably leads to an increased production of ROS, and we have observed a burst of oxidative stress inside the placenta between 9 and 10 wk of gestation (5).

The cytotrophoblast shell and plugs, which limit the entry of maternal blood to the placenta during the first trimester (4, 5), will also limit direct exchange of nutrients between the maternal and the fetal circulations, suggesting that alternative nutritional pathways must operate. We have recently shown that histiotrophic pathways may contribute to human fetal nutrition during the first trimester (6). The uterine glands are active during early pregnancy and remain so until at least the 10th week of pregnancy, and their secretions are delivered freely into the placental intervillous space. We have also demonstrated phagocytic uptake by the placental syncytiotrophoblast of two glycoproteins, epithelial mucin MUC-1 and glycodelin A, synthesized in the maternal glands (6). By contrast to the arrangement in most mammalian species, in primates, and in the human in particular, the secondary yolk sac floats within the exocoelomic cavity (ECC) lying between the placenta and the amniotic cavity (Fig. 1). The secondary yolk sac is directly connected to the embryonic gut and possesses a rich vascular plexus at an earlier stage of pregnancy than placental villi. Glycodelin is also detected within the epithelium of the secondary yolk sac lining the exocoelomic cavity, indicating that the yolk sac may play an important role in nutrient exchange before vascularization of the chorionic villi (7, 8, 9). These findings demonstrate that the uterine glands are an important potential source of nutrients during organogenesis when metabolism is essentially anaerobic and the oxygen tension within the fetus must be maintained at a low level for correct cell differentiation.

View larger version (89K): [in this window] [in a new window]   FIG. 1. Diagram of a gestational sac at the end of the second month (8–9 wk) showing the myometrium (M), decidua (D), placenta (P), ECC, amniotic cavity (AC), and secondary yolk sac (SYS). Note the utero-placental blood circulation starting in the periphery of the placenta (arrows).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subjects and samples

We studied 24 series of matched samples of coelomic fluid (CF), amniotic fluid (AF), and maternal plasma at 5–12 wk of gestation. The samples were collected before elective surgical termination of pregnancy under general anesthesia. Gestational age was determined from the first day of the last menstrual period and confirmed by ultrasound measurement of the fetal crown-rump length. Written consent was obtained from each woman after receiving complete information on the procedure. The study included only uncomplicated pregnancies and was approved by The University College London Hospitals Committee on the Ethics of Human Research.

CF (n = 24) and AF (n = 18) samples were obtained by transvaginal puncture under sonographic guidance as described previously (7). AF samples could not be obtained before 8 wk of gestation. The first 0.2 ml of each fluid was discarded to decrease the risk of contamination by maternal blood. In all cases maternal venous blood was obtained during the surgical procedure and placental tissue was collected at the end of the procedure. Samples of maternal plasma and fetal fluids were stored at -80 C until assayed.

Three samples of placental villi and decidual tissue and three intact secondary yolk sacs between 6 and 10 wk of gestation were collected at the end of the surgical procedure and processed for immunohistochemical investigations.

Fluid assays methodology

Determination of total glutathione in fetal fluid was performed using the 5,5'dithiobis-2-2-nitrobenzoic acid-glutathione reductase recycling method based on previously reported methodology (13). Briefly, 50 µl of fluid sample was placed into a well of a 96-well plate and 100 µl of substrate added [0.3 mM nicotinamide adenine dinucleotide phosphate reduced, 0.22 mM 5,5'dithiobis-2-2-nitrobenzoic acid, and 19 U glutathione reductase in 100 mM phosphate buffer (pH 7.5)]. The glutathione content was monitored as an increase in tetrazolium nitroblue color over 2 min at 30 C in a multiplate reader. Reduced glutathione (GSH) in the samples was extracted with 2-vinyl pyridine and the method repeated to measure the glutathione disulfide (GSSG). External standards were run simultaneously.

- and -tocopherol were measured in a plasma using HPLC with UV detection. Then 100 µl of thawed plasma was mixed with 5 µg internal standard (-tocopherol acetate) in 100 µl ethanol. Ice-cold HPLC grade hexane (400 µl) was added to the plasma, and samples were vortexed for 2 x 40 sec. After centrifugation at 3000 rpm for 5 min at 4 C, the hexane layer was carefully removed into 0.8 ml HPLC vial and evaporated to dryness under a stream of nitrogen. The extract was then redissolved into 400 µl HPLC-grade methanol and vortexed for 2 x 40 sec. Aliquots of 100 µl were injected onto HPLC with UV detector. Final concentrations for - and -tocopherol were calculated with external standards and adjusted for recoveries with the internal vitamin E standard (14). For fetal fluid samples, 100 pmol deuterated -tocopherol internal standard was added to a 1-ml sample followed by vortexing. Then 50% sodium dodecyl sulfate was added 1:1, the sample was vortexed, 3 ml of ethanol was added, and the tocopherols extracted into hexane after 10 min of inversion. After centrifugation at 3000 rpm for 10 min at 10 C, the hexane layer was transferred into a glass vial and evaporated to dryness under a stream of nitrogen. The dried residue was then silylated in the presence of 100 µl anhydrous pyridine and 50 µl bistrimethylsilyltrifluoroacetamide containing 1% trimethylchlorosilane for 1 h at 65 C. Vials were removed from the heat, allowed to cool, and evaporated to dryness under a stream of nitrogen. Once dry, 100 µl gas chromatography (GC)-grade octane was added to the vial, and this was capped and vortexed for 40 sec twice over a 20-min period. The octane was transferred to GC vials and capped, and 1 µl was injected onto GC with a mass spectrometry detector. Final concentrations for - and -tocopherol were calculated with external standards and adjusted for recoveries with the internal deuterated vitamin E standard.

HPLC was performed to measure uric acid and ascorbic acid (vitamin C) in maternal plasma and fetal fluids. Plasma samples were acidified 1:1 with ice-cold 10% metaphosphoric acid, vortexed, and diluted 2:3 with ice-cold 5% metaphosphoric acid so that the final dilution of the plasma was 1:5. The fetal fluid samples were diluted 2:3, 100 µl HPLC-grade heptane were added, and samples were vigorously vortexed 40 sec. The samples were subsequently centrifuged at 13,000 x g for 5 min and the lower (aqueous) layer removed and treated with heptane again until the supernatant was clear. The clear supernatant was transferred to a 0.8 ml HPLC vial. Dehydroascorbic acid (DHA) was calculated from measuring the total vitamin C in samples incubated with dithioreitol before acidification. Aliquots of 20 µl were injected onto a HPLC with electrochemical detector, working electrode set at 400 mV, and sensitivity of 1 µA. Final concentrations for ascorbic acid and uric acid were calculated with external standards, which were run simultaneously (15).

Immunofluorescent staining

Samples of three placental villi and decidua were fixed for immunohistochemistry in 4% formaldehyde and embedded in paraffin wax. Sections were cut at 5–7 µm, dewaxed in xylene, and rehydrated in a graded series of ethanol. Sections were subjected to antigen retrieval by proteinase K (20 µg/ml for 30 min), permeabilized in Tris-buffered saline containing Triton X-100 (0.1%) and Tween 20 (0.1%) (TBS-TT) for 30–60 min, and blocked in 5% goat serum for 30 min at room temperature. Excess serum was wiped away and a mixture of rabbit polyclonal antitocopherol transfer protein (TTP) antibody (used 1/300; kind donation from Dr Kaempf-Rotzoll) and mouse monoclonal anticytokeratin 7 antibody (used 1/100; from Dako, Ely, UK) diluted in TBS-TT was applied, and sections were incubated overnight at 4 C. Negative control sections were left at the blocking stage and not covered with primary antibodies. After three 10-min washes in TBS-TT, sections were incubated for 1 h at room temperature with a mixture of fluorescent secondary antibodies, containing goat antirabbit Alexa 488 and goat antimouse Alexa 568 (both used 1/200; from Molecular Probes, Eugene, OR) in TBS-TT. Sections were washed in TBS-TT as before and then twice in distilled water for 5 min and subsequently mounted in Vectashield mounting medium containing 4,6-diamidino-2-phenylindoladihydrochloride (Vector, Peterborough, UK). The yolk sacs were frozen in cryoembedding medium. Sections were cut with Reichert cryomicrotome (at 10–12 µm), air dried, and fixed briefly in cold methanol/acetone and permeabilized in TBS-TT for 30–60 min. All subsequent immunolabeling steps were carried out as with the paraffin-embedded sections. Images were captured using a Leica confocal microscope (LeicaTCS-NT, Leica Instruments GmbH, Wetzlar, Germany).

Statistical analysis

A biomedical data-processing statistical package (Statgraphics, Manugistics, Rockville, MD) was used for the analysis, and data are presented as mean and SEM. Differences in mean of fetal fluids and maternal values were tested using the ANOVA, the multiple range tests, and the Fisher’s least significant difference procedure. Individual linear regression between fetal fluid values and gestational age and between fetal fluid and maternal plasma values were calculated by the least squares method, and their slopes tested for significance by the F ratio test. Multiple linear regression analysis was performed to evaluate the relations between the CF and maternal plasma values and gestational age. Results were considered statistically significant at P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fluid analysis

Table 1 presents the mean level of the different antioxidant molecules measured in maternal plasma and fetal fluids. The mean level of GSH was significantly (P <0.001) lower in CF than in maternal plasma. GSSG was detected in only four coelomic samples (mean = 0.04 µmol/liter; SEM = 0.02 µmole/liter). GSH and GSSG were not detectable in amniotic fluid. There were significantly (P < 0.001) higher levels of -tocopherol in CF than in AF and significantly (P < 0.001) lower levels in CF than in maternal plasma. The mean -tocopherol level was significantly (P < 0.001) lower in CF than in maternal plasma, and this vitamin was not detectable in AF samples. The mean ascorbic acid level was significantly (P < 0.05) higher in CF than in AF but was similar in CF and maternal plasma, whereas the mean level of DHA was similar in CF and AF but significantly lower (P < 0.01) in CF than in maternal plasma. There were significantly (P < 0.001) higher concentrations of uric acid in CF than in AF and no difference between CF and maternal plasma.

Immunofluorescent staining

Fluorescent immunolabeling for TTP and cytokeratin 7 was used to identify TTP-positive cells in the first-trimester villi, decidua, and secondary yolk sac. In the placental villi, the TTP antibody labeled the trophoblastic covering, whereas the stroma and fetal capillaries showed little or no staining (Fig. 2a). Both the syncytiotrophoblast and cytotrophoblast cells appeared to be immunoreactive, and this was confirmed using other sections visualized colorimetrically (Fig. 3). The cytoplasm of both cell types stained uniformly, but whereas all cytotrophoblastic nuclei were stained, only some of the syncytial nuclei reacted positively. The TTP antibody also strongly labeled the cell columns formed by invading extravillous trophoblast cells (not shown). These cells also stained with cytokeratin 7.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Immunofluorescent staining with TTP antibody (green) and cytokeratin 7 antibody (red) in the syncytiotrophoblast and cytotrophoblast cells of first-trimester placenta (8 wk; scale bar, 30 µm) (a); the glandular epithelium of first-trimester decidua (8 wk; scale bar, 20 µm) (b); and the mesothelial layer of the secondary yolk sac (8 wk; scale bar, 20 µm) (c). Nuclei are stained blue.

View larger version (146K): [in this window] [in a new window]   FIG. 3. A high-power photomicrograph of an 8-wk villus in which TTP binding was visualized using diaminobenzidine. The cytoplasm of both the syncytiotrophoblast (arrows) and the cytotrophoblast cells (arrowheads) stained uniformly. The nuclei of the cytotrophoblast cells were also immunoreactive, but the syncytiotrophoblastic nuclei appeared less so.

TTP staining was also apparent in the yolk sac. The TTP antibody labeled the cytoplasm of the external mesothelial layer, which also stained with cytokeratin 7 (Fig. 2c). The endoderm, mesenchyme, blood islands, and ducts did not show TTP labeling. Negative control sections, which were not exposed to any primary antibodies, showed no fluorescent staining (not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of our investigation indicate that antioxidant molecules are transferred into the exocoelomic cavity and subsequently into the fetal gut and circulation via the secondary yolk sac.

The ECC is the largest anatomical space inside the gestational sac between 5 and 9 wk of gestation, and it contains no oxygen transport system (8). There is no anatomical barrier between the mesenchyme of the placental fetal plate and the ECC. Protein electrophoresis has shown that the CF results from an ultrafiltrate of maternal serum with the addition of specific placental and secondary yolk sac bioproducts (8). The higher concentrations of human chorionic gonadotrophin, estradiol, oestriol, and progesterone in the CF than in maternal serum strongly suggest the presence of a direct pathway between the trophoblast and the ECC. Morphologically, this may be via the villous stromal channels and the loose mesenchymal tissue of the chorionic plate. These findings suggest that the ECC is a physiologic liquid extension of the early placenta and an important interface in fetal nutritional pathways.

The glutathione peroxidase enzyme and glutathione S-transferase play an important role in the detoxification of numerous toxic compounds including xenobiotics, carcinogens, and ROS (16). Glutathione is the major cellular thiol redox buffer in cells and is synthesized in the cytosol from L-glutamate, L-cysteine, and glycine by the sequential action of -glutamylcysteine synthetase and glutathione synthetase, of which the former is rate limiting (17). GSH levels in erythrocytes are as high as 1000 µmol/liter (17), whereas in plasma GSH levels are usually less than 20 µmol/liter (18). Levels of 50 µmol/liter or more have been reported in cord blood at term (19, 20). L-Glutamate, L-cysteine, and glycine are found in higher concentrations in CF than in maternal plasma (21). The ECC contains a very low concentration of enzymes except of -glutamyl-transpeptidase (22), which is the only peptidase that can cleave the -glutamyl bound of GSH. This can explain the very low mean GSH level that we found in the CF samples. Together with the absence of detectable level of GSSG in most CF samples and in all AF samples, this suggests that the role of the glutathione-related detoxification system is limited in the fetal fluid compartments.

Vitamins E and C have been shown to function as antioxidant in various settings, but their rate and mechanisms of transport by the human placenta remain uncertain. The transport of these molecules across the human placenta has been studied directly using the isolated cotyledons system in vitro (23, 24) and by comparing fetal and maternal level around term (25, 26, 27, 28, 29, 30) or indirectly by dietary supplements to diabetic pregnant rats (31, 32, 33).

Vitamin E is a generic term referring to all tocol compounds exhibiting the biological activity of -tocopherol. Vitamin E is an essential lipid component of biological membranes, which is known to interact with peroxyl radicals and interrupts the propagative reaction of the peroxidative process (34, 35, 36). Vitamin E also has a role in the modulation of cellular signaling and immune function, gene regulation, and induction of apoptosis (36). -Tocopherol is the most prevalent form in the Western diet, whereas -tocopherol is the form most commonly artificially supplemented (36). After absorption by the maternal gut and uptake by the maternal liver, vitamin E is secreted into very low-density proteins and carried to various tissues including the placenta (37). Fetal blood levels of vitamin E are lower than in maternal blood (28, 29, 30). In this study, we found very low levels of both - and -tocopherol in CF, compared with maternal plasma (Table 1). Vitamin E is poorly soluble in hydrophilic milieu of plasma, extracellular fluids, and cytosol (36). Very low lipoprotein concentrations in fetal blood (29) and fluids (8) can also partially explain the discrepancy between the maternal and fetal compartments.

Both a 15-kDa cytosolic -tocopherol binding protein and the 30-kDa transfer protein (-TTP) have previously been identified in the cytosol of the human placental syncytiotrophoblast and extravillous trophoblast cells (38, 39). Unlike liver tissue, placental tissue does not break down the acetate esters of -tocopherol to any extent, and so the free form, not the acetate, is preferentially transferred (23). Adequate fetal tocopherol levels before delivery and fetal tocopherol supply are largely maintained by -TTP in the trophoblastic cells of the human placenta (39). In later pregnancy the source of the tocopherol transported will be the maternal blood perfusing the placenta. However, in early pregnancy there is only a very limited maternal blood flow through the intervillous space (4, 5, 6). In the present study, we report the identification of -TTP in the secretory epithelium of the uterine glands during the first trimester. The secretions from these glands may thus represent an alternative source of the vitamin, for they are delivered into the intervillous space of the placenta in which they diffuse between the villi. We also extend the observations of Kaempf-Rotzoll et al. (39) and report the localization of -TTP in the syncytiotrophoblast and cytotrophoblast cells of first trimester villi. In addition, we localized -TTP to the mesothelial layer of the secondary yolk sac. Morphologically, the yolk sac mesothelial layer resembles an absorptive epithelium because it has a well-developed microvillous border (7). It serves as the interface between the coelomic fluid and the vitelline capillaries, which reach the embryo via the vitelline duct. Although kinetic studies are not yet available, these findings strongly suggest that the uterine glands and the secondary yolk sac play key roles in supplying this essential molecule to the developing fetus before the chorioallantoic and uteroplacental circulations are established.

Vitamin C is a hydrophilic molecule, which exists in three forms: ascorbic acid, DHA, and diketogulonic acid. Like vitamin E, vitamin C is not synthesized by primates and so must be constantly absorbed from the diet (40). Vitamin C protects against the oxidative degradation of collagen and proteins and lipid peroxidation by directly scavenging ROS. In addition, vitamin C can recycle -tocopherol from the tocopheroxyl free radical generated in lipid bilayers, so increasing its effectiveness (40). Unlike vitamin E, vitamin C is present in higher concentration in the fetal than in the maternal circulation (23, 26, 30). Vitamin C is transferred into the human placenta as DHA, possibly using the glucose carrier (23). The mechanism of transport from the intracellular trophoblastic pool to the fetal circulation is unknown. We found similar ascorbic acid and lower DHA levels in CF than in maternal plasma, suggesting that the transport of vitamin C to the fetal compartments starts as early as the first month of pregnancy.

A potentially important source of superoxide is the enzyme xanthine dehydrogenase/oxidase, which is present in the placenta (41). Under normal conditions the enzyme exists in the dehydrogenase form and degrades purines, xanthine, and hypoxanthine to uric acid, using nicotinamide adenine dinucleotide⁺ as the electron recipient. Uric acid has the ability to neutralize the oxidative properties of peroxynitrite in the presence of free iron ions (42, 43). Maternal and neonatal uric acid levels are similar at birth and are strongly correlated, suggesting a free transfer of uric acid through the human placenta in both directions (44, 45). Our data showing similar maternal and CF uric acid levels and a strong correlation between the two compartments support this concept. The trophoblastic activity of superoxide dismutase and the other antioxidant enzymes is limited during the first 2 months of pregnancy (5, 46, 47), and during that period the placenta degenerates rapidly when cultured under conventional normoxic (21%) conditions (48). These findings suggest that uric acid may be an essential antioxidant molecule inside the first-trimester gestational sac.

There are no correlations between the ECC and maternal concentrations of most proteins and in particular of prealbumin, suggesting that the CF composition is not directly influenced by maternal protein intake (49). However, the CF levels of smaller molecules such as some amino acids (21) and thyroid hormones (50), which are not synthesized by fetal tissues in early pregnancy, are correlated with the corresponding maternal levels. We found relationships between maternal and CF -tocopherol levels. Similar correlations have been found between maternal and fetal blood -tocopherol levels at term (28, 29, 30), suggesting that vitamin E supplementation could be beneficial from conception onward in reducing the incidence of fetal malformations in diabetic mothers.

Most vertebrates have evolved complex mechanisms to protect the tissues of their developing offspring from possible damage caused by the actions of free radicals. The anatomical features of the first-trimester human gestational sac provide indirect evidence that this architecture limits fetal exposure to O₂ to what is strictly necessary for its development (51). This may serve to protect the fetal tissues from damage by O₂ free radicals and prevent disruption of signaling pathways during the crucial stages of embryogenesis and organogenesis. The presence in the ECC of molecules with a well-established antioxidant role such as vitamin C, vitamin E, and uric acid support this concept.

	Acknowledgments

 
We thank Dr. Daisy Kaempf-Rotzoll (Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan) for providing the -TTP antibody and Ms. Angela Scott (UCL illustration center) for her assistance in preparing Fig. 1.

	Footnotes

 
This work was supported by a grant from Wellbeing.

Abbreviations: AF, Amniotic fluid; CF, coelomic fluid; DHA, dehydroascorbic acid; ECC, exocoelomic cavity; GC, gas chromatography; GSH, reduced glutathione; GSSG, glutathione disulfide; O₂, oxygen; ROS, reactive oxygen species; TBS-TT, Tris-buffered saline containing Triton X-100 and Tween 20; TTP, tocopherol transfer protein.

Received July 31, 2003.

Accepted December 1, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Burton GJ, Hempstock J, Jauniaux E 2003 Oxygen, early embryonic metabolism and radical mediated embryopathies. Reprod Biomed Online 6:84–96[Medline]
2. Wiznitzer A, Furman B, Mazor M, Reece EA 1999 The role of prostanoids in the development of diabetic embryopathy. Sem Reprod Endocrinol 17:175–181[Medline]
3. Parman T, Wiley MJ, Wells PG 1999 Free radical-mediated oxidative DNA damage in the mechanism of thalidomide teratogenicity. Nat Med 5:582–585[CrossRef][Medline]
4. Jauniaux E, Hempstock J, Greenwold N, Burton GJ 2003 Trophoblastic oxidative stress in relation to temporal and regional differences in maternal placental blood flow in normal and abnormal early pregnancy. Am J Pathol 162:115–125[Abstract/Free Full Text]
5. Jauniaux E, Watson AL, Hempstock J, Bao Y-P, Skepper JN, Burton GJ 2000 Onset of placental blood flow and trophoblastic oxidative stress: a possible factor in human early pregnancy failure. Am J Pathol 157:2111–2122[Abstract/Free Full Text]
6. Burton GJ, Watson AL, Hempstock J, Skepper JN, Jauniaux E 2002 Uterine glands provide histiotrophic nutrition for the human fetus during the first trimester of pregnancy. J Clin Endocrinol Metab 87:2954–2959[Abstract/Free Full Text]
7. Jones CPJ, Jauniaux E 1995 Ultrastructure of the materno-embryonic interface in the first trimester of pregnancy. Micron 2:145–173
8. Jauniaux E, Gulbis B 2000 Fluid compartments of the embryonic environment. Hum Reprod Update 6:268–278[Abstract/Free Full Text]
9. Gulbis B, Jauniaux E, Cotton F, Stordeur P 1998 Protein and enzyme pattern in fluid cavities of the first trimester gestational sac: relevance to the absorptive role of the secondary yolk sac. Mol Hum Reprod 4:857–862[Abstract/Free Full Text]
10. Therond P, Auger J, Legrand A, Jouannet P 1996 -Tocopherol in human spermatozoa and seminal plasma: relationship with motility, antioxidant enzymes and leucocytes. Mol Hum Reprod 2:739–744[Abstract/Free Full Text]
11. Raijmakers MT, Roelofs HM, Steegers EA, Steegers-Theunissen RP, Mulder TP, Knapen MF, Wong WY, Peeters WH 2003 Glutathione and glutathione transferase A1-1 and P1-1 in seminal plasma may play a role in the protection against oxidative damage to spermatozoa. Fertil Steril 79:162–172
12. Schwigert FJ, Steinhagen B, Raila J, Siemann A, Peet D, Busher U 2003 Concentrations of carotenoids, retinol and -tocopherol in plasma and follicular fluid of women undergoing IVF. Hum Reprod 18:1259–1264[Abstract/Free Full Text]
13. Baker MA, Cerniglia CJ, Zaman A 1990 Microtiter plate assay for the measurements of glutathione and glutathione disulfide in large numbers of biological samples. Anal Biochem 190:360–365[CrossRef][Medline]
14. Kelly FJ, Rodgers W, Handel J, Smith S, Hall MA 1990 Time course of vitamin E repletion in the premature infant. Br J Nutr 63:631–638[CrossRef][Medline]
15. Iriyama K, Yoshiura M, Iwamoto T, Ozaki Y 1984 Simultaneous determination of uric and ascorbic acids in human serum by reversed-phase high performance liquid chromatography with electrochemical detection. Anal Biochem 141:238–243[CrossRef][Medline]
16. Griffiths OW 1999 Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic Biol Med 27:922–935[CrossRef][Medline]
17. Knapen MFCM, Zusterzeel PLM, Peters WHM, Steegers EAP 1999 Glutathione and glutathione-related enzymes in reproduction. A review. Eur J Obstet Gynecol Reprod Biol 82:171–184[CrossRef][Medline]
18. Andersen ME, Meister A 1980 Dynamic state of glutathione in blood plasma. J Biol Chem 255:9530–9533[Abstract/Free Full Text]
19. Qanungo S, Sen A, Mukherjea M 1999 Antioxidant status and lipid peroxidation in human feto-placental unit. Clin Chim Acta 285:1–12[CrossRef][Medline]
20. Vento M, Asensi M, Sastre J, Lloret A, Garcia-Sala F, Vina J 2003 Oxidative stress in asphyxiated term infants resuscitated with 100% oxygen. J Pediatr 142:240–246[CrossRef][Medline]
21. Jauniaux E, Gulbis B, Gerlo E, Rodeck CH 1998 Free amino acid distribution in the first trimester human gestational sac. Early Hum Dev 51:159–169[CrossRef][Medline]
22. Jauniaux E, Gulbis B, Hyett J, Nicolaides KH 1998 Biochemical analyses of mesenchymal fluid in early pregnancy. Am J Obstet Gynecol 178:765–769[CrossRef][Medline]
23. Rybakowski C, Mohar B, Wohlers S, Leichtweiss HP, Schroder H 1995 The transport of vitamin C in the isolated human near-term placenta. Eur J Obstet Gynecol Reprod Biol 62:107–114[CrossRef][Medline]
24. Schenker S, Yang Y, Perez A, Acuff RV, Papas AM, Henderson G, Lee MP 1998 Antioxidant transport by the human placenta. Clin Nutr 17:159–167[CrossRef][Medline]
25. Cachia O, Leger CL, Boulot P, Vernet MH, Michel F, Crastes de Paulet A, Descomps B 1995 Red blood cell vitamin E concentrations in fetuses are related to but lower than those in mothers during gestation. Am J Obstet Gynecol 173:42–51[CrossRef][Medline]
26. Guajardo I, Beharry KD, Modanlou HD, Aranda JV 1995 Ascorbic acid concentrations in umbilical cord veins and arteries of preterm and term newborns. Biol Neonate 68:1–9[Medline]
27. Leger CL, Dumontier C, Fouret G, Boulot P, Descomps B 1998 A short-term supplementation of pregnant women before delivery does not improve significantly the vitamin E status of neonates: low efficiency of the vitamin E placental transfer. Int J Vitam Nutr Res 68:293–299[Medline]
28. Oostenbrug GS, Mensink RP, van Houwelingen AC, Hornstra G 1998 Maternal and neonatal plasma antioxidant levels in normal pregnancy, and the relationship with fatty acid unsaturation. Br J Nutr 80:67–73[Medline]
29. Kiely M, Cogan PF, Kearney PJ, Morrissey PA 1999 Concentrations of tocopherols and carotenoids in maternal and cord blood plasma. Eur J Clin Nutr 53:711–715[CrossRef][Medline]
30. Baydas G, Karatas F, Gursu MF, Bozkurt HA, Ilhan N, Yasar A, Canatan H 2002 Antioxidant vitamin levels in term and preterm infants and their relation to maternal vitamin status. Arch Med Res 33:276–280[CrossRef][Medline]
31. Sivan E, Reece EA, Wu YK, Homko CJ, Polansky M, Borenstein M 1996 Dietary vitamin E prophylaxis and diabetic embryopathy: morphology and biochemical analysis. Am J Obstet Gynecol 175:793–799[CrossRef][Medline]
32. Siman CM, Gittenberger-DeGroot AC, Wisse B, Eriksson UJ 2000 Malformations in offspring of diabetic rats: morphometric analysis of neural crest-derived organs and effects of maternal vitamin E treatment. Teratology 61:355–367[CrossRef][Medline]
33. Cederberg J, Siman CM, Ericksson UJ 2001 Combined treatment with vitamin E and vitamin C decreases oxidative stress and improves fetal outcome in experimental diabetic pregnancy. Pediatr Res 49:755–762[Medline]
34. Burton GW, Traber MG 1990 Vitamin E: antioxidant activity, biokinetics, and bioavailability. Annu Rev Nutr 10:357–382[CrossRef][Medline]
35. Wagner BA, Buettner GR, Burns CP 1996 Vitamin E slows the rate of free radical-mediated lipid peroxidation in cells. Arch Biochem Biophys 334:261–267[CrossRef][Medline]
36. Brigelius-Flohe R, Kelly FJ, Salonen JT, Neuzil J, Zingg JM, Azzi A 2002 The European perspective on vitamin E: current knowledge and future research. Am J Clin Nutr 76:703–716[Abstract/Free Full Text]
37. Traber MG, Cohn W, Muller DPR 1992 Absorption transport and delivery to tissues. In: Parker L, Fuchs J, eds. Vitamin E in health and disease. New York: Marcel Dekker; 35–51
38. Gordon MJ, Campbell FM, Dutta-Roy AK 1996 -Tocopherol-binding protein in cytosol of the human placenta. Biochem Soc Trans 24:202S[Medline]
39. Kaempf-Rotzoll DE, Horiguchi M, Hashiguchi K, Aoki J, Tamai H, Linderkamp O, Arai H 2003 Human placental trophoblast cells express -tocopherol transfer protein. Placenta 24:439–444[CrossRef][Medline]
40. Nandi A, Mukhopadhyay CK, Ghosh MK, Chattopadhyay DJ, Chatterjee IB 1997 Evolutionary significance of vitamin C biosynthesis in terrestrial vertebrates. Free Radic Biol Med 22:1047–1054[CrossRef][Medline]
41. Many A, Westerhausen-Larson A, Kanbour-Shakir A, Roberts JM 1996 Xanthine oxidase/dehydrogenase is present in human placenta. Placenta 17:361–365[CrossRef][Medline]
42. Spitsin SV, Scott GS, Mikheeva T, Zborek A, Kean RB, Brimer CM, Koprowski H, Hooper DC 2002 Comparison of uric acid and ascorbic acid in protection against EAE. Free Radic Biol Med 33:1363–1371[CrossRef][Medline]
43. Teng RJ, Ye YZ, Parks DA, Beckman JS 2002 Urate produced during hypoxia protects heart proteins from peroxynitrate-mediated protein nitration. Free Radic Biol Med 33:1243–1249[CrossRef][Medline]
44. Chang FM, Chow SN, Huang HC, Hsieh FJ, Chen HY, Lee TY, Ouyang PC, Chen YP 1987 The placental transfer and concentration difference in maternal and neonatal serum uric acid at parturition: comparison of normal pregnancies and gestosis. Biol Res Pregnancy Perinatol 8:35–39[Medline]
45. Kiely M, Morrissey PA, Cogan PF, Kearney PJ 1999 Low molecular weight plasma antioxidants and lipid peroxidation in maternal and cord blood. Eur J Clin Nutr 53:861–864[CrossRef][Medline]
46. Watson AL, Palmer ME, Jauniaux E, Burton GJ 1997 Variations in expression of copper/zinc superoxide dismutase in villous trophoblast of the human placenta with gestational age. Placenta 18:295–299[CrossRef][Medline]
47. Watson AL, Skepper JN, Jauniaux E, Burton GJ 1998 Changes in the concentration, localisation and activity of catalase within the human placenta during early gestation. Placenta 19:27–34[CrossRef][Medline]
48. Watson AL, Palmer ME, Skepper JN, Jauniaux E, Burton GJ 1998 Susceptibility of human placental syncytiotrophoblast mitochondria to oxygen-mediated damage in relation to gestational age. J Clin Endocrinol Metab 83:1697–1705[Abstract/Free Full Text]
49. Jauniaux E, Gulbis B, Jurkovic D, Campbell S, Collins WP, Ooms HA 1994 Relationship between protein levels in embryological fluids and maternal serum and yolk sac size during early human pregnancy. Hum Reprod 9:161–166[Abstract/Free Full Text]
50. Calvo RM, Jauniaux E, Gulbis B, Asuncion M, Gervy C, Contempre B, Morreale de Escobar M 2002 Fetal tissues are exposed to biologically relevant free tryroxine concentrations during early phases of development. J Clin Endocrinol Metab 87:1768–1777[Abstract/Free Full Text]
51. Jauniaux E, Gulbis B, Burton GJ 2003 The human first trimester gestational sac limits rather than facilities oxygen transfer to the foetus: a review. Placenta 24:S86–S93

This article has been cited by other articles:

				I. Cetin, C. Berti, and S. Calabrese Role of micronutrients in the periconceptional period Hum. Reprod. Update, June 30, 2009; (2009) dmp025v1. [Abstract] [Full Text] [PDF]

				H. Dassen, R. Kamps, C. Punyadeera, F. Dijcks, A. de Goeij, A. Ederveen, G. Dunselman, and P. Groothuis Haemoglobin expression in human endometrium Hum. Reprod., March 1, 2008; 23(3): 635 - 641. [Abstract] [Full Text] [PDF]

				Y. Hagos, D. Stein, B. Ugele, G. Burckhardt, and A. Bahn Human Renal Organic Anion Transporter 4 Operates as an Asymmetric Urate Transporter J. Am. Soc. Nephrol., February 1, 2007; 18(2): 430 - 439. [Abstract] [Full Text] [PDF]

				C. Biondi, B. Pavan, A. Dalpiaz, S. Medici, L. Lunghi, and F. Vesce Expression and characterization of vitamin C transporter in the human trophoblast cell line HTR-8/SVneo: effect of steroids, flavonoids and NSAIDs Mol. Hum. Reprod., January 1, 2007; 13(1): 77 - 83. [Abstract] [Full Text] [PDF]

				M. A. Underwood and M. P. Sherman Nutritional Characteristics of Amniotic Fluid NeoReviews, June 1, 2006; 7(6): e310 - e316. [Full Text] [PDF]

				E. Jauniaux, J. Hempstock, C. Teng, F. C. Battaglia, and G. J. Burton Polyol Concentrations in the Fluid Compartments of the Human Conceptus during the First Trimester of Pregnancy: Maintenance of Redox Potential in a Low Oxygen Environment J. Clin. Endocrinol. Metab., February 1, 2005; 90(2): 1171 - 1175. [Abstract] [Full Text] [PDF]

				G. J. Burton and E. Jauniaux Placental Oxidative Stress: From Miscarriage to Preeclampsia Reproductive Sciences, September 1, 2004; 11(6): 342 - 352. [Abstract] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jauniaux, E. Articles by Burton, G. J. Search for Related Content PubMed PubMed Citation Articles by Jauniaux, E. Articles by Burton, G. J.