Fetal and maternal plasma endothelin levels during the second half of pregnancy

Fetal and maternal plasma endothelin levels during the second half of pregnancy Nebojsa Radunovic, MD, PhD, Charles J. Lockwood, MD, Manny Alvarez, MD, Danica Nastic, PhD, Spasoje Petkovic, MD, PhD, and Richard L. Berkowitz, MD New York, New York, and Belgrade,

Yugoslavia

OBJECTIVE: Our objective was to evaluate maternal and fetal endothelin concentrations in uncomplicated pregnancies across the second half of pregnancy. STUDY DESIGN: Paired (n = 64) maternal venous and fetal umbilical venous or arterial samples were obtained during cordocentesis. In addition, eight neonatal umbilical vein samples were obtained immediately after delivery. Samples were assessed for hematocrit and pH, and concentrations of endothelin were measured by sensitive enzyme immunoassay. RESULTS: No significant correlation was found between either fetal or maternal endothelin levels and gestational age (r = 0.01, p = 0.91 and r = 0.07, p = 0.5, respectively). Fetal plasma endothelin concentrations were significantly lower than neonatal umbilical vein endothelin levels [median 2.5 pg/ml (range 0.9 to 5.73) vs 15.77 pg/ml (8.12 to 19.58), respectively; p < O.OOOl] but significantly higher than maternal levels [1.3 pg/ml (0.8 to 3.25); p < O.OOOl]. In addition, endothelin values were higher in the umbilical artery than in the umbilical vein, but this difference failed to achieve statistical significance [2.89 pg/ml (1.61 to 5.73) vs 2.29 pglml (0.9 to 5.70) respectively; p = 0.061. No correlation was noted between fetal and maternal endothelin levels (r = 0.12, p = 0.36). CONCLUSION: Fetal endothelin levels were significantly higher than maternal levels, but neither correlated with gestational age across the second half of pregnancy. (AM J OBSTET GYNECOL 1995;172:28-32.)

Key words:

Feal blood,

maternal

blood,

endothelin

It is now known that endothelial cells synthesize many vasoactive substances, which act locally to regulate vascular smooth muscle tone and maintain blood flow. One such substance, endothelin, is a recently discovered 2 l-amino-acid peptide whose gene has been mapped to chromosome 6.’ Endothelin is synthesized and preferentially secreted toward the basal side of the endothelial cell where it is strategically placed to bind to specific receptors on vascular smooth muscle.3 The binding of endothelin to vascular smooth muscle endothelin-A receptors results in vasoconstriction, whereas binding to endothelin-B receptors mediates generation of the potent vasodilator nitric oxide.3 The predominant effect of endothelin infusion into the circulation is vasoconstriction and hypertension after a transient period of nitric oxide-induced hypotension.4, ’ Endothelin also exerts its hypertensive effects by enhancing renin activity and aldosterone secretion.’ Whereas endothelin acts From the Department of Obstetrics, Gynecology and Reproductive Science, Mount Sinai School ofMedicine, and the Clinic of Obstetrics and Gynecology, University of Belgrade, School of Medic&e. Received for bublicution March 4. 1994: revised Mav 31. 1994: acceptedjune’21, 1994. ’ ’ Refirint requests: Charles J. Lockwood, MD, Department of Obstetrics, Gynecology and Reproductive Science, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029.6574. Copyright 0 1995 by Mosby-Year Rook, Inc. 0002-9378/95 $3.00 + 0 6/1/58507 ,I

28

,

at a local or paracrine level, measurements of plasma concentrations of this peptide may allow a general assessment of its impact on endothelial homeostasis. Given the unique hemodynamic properties of the fetoplacental circulation, endothelin may play a crucial role in the maintenance of its vascular tone.7S 8 The messenger ribonucleic acid for endothelin is expressed in the placenta, as well as in cultured human umbilical endothelial cellsgS lo However, there is a paucity of data on the ontogeny of endothelin synthesis, secretion, and regulation during intrauterine life. Prior reports of neonatal umbilical cord endothelin levels do not reflect the physiologic state in utero given the putative effects of labor on systemic vascular and placental endothelin of cordocentesis now expression.11m’4 The availability permits access to the fetal circulation in a relatively undisturbed state.” Schiff et aLI6 have demonstrated the presence of detectable plasma endothelin levels in 8 fetuses evaluated between 19 and 22 weeks. The purpose of this study was to examine concentrations of endothelin in paired fetal and maternal plasma samples during the second half of pregnancy and to evaluate possible associations between maternal and fetal endothelin levels, as well as gestational age. Material

and

methods

Study population.

Paired maternal

and fetal samples

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Fig. 1. Box-and-whisker plot of fetal plasma endothelin concentrations during second half of gestation. Gestational ages are grouped in 4-week intervals from 20 to 39 weeks. No correlation was observed between fetal endothelin levels and gestational age.

were obtained between September 1992 and September 1993, at the time of diagnostic cordocentesis to assess karyotype (n = 36), rule out congenital infections (n = 17), evaluate hematocrit and Rh antigen status in isoimmunized pregnancies (n = 7), or obtain fetal blood gas analysis after nonreactive nonstress tests (n = 4). None of these fetuses had abnormal karyotypes, infection, anemia, or acidosis and all had birth weights appropriate for gestational age at delivery. None of the mothers smoked cigarettes or subsequently had preeclampsia, pregnancy-induced hypertension, or diabetes, Gestational age was established by menstrual age confirmed by ultrasonographic examination at < 20 weeks. Informed consent was obtained and the project was approved by the hospital ethics committee. Blood sampling. Initial maternal venous blood samples were obtained from an antecubital vein before cordocentesis. Cordocenteses were performed under strict aseptic conditions with a 20-gauge 15 cm spinal needle that was guided to the umbilical cord at the placental insertion by means of a 3.75 MHz convex ultrasound probe (Toshiba SSA 90, Toshiba, Tokyo). Samples were included in this study only if access to the umbilical circulation was achieved with one puncture, the total duration of the procedure was < 2 minutes, and no fetal bradycardia was observed. Local anesthesia (1% lidocaine) was used without maternal sedation. After satisfactory umbilical vein or artery access was

achieved, 2 to 4 ml of pure fetal blood was collected for diagnostic purposes. After fetal blood was obtained, the umbilical vein was differentiated from the artery by assessing the direction of flow after the injection of 0.5 ml of normal saline solution. The absence of maternal blood or amniotic fluid contamination was confirmed by Kleihauer-Betke analysis and assay for coagulation factor V. In addition, 2 ml of umbilical vein blood was obtained after spontaneous vaginal delivery at term in eight appropriately grown neonates. A 1 ml aliquot of maternal, fetal, or neonatal cord blood was used for measurement of endothelin levels. These samples were collected in tubes containing 5 mmoVL ethylenediaminetetraacetic acid and aprotinin. Plasma was extracted by centrifugation at 1OOOg for 10 minutes at 4” C, and the samples were then placed in polypropylene cryotubes and stored at -70” C until assayed. Endothelin immunoassays. Determination of endothelin concentrations was carried out with a commercially available enzyme immunoassay according to the manufacturer’s specifications (Cayman Chemical Co, Ann Arbor, Mich.). Plasma samples were first acidified with 4% acetic acid in a ratio of 1: 4. Samples were then extracted through 100 mg Amprep C-l 8 cartridges per the manufacturer’s specifications (Amersham Int., United Kingdom). Eluates were dried under nitrogen and resuspended in 120 ul of enzyme immunoassay

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n=4

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Fig. 2. Box-and-whisker gestation. observed

Gestational ages between maternal

plot of maternal plasma are grouped in 4-week endothelin levels and

Table I. Concentrations of plasma endothelin in fetal, maternal, and neonatal samples Plasma

endothelin

Median Maternal Fetal Neonatal

1.30 2.50 15.77

@g/ml) Range 083.25 0.9-5.73 8.12-19.58

buffer. This method allowed 80% recovery of a known quantity of endothelin. The enzyme immunoassay used an immunometric “sandwich” technique with antihuman monoclonal antibody to endothelin-1,2 employed in the capture phase and a second monoclonal antibody linked to acetylcholinesterase used to amplify the signal. A calorimetric reaction was generated with the Ellman reagent and the intensity of the resultant change in optical density directly proportional to the amount of bound endothelin. The intraassay coefficient of variation was < 8% at concentrations of 2.7 pgiml. The sensitivity of detection of this assay was 0.8 pgiml. This assay had c I% cross-reactivity

to human

big-endothelin.

o

II

I

k

endothelin concentrations during second half of intervals from 20 to 39 weeks. No correlation was gestational age.

Statistical analysis. All statistical analyses were performed with the Statgraphics (Version 3.0) statistical software package (STSC, Rockville, MD.) and included Wilcoxon rank, and MannSpearman correlations, Whitney U tests. A p value of ~0.05 was considered significant. “Box-and-whisker” plots were used to demonstrate the interquartile range (box), median value (horizontal line), and absolute range (vertical line) for endothelin levels across gestation.

Results There were no procedure-related complications and all fetuses were subsequently delivered uneventfully after 36 weeks. Fig. 1 presents fetal endothelin values during the second half of gestation. No significant correlation was found between fetal endothelin levels and gestational age (r = 0.01, p = 0.91). Fetal plasma endothelin concentrations were significantly lower than neonatal umbilical vein endothelin levels (p = 0.0001) but

significantly

higher

than

the

overall

maternal

levels

values (p = 0.0001) (Table I). I n addition, endothelin were higher in the umbilical artery compared with the umbilical vein, but this difference failed to achieve statistical significance [median 2.89 pgiml (range 1.61

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to 5.73) vs 2.29 pgiml (0.9 to 5.70), respectively; p = 0.061. Maternal endothelin values did not correlate with gestational age (r = 0.08, p = 0.51) (Fig. 2) or with fetal endothelin levels (r = 0.12, p = 0.36). Neonatal umbilical cord values were also significantly higher than maternal values (p = 0.0001) (Table I).

Comment The fetal circulation is characterized by relatively low systemic and high pulmonary vascular resistance. Thus our findings of a significant elevation in fetal plasma endothelin levels compared with maternal levels are surprising. This phenomenon may reflect selective endothelin binding to vasodilator endothelin-B vascular smooth muscle receptors. Alternatively, elevated endothelin levels in the fetal circulation may represent a compensatory response to increased sheer stress induced by vasodilatory substances (e.g., nitric oxide). Of interest, the placenta and umbilical vessels have been found to be discordant for endothelin receptor subtypes, with the vasoconstrictor endothelin-A forms predominating in the umbilical vessels and the vasodilatory endothelin-B forms predominating in the placenta.* The precise source(s) of endothelin in the fetal circulation remain obscure. Hemsen et al.* observed that the bulk of umbilical vessel-derived endothelin is endothelin-1. Schiff et al.‘” demonstrated endothelin in the fetal circulation as easly as 19 weeks. In this study we report similar findings in a larger population. In addition, we observed a trend toward higher concentrations of endothelin in fetal umbilical arterial plasma compared with venous plasma, a finding previously reported in neonatal umbilical cord blood.“, l’-” The latter supports the hypothesis that fetal endothelial cells rather than placental endothelium is the primary source of endothelin released into the circulation. Furthermore, this hypothesis is consistent with a report that cultured human umbilical endothelial cells produce endothelin1.‘. I1 Given that the half-life of circulating endothelin is approximately 1 minute,” observed umbilical arterial-venous endothelin differences could reflect net placental degradation. However, endothelin messenger ribonucleic acid has been detected in placental tissue,” and endothelin immunoreactivity has been localized to the vascular endothelium of the placenta.* Thus placental endothelin production may also contribute to the total circulating pool. The absence of a significant association between fetal and maternal levels suggests that endothelin does not cross the placenta. Cordocentesis offers the only practical means for assessing the physiologic mediators of fetal vascular tone. Moreover, because all the pregnancies evaluated in this study were not associated with maternal or fetal

et al.

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conditions linked to abnormal endothelin expression, the observed maternal and fetal endothelin levels should reflect relatively unperturbed maternal and fetal states. Moreover, the restriction of analysis to those umbilical blood sampling procedures that required only one puncture and were terminated within 2 minutes should have minimized the potential for cordocentesisrelated umbilical endothelial trauma and effects related to fetal stress.” A number of investigators have reported an increase in endothelin levels with advancing gestation’” Is; however, others have not found any change.‘” We were unable to detect any clear association between maternal endothelin levels and gestational age. However, the low numbers of available samples after 36 weeks (n = 4) does not permit us to rule out an increase in maternal endothelin levels late in pregnancy. The finding that neonatal endothelin levels were also higher than maternal levels is consistent with the findings of previous studies performed on neonatal cord blood.“, ” The elevation in neonatal endothelin levels compared with fetal values observed in our study may reflect enhanced endothelin production associated with labo? and play a role in the dramatic increase in neonatal systemic vascular resistance that follows delivery. In vitro human umbilical vessels constrict in response to exogenous endothelin in a dose-dependent manner with a threshold concentration of 100 pmol/L.“’ This suggests a possible contribution of endothelin to the closure of human umbilical vessels after delivery.” However, plasma endothelin concentrations in the neonatal circulation measured in this and other studiesI are about 10 times less than those used to induce such pharmacologic effects. It is possible that endogenous endothelin may play a physiologic role in the redistribution of blood volume to vital organs and/or constriction of placental vessels that takes place at the time of birth. REFERENCES

1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:41 l-5. 2. Inoue A, Yanagisawa M, Kimura S, et al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Nat1 Acad Sci U S A 1989;86:2863-7. 3. Remuzzi G, Benigni A. Endothelins in the control of cardiovascular and renal function. Lancet 1993;342:58993. 4. Miller WL, Redfield MM, Burnett JC. Integrated cardiac, renal, and endocrine actions of endothelin. J Clin Invest 1989;83:317-20. 5. Vierhapper H, Wagner 0, Nowothy P, Waldhause W. Effect of endothelin in man. Circulation 1990;81:1415-8. 6. Kawaguchi H, Ito K, Yasunda H. Endothelin stimulates angiotensin converting enzyme activity [Abstract]. In: Proceedings of the thirteenth scientific meeting of the International Society of Hypertension, Montreal, Quebec,

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Canada, December 1990. Montreal: International Society of Hypertension, 1990. Wilkes BM, Mento PF, Hollander AM, Maita ME, Sung S, Giradi EP. Endothelin receptors in human placenta: relationship to vascular resistance and thromboxane release. Am J Physiol 1990;258:E864-70. Hemsen A, Gillis C, Larsson 0, Haegerstrand A. Characterization, localization and action of endothelins in umbilical vessels and placenta of man. Acta Physiol Stand 1991; 143:395-404. Nunez DJR, Brown MJ, Davenport AP, Neylon CB, Schoefield JP, Wyse RK. Endothelin-1 mRNA is widely expressed in porcine and human tissues. J Clin Invest 1990;85:153’741. Benigni A. Gasnari F. Orisio S. et al. Human nlacenta expresses the endothelin-1 gene and the corresponding protein is excreted in the urine in increased amounts during the course of normal pregnancy. AM J OBSTET GYNECOL 1991;164:844-8. Hagerstrand A, Hemsen A, Gillis C, Larsson 0, Lundberg JM. Endothelin: presence in human umbilical vessels, high levels in fetal blood and potent constrictor effect. Acta Physiol Stand 1989;137:541-2. Nisell H, Hemsen A, Lunell NO, Wolff K, Lundberg MJ. Maternal and fetal levels of a novel polypeptide, endothelin: evidence for release during pregnancy and delivery. Gynecol Obstet Invest 1990;30:129-32. Iwata I, Takagi T, Yamaji K, Tanizawa 0. Increase in the concentration of immunoreactive endothelin in human pregnancy. J Endocrinol 1991;129:301-7. Isozaki-Fukuda Y, Kojima T, Hirata Y, et al. Plasma im”

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munoreactive endothelin-1 concentration in human fetal blood: its relation to asphyxia. Pediatr Res 1991;30:244-7. Radunovic N, Lockwood CJ, Ghidini A, Alvarez M, Berkowitz RL. Is fetal blood sampling associated with increased beta-endorphin release into the fetal circulation? Am J Perinatol 1993;lO: 112-4. Schiff E, Zael Y, Friedman SA, Shalev E. Fetal circulatory endothelin1,2 in the midtrimester. Gynecol Obstet Invest 1993;35:185-6. Nakamura T, Kasai K, Konuma S, et al. Immunoreactive endothelin concentrations in maternal and fetal blood. Life Sci 1990;46:1045-50. Usuki S, Saitoh T, Sawamura T, et al. Increased maternal plasma concentration of endothelin-1 during labor pain or on delivery and the existence of large amount of endothelin-1 in amniotic fluid. Gynecol Endocrinol 1990;4:85-97. McQueen J, Kingdom JCP, Connell JMC, Whittle MJ. Fetal endothelin levels and placental vascular endothelin receptors in intrauterine growth retardation. Obstet Gynecol 1993;82:992-8. Hakkinen LM, Vuolteenaho OJ, Leppaluoto JP, Laatikainen TJ. Endothelin in maternal and umbilical cord blood in spontaneous labor and at elective cesarean delivery. Obstet Gynecol 1992;80:72-5. Hartikainen-Sorri AL, Vuolteenaho 0, Leppaluoto J, Rushoaho H. Endothelin in umbilical artery vasospasm. Lancet 1991;337:619. Haugen G, Stray-Pedersen SS. Effects of endothelin-1 on vascular tension in human umbilical vessels. Early Hum Dev 1991;27:25-32.

Crown-rump length in chromosomally at 10 to 13 weeks’ gestation Peter Kuhn, MD, Maria de Lourdes Brizot, MD, Pranav P. Pandya, Rosalinde J. Snijders, PhD, and Kypros H. Nicolaides, MD London,

abnormal fetuses

MD,

United Kingdom

OBJECTIVE: Our purpose was to investigate whether fetuses with aneuploidies demonstrate evidence of growth retardation during the first trimester. STUDY DESIGN: This was a retrospective, cross-sectional study of singleton pregnancies undergoing fetal karyotyping at 10 to 13 weeks’ gestation. Measurements of crown-rump length in 135 chromosomally abnormal fetuses were compared with those in 700 chromosomally normal fetuses. RESULTS: The median crown-rump length of fetuses with trisomy 18 (n = 32) was significantly reduced. In contrast, in fetuses with trisomy 21 (n = 72), trisomy 13 (n = 1 l), 47,XxX (n = 6), 47,XXY (n = 6), 45,X (n = 5), and triploidy (n = 3) the crown-rump length was not lower than normal. CONCLUSION: At 10 to 13 weeks’ gestation fetuses with trisomy 18 are growth retarded, whereas in trisomy 21, trisomy 13, and sex chromosome aneuploidy growth is normal. (AM J Oesm GYNECOL 1995;172:32-5.)

Key words:

Crown-rump

length,

chromosomal

abnormality,

fetal

karyotyping,

maternal

serum

biochemistry From the Harris Birthright Research Centre for King’s College Hospital Medical School. Supported by the Swiss National Science Foundation Received for publication March 22, 1994; revised accepted June 21, 1994. 32

Fetal

Medicine,

(P.K.) May IO, 1994;

Reprint requests: K.H. Nicolaides, MD, Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital Medical School, London, United Kingdom SE5 8RX. Copyright 0 1995 by Mosby-Year Book, Inc. 0002.9378/95 $3.00 + 0 6/l/58505