Amniotic fluid 5-hydroxyeicosatetraenoic acid in preterm labor

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonate lipoxygenase product capable of stimulating human uterine contractility in a dose-dependent manner in vitro. The purpose of this study was to determine if preterm labor is associated with changes in the concentration of this metabolite in amniotic fluid. Amniotic fluid was obtained by transabdominal amniocentesis from three groups of women with preterm labor: group 1 - women without intraamniotic infection who responded to tocolysis (n = 32); group 2 - women without intraamniotic infection who failed to respond to tocolysis (n = 22); and group 3 - women with intraamniotic infection (n = 14). 5-HETE was determined by radioimmunoassay. The median amniotic fluid concentration of 5-HETE in women who responded to tocolysis (median = 1412 pg/ml; range: 111-3547) was significantly lower than in women who delivered despite tocolysis (median = 2052 pg/ml; range: 136-7774) and women with intraamniotic infection (median = 1876 pg/ml; range: 543-7033) [p less than 0.05]). No difference in amniotic fluid concentrations' of 5-HETE were found between women in groups 2 and 3 (p greater than 0.05).     

Amniotic fluid 5-hydroxyeicosatetraenoic acid in preterm labor

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonate lipoxygenase product capable of stimulating human uterine contractility in a dose-dependent manner in vitro. The purpose of this study was to determine if preterm labor is associated with changes in the concentration of this metabolite in amniotic acid. Amniotic fluid was obtained by transabdominal amniocentesis from three groups of women with preterm labor: group 1 — women without intraamniotic infection who responded to tocolysis (n = 32); group 2 — women without intraamniotic infection who failed to respond to tocolysis (n = 22); and group 3 — women with intraamniotic infection (n = 14). 5-HETE was determined by radioimmunoassay. The median amniotic fluid concentration of 5-HETE in women who responded to tocolysis (median = 1412 pg/ml; range: 111–3547) was significantly lower than in women who delivered despite tocolysis (median = 2052 pg/ml; range 136–7774) and women with intraamniotic infection (median = 1876 pg/ml; range: 543–7033) [p < 0.05]). No difference in amniotic fluid concentrations' of 5-HETE were found between women in groups 2 and 3 (p > 0.05).     

5-Hydroxyeicosatetraenoic acid and human parturition

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonic acid (AA) metabolite derived from the lipoxygenase pathway which is capable of inducing uterine contractions. The purpose of this study was to determine a). whether 5-HETE concentrations in amniotic fluid increase before or after the onset of labor and b). whether acetylsalicylic acid (ASA) could modulate the production of 5-HETE by human amnion cells. 5-HETE concentrations are increased in amniotic fluid before the onset of labor. Furthermore, ASA treatment as expected inhibited PGE₂, but also significantly increased 5-HETE production by amnion cells. 5-HETE concentrations on average increased by greater than 2.5 fold (p < 0.001) in amniotic fluid prior to spontaneous labor when compared with samples obtained from the same patients earlier in gestation and therefore may be important in mechanisms regulating the onset of labor. ASA provokes an increase in 5-HETE biosynthesis by amnion cells: control media 2.60 ± 1.5, ASA treatment alone 5.17 ± 0.20, IL-1β alone 6.39 ± 2.1, and ASA + IL-1β 8.95 ± 1.2 (mean ± SEM) picograms per microgram protein per 16 hours. These findings may explain in part why cyclooxygenase inhibitors are not always successful in treating women with preterm labor.     

Amniotic fluid lipoxygenase metabolites during spontaneous labor and after RU486 treatment during late pregnancy in rhesus macaques

To determine changes in amniotic fluid (AF) lipoxygenase metabolites prior to spontaneous labor and after RU486 administration, we implanted AF and vascular catheters and myometrial electromyographic (EMG) electrodes in 8 rhesus macaques at 120-130 days of pregnancy (term = 167 days). Four animals had AF samples taken serially until they delivered their infants normally at term. The other four animals received RU486 (20 mg/kg/day) for 3 days. AF samples were collected every 2-3 days and at 12 hour intervals for 72 hours before and after treatment with RU486. Uterine activity was monitored continuously. LTB4, 5-HETE and 15-HETE were measured by radioimmunoassay. In untreated animals, LTB4 and 5-HETE concentrations in AF increased significantly (P less than 0.05) 4 days before delivery with no change in 15-HETE. After RU486, mean levels of LTB4 and 5-HETE were increased although the difference was not statistically significant. No change in 15-HETE levels was observed. In conclusion, LTB4 and 5-HETE increase in AF before the onset of spontaneous labor. Progesterone receptor blockade by RU486 does not reproduce the changes in AF lipoxygenase metabolites observed during normal parturition.     

Hydroxyeicosatetraenoic acids are increased in bronchoalveolar lavage fluid of endotoxemic pigs

We hypothesized that lipoxygenase metabolites of arachidonic acid might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA) to determine the presence of 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE in bronchoalveolar lavage fluid (BALF) of saline (n = 12)- and endotoxin (n = 18)-treated pigs. Endotoxin, infused at 5 micrograms/kg for 1 hr followed by 2 micrograms/kg/hr for an average of 3 hrs, caused pulmonary hypertension, a biphasic increase in pulmonary vascular resistance, hypoxemia, bronchoconstriction, leukopenia, and thrombocytopenia. Relative to saline controls, the levels of immunoreactive (i)-5-HETE (816 +/- 209 pg/ml), i-12-HETE (1589 +/- 517 pg/ml), and i-15-HETE (448 +/- 78 pg/ml) were significantly (P less than 0.05) increased in BALF recovered from endotoxemic pigs at postmortem. Relative to control BALF i-HETE concentrations, the endotoxin values were 3.5x, 5.1x, and 2.8x higher for i-5-HETE, i-12-HETE, and i-15-HETE, respectively. We conclude that during porcine endotoxemia, the 5-, 12-, and 15-lipoxygenase pathways are activated and that HETES might be involved in the pathophysiology of endotoxin-induced ARF.     

Hydroxyeicosatetraenoic acids are increased in bronchoalveolar lavage fluid of endotoxemic pigs

We hypothesized that lipoxygenase metabolites of arachidonic acid might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA) to determine the presence of 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE in bronchoalveolar lavage fluid (BALF) of saline (n=12)- and endotoxin (n=18)- treated pigs. Endotoxin, infused at 5 μg/kg for 1 hr followed by 2 μg/kg/hr for an average of 3 hrs, caused pulmonary hypertension, a biphasic increase in pulmonary vascular resistance, hypoxemia, bronchoconstriction, leukopenia, and thrombocytopenia. Relative to saline controls, the levels of immunoreactive (i)-5-HETE (816 ± 209 pg/ml), i-12-HETE (1589 ± 517 pg/ml), and i-15-HETE (448 ± 78 pg/ml) were significantly ) increased in BALF recovered from endotoxemic pigs at postmortem. Relative to control BALF i-HETE concentrations, the endotoxin values were 3.5x, 5.1x, and 2.8x higher for i-5-HETE, i-12-HETE, and i-15-HETE, respectively. We conclude that during porcine endotoxemia, the 5-, 12-, and 15-lipoxygenase pathways are activated and that HETES might be involved in the pathophysiology of endotoxin-induced ARF.     

The Association between Intrauterine Inflammation and Spontaneous Vaginal Delivery at Term: A Cross-Sectional Study

Background

Different factors contribute to the onset of labor at term. In animal models onset of labor is characterized by an inflammatory response. The role of intrauterine inflammation, although implicated in preterm birth, is not yet established in human term labor. We hypothesized that intrauterine inflammation at term is associated with spontaneous onset of labor.

Methods/Results

In two large urban hospitals in the Netherlands, a cross-sectional study of spontaneous onset term vaginal deliveries and elective caesarean sections (CS), without signs of labor, was carried out. Placentas and amniotic fluid samples were collected during labor and/or at delivery. Histological signs of placenta inflammation were determined. Amniotic fluid proinflammatory cytokine concentrations were measured using ELISA. A total of 375 women were included. In term vaginal deliveries, more signs of intrauterine inflammation were found than in elective CS: the prevalence of chorioamnionitis was higher (18 vs 4%, p = 0.02) and amniotic fluid concentration of IL-6 was higher (3.1 vs 0.37 ng/mL, p<0.001). Similar results were obtained for IL-8 (10.93 vs 0.96 ng/mL, p<0.001) and percentage of detectable TNF-α (50 vs 4%, p<0.001).

Conclusions

This large cross-sectional study shows that spontaneous term delivery is characterized by histopathological signs of placenta inflammation and increased amniotic fluid proinflammatory cytokines.     

Reference values of concentrations of matrix metalloproteinases-1, -2, -9 and the tissue inhibitor of matrix metalloproteinases (TIMP-1) in the amniotic fluid during physiological pregnancy and delivery

The aim of this study was to determine reference values of matrix metalloproteinases (MMPs) MMP-1, MMP-2, MMP-9 and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in the amniotic fluid at the first stage of labor in physiological pregnancy. Eighty nine women at the first stage of term labor have been examined. Samples of amniotic fluid were taken at the first period of labor by vaginal amniotomy. Concentrations of MMP-1, MMP-2, MMP-9, and TIMP-1 have been investigated in amniotic fluid samples by ELISA kits. The following normal concentration ranges for MMP-1, MMP-2, MMP-9, TIMP-1, and ratios of concentrations of MMPs and TIMP-1 (MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-9/TIMP-1) have been determined for amniotic fluid samples obtained during the first period of labor in physiological pregnancy. These included: MMP-1: 5.1–16.8 pg/mg of protein; MMP-2: 238.3–374.1 pg/mg of protein; MMP-9: 66.1–113.3 pg/mg of protein, TIMP-1: 4.7–13.6 pg/mg of protein, MMP-1/TIMP-1 ratio: 0.1–2.2, MMP-2/TIMP-1 ratio: 19.9–55.7; MMP-9/TIMP-1 ratio: 4.2–17.2.     

Cytokines in normal and abnormal parturition: elevated amniotic fluid interleukin-6 levels in women with premature rupture of membranes associated with intrauterine infection

The participation of interleukin-6 (IL-6) in the pathophysiology of normal and abnormal human parturition was evaluated by determining IL-6 concentrations in amniotic fluid (AF). Biologically active IL-6 was determined (in U/ml) using the B9 hybridoma growth factor assay, while the concentrations of immunoreactive IL-6 species (in pg/ml) were assessed using a monoclonal antibody (moAb)-based ELISA. Two hundred and twenty-seven AF samples from women in normal labor and from those presenting with a clinical diagnosis of premature rupture of membranes (PROM) were assayed. In selected instances, IL-6 levels were evaluated simultaneously in AF and in maternal and fetal plasma. Women with a normal pregnancy had low titers of biologically active IL-6 in AF both at midtrimester (group 1, n = 27; median IL-6 concentration = 16 U/ml) and at term (group 2, n = 33; median = 15 U/ml). There was an increase in the IL-6 bioactivity in AF from women in normal labor at term (group 3, n = 40; median = 74 U/ml; p less than 0.001). In order to distinguish between the relative contributions of parturition per se and of intrauterine infection to the elevation of biologically active IL-6 levels in AF, IL-6 titers were compared in four different groups of women with PROM.(ABSTRACT TRUNCATED AT 250 WORDS)     

F prostaglandin levels in amniotic fluid during balloon-induced cervical softening and labor at term

Mechanical stretching of the uterus was applied to seven term patients not in labor with unripe cervix by means of intra-uterine application of a rubber balloon. Significant cervical softening, and initiation and progress of labor was achieved in all cases. Serial analyses of the F prostaglandins (PGF) in amniotic fluid were performed before and at given intervals of cervical dilatation. Before the application of stretch, the level of PGF varied greatly; the values in 5 cases were measureable but low (less than 645 pg/ml) and in two cases the levels were below the sensitivity of the assay (less than 50 pg/ml). Large fluctuations of the levels were noted in many cases during the treatment. However, the rise of the PGF values was significantly with the progress of cervical dilatation compared to the pre-treatment values (P<0.05 at both 3–4 and 5–6 cm, and P<0.01 at 9–10 cm). It was concluded that the increased release of PGF in amniotic fluid is mediated by uterine stretching, and that upon being released PGF is probably involved in the cervical softening and progress of labor.     

F prostaglandin levels in amniotic fluid in premature labour

The concentration of prostaglandin F (PGF) in amniotic fluid was measured by radioimmunoassay in 27 patients admitted in premature labour. There was a strong correlation between PGF levels in amniotic fluid and both cervical dilatation (r = 0.81; P < 0.001) and duration of labour (r = 0.79; P < 0.001). Cervical dilatation greater than 7 cm was associated with levels exceeding 2000 pg/ml. When contractions were present for less than one hour, levels of PGF were below 50 pg/ml. Low levels of PGF were found in amniotic fluid from a separate group of three patients, of whom two had cervical incompetence. It is concluded that the onset of premature labour is not associated with elevated levels of PGF in amniotic fluid. During premature labour, concentrations rise to an extent comparable to that observed in labour at term.     

The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility

The effects of the lipoxygenase products of arachidonic acid, 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B4 (LTB4), on the spontaneous contractility of lower uterine segment human myometrial strips obtained prior to labour have been studied in vitro. 5-HETE gave a dose- dependent (10-500ng) increase in both the rate of contractions and overall contractility of myometrial strips while 12-HETE and LTB4 had no effect at the same concentrations. Prostaglandin F2 alpha (50ng) contracted all myometrial strips in a similar pattern to 5-HETE but was approximately 10 times more potent. The effect of 5-HETE may be direct or perhaps indirect via interaction with the cyclo-oxygenase pathway. The findings do not disprove the contention that the onset of parturition may be characterised by a switch in arachidonic acid metabolism in intra-uterine tissues from lipoxygenase to cyclo-oxygenase products.     

Reduction of 15-hydroxyeicosatetraenoic acid (15-HETE) in tracheal fluid by high frequency oscillatory ventilation

The effects of high frequency oscillatory ventilation (HFOV) and conventional mechanical ventilation (CMV) on tracheal secretion were compared in 6 anesthetized dogs. Using a double-balloon endotracheal catheter, 5 ml of saline was instilled into an isolated tracheal segment during HFOV and CMV for 10 min respectively. Two eicosanoids, 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-dehydrothromboxane B2 (11-dehydro-TXB2) were measured by radioimmunoassay in each sample. HFOV (stroke volume: 6 ml/kg, f: 10 Hz, bias flow: 5 l/min) and CMV (stroke volume: 12 ml/kg, f: 15/min) were performed in random sequence and achieved comparable gas exchange. The concentration of 15-HETE in tracheal fluid during HFOV (87 +/- 67 pg/ml) was decreased to less than half of that during CMV (286 +/- 184 pg/ml, P less than 0.05), while there was no significant change of 11-dehydro-TXB2 either in tracheal fluid or in plasma. This reduction of 15-HETE was tended to be enhanced by vagotomy (HFOV: 42 +/- 14, CMV: 120 +/- 103 pg/ml) with the concentration ratio of CMV/HFOV remaining unchanged. HFOV may provide hitherto unrecognized advantage over CMV by reducing airway secretion of 15-HETE, a potent inflammatory mediator.     

Amniotic fluid levels of tumor necrosis factor-alpha and soluble tumor necrosis factor receptor 1 before and after the onset of labor in normal pregnancies.

Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells and promotes the regulation of trophoblast growth and invasion. Tumor necrosis factor receptor 1 (TNF-R1) is a receptor for TNF-alpha, and soluble TNF-R1 (sTNF-R1) is present in amniotic fluid after receptor shedding. We evaluated whether amniotic fluid TNF-alpha and sTNF-R1 levels during labor differed from those before the onset of labor in normal pregnancies. This study enrolled 34 Japanese women experiencing normal pregnancies with single fetuses who had no infection. Of these subjects, 17 went into labor and had subsequent term deliveries (the labor group), and the other 17 underwent cesarean section without labor (the nonlabor group). The average gestational age at entry was 38-39 weeks of gestation. Maternal ages and gestational ages did not differ significantly between the two groups. Amniotic fluid was collected and the TNF-alpha and sTNF-R1 levels were determined by the ELISA method. Each of these levels was compared between the two groups. There was a significant increase in amniotic fluid TNF-alpha levels in the labor group. However, amniotic fluid sTNF-R1 levels did not differ significantly between the two groups. Amniotic fluid TNF-alpha may promote the onset of labor at term and/or term labor contributing to subsequent delivery may induce the production and secretion of TNF-alpha into the amniotic cavity. There was no pregnancy-associated increase in receptor shedding or cell apoptosis at the onset of labor.     

Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

A role for lipoxygenase metabolites of arachidonic acid in porcine ovulation

Prostaglandins, products of arachidonic acid via the cyclooxygenase pathway, are essential to the porcine ovulatory process in that inhibition of their synthesis results in ovulation failure. Studies in the rat have shown that ovulation is also preceded by a rise in three ovarian hydroxyeicosatetraenoic acids, products of the lipoxygenase pathway, and inhibition of this pathway also inhibits ovulation. Experiments were designed, using a pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-treated prepuberal gilt model, to measure pre-ovulatory changes in follicular fluid concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), and to compare the effects of indomethacin and nordihydroguaiaretic acid (NDGA) on ovulation in the pig and on 15-HETE and prostaglandin F₂α synthesis both in vivo and in vitro. Follicular fluid concentrations of 15-HETE were elevated significantly just prior to the expected time of ovulation (40 h after hCG). When indomethacin (10 mg) was injected into the ovarian stalk at 24 h after hCG, follicular fluid concentrations of both 15-HETE and prostaglandin F₂α were lower (P<0.01) than controls at 40 h and ovulation rate was suppressed (P<0.01). When NDGA (5 mg) was administered in the same manner, ovulation rate was suppressed (P<0.01), but the levels of 15-HETE and prostaglandin F₂α were not altered. Synthesis of 15-HETE by cultured granulosa and theca interna cells was reduced by the presence of NDGA (1 mg/ml), whereas indomethacin (100 ng/ml) lowered 15-HETE production in theca interna cells only. These results clearly demonstrate that indomethacin can block the lipoxygenase as well as the cyclooxygenase pathways, depending on the dose used, and suggest that lipoxygenase metabolites of arachidonic acid are involved in the ovulatory process in the pig.     

Amniotic fluid concentrations of prostaglandin F2 alpha, 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) and 11-deoxy-13,14-dihydro-15-keto-11, 16-cyclo-prostaglandin E2 (PGEM-LL) in preterm labor

Although prostaglandins (PGs) are considered the key mediators of human parturition at term, there is a paucity of data regarding their participation in the mechanisms responsible for preterm labor. The purpose of this study was to establish if preterm labor is associated with changes in the amniotic fluid concentrations of prostaglandins. PGF2 alpha, 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) and 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-prostaglandin E2 (PGEM-ll) were measured by using specific and sensitive radioimmunoassays. Amniotic fluid was retrieved by transabdominal amniocentesis from 55 women with preterm labor and intact membranes. Patients were divided into three groups according to the response to tocolysis and the presence or absence of an intra-amniotic infection. Amniotic fluid concentrations of PGFM and PGEM-ll were significantly greater in women with preterm labor and intra-amniotic infection than in women without infection. In addition, patients unresponsive to tocolysis without intra-amniotic infection also had a significantly greater concentration of PGFM and PGEM-ll in amniotic fluid than those responsive to tocolysis. Amniotic fluid concentrations of PGF2 alpha were greater in women with intra-amniotic infection than in women without intra-amniotic infection. In the absence of intra-amniotic infection, no difference in amniotic fluid PGF2 alpha concentrations could be found between women who responded to tocolytic treatment and those who did not.     

The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility

The effects of the lipoxygenase products of arachidonic acid, 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B₄ (LTB₄), on the spontaneous contractility of lower uterine segment human myometrial strips obtained prior to labour have been studied . 5-HETE gave a dose- dependent (10–500ng) increase in both the rate of contractions and overall contractility of myometrial strips while 12-HETE and LTB₄ had no effect at the same concentrations. Prostaglandin F₂ (50ng) contracted all myometrial strips in a similar pattern to 5-HETE but was approximately 10 times more potent. The effect of 5-HETE may be direct or perhaps indirect via interaction with the cyclo-oxygenase pathway. The findings do not disprove the contention that the onset of parturition may be characterized by a switch in arachidonic acid metabolism in intra-uterine tissues from lipoxygenase to cyclo-oxygenase products.     

Amniotic fluid prostaglandin D2 in spontaneous and augmented labor

Prostaglandin D2 (PGD2) was quantified in human amniotic fluid obtained from women in various phases of the first stage of spontaneous labor, augmented labor, and induced labor. PGD2 increased significantly only in late labor in the spontaneous and augmented labor groups.     

Identification and characterization of cyclo(His-Pro)-like immunoreactivity in amniotic fluid

Amniotic fluid (AF) from 25 term pregnancies was analyzed for cyclo(His-Pro)-like immunoreactivity (CHP-LI). CHP-LI was detected in all AF samples and was indistinguishable from synthetic CHP by immunoidentity, by gel chromatography on Sephadex G-25, and by high pressure liquid chromatography. The mean concentration of CHP-LI in AF was 13,622 +/- 1288 pg/ml (+/- SE) and concentrations were not altered by maternal labor. Plasma concentrations of CHP-LI were similar in 4 pregnant and 4 control subjects [2260 +/- 432 pg/ml vs. 2162 +/- 419 pg/ml (+/- SE), respectively]. We conclude that 1) CHP-LI is readily detected in AF from term pregnancies and is indistinguishable from synthetic CHP, and 2) concentrations of CHP-LI in human AF are significantly higher than concentrations of maternal plasma CHP-LI, suggesting CHP AF originates by mechanisms other than diffusion from maternal plasma.