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Obstetrics & Gynecology 2000;95:561-564
© 2000 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Maternal Smoking and Fetal Erythropoietin Levels

ANDRÉE GRUSLIN, MD, FRCSC, SHERRY L. PERKINS, PhD, FCACB, RAMAN MANCHANDA, MSc, NATHALIE FLEMING, MD and JENNIFER J. CLINCH, MA

From the Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, and the Department of Pathology and Laboratory Medicine, The Ottawa Hospital, University of Ottawa; and Clinical Epidemiology Unit, Ottawa Hospital Research Institute, Ottawa Hospital—General Campus, Ottawa, Ontario, Canada.

Address reprint requests to: Andrée Gruslin, MD, FRCSC, The Ottawa Hospital—General Campus, Department of Obstetrics and Gynecology, 501 Smyth Road, Room 8420, Ottawa, ON K1H 8L6, Canada, E-mail: agruslin{at}ogh.on.ca


   Abstract
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Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: To determine the influence of maternal smoking on fetal erythropoietin concentrations in health term pregnancies and test the correlation between cotinine, a biomarker of maternal smoking, and erythropoietin levels in fetuses.

Methods: We invited women with healthy term pregnancies to participate in the study, excluding those with conditions previously known to be associated with elevated fetal erythropoietin levels. We recorded demographic data, smoking status, and labor outcome prospectively for each patient. Umbilical venous samples were collected, and serum was stored at -70C to be analyzed later for erythropoietin and cotinine. Umbilical arterial samples were tested for pH and base excess determination. We compared fetal erythropoietin and cotinine between smokers and nonsmokers and examined correlations between erythropoietin and cotinine. Kruskal-Wallis test, t test, median test, and Spearman rank correlation test were used when appropriate. Statistical significance was P < .05.

Results: We recruited 35 nonsmokers and 26 smokers and analyzed their samples. The two groups were comparable in demographics and birth outcomes, except for birth weights, which were lower in smokers. Fetal erythropoietin concentrations increased significantly with increasing maternal cigarette consumption, ranging from none to more than 15 cigarettes per day (P = .03). There was positive correlation between fetal erythropoietin and cotinine concentrations (r = .41; P = .04), suggesting a dose-response relationship.

Conclusion: Fetuses of smokers had increased erythropoietin concentrations that correlate positively with fetal cotinine levels; which suggests an increased risk of subacute hypoxia related to degree of maternal cigarette consumption.

Erythropoietin is a glycoprotein hormone involved in regulating erythropoiesis during adult and fetal life.1 The only known stimulus for its production is decreased partial oxygen pressure.2–4 Erythropoietin does not cross the placenta, therefore, in cord blood, it is believed to be of fetal origin.5 There have been elevations of erythropoietin concentrations in various maternal and fetal conditions associated with underlying chronic hypoxia, including maternal hypertension,6 diabetes,6 alcohol abuse,7 prolonged pregnancies,8 fetal growth restriction (FGR),9 and Rh isoimmunization.10

Maternal smoking is associated with various perinatal complications such as low birth weight, and placental abnormalities such as decreased intervillous blood flow and increased neonatal morbidity. Suggested pathophysiologic mechanisms include increased fetal carboxyhemoglobin levels,11 altered uteroplacental flow,12 and fetal hypoxia.13 The goal of this study was to determine whether maternal smoking is associated with elevated fetal erythropoeitin concentrations in otherwise healthy term pregnancies and investigate whether fetal cotinine, an objective marker of maternal smoking, correlates with fetal erythropoietin.


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
Women with uncomplicated, healthy singleton term pregnancies (37–41 weeks) who presented to the Ottawa Hospital General Campus Labor and Delivery Unit were invited to participate. We excluded those with conditions previously associated with increased erythropoietin, including FGR, preeclampsia, hypertension, maternal diabetes or other chronic disease, alcohol abuse, meconium-stained amniotic fluid (AF), Rh isoimmunization, hydrops, aneuploidies,14 and fetal asphyxia at delivery. To remove factors that potentially lead to acute hypoxia or asphyxia, we excluded women whose fetuses had congenital anomalies, macrosomia, abnormal intrapartum tracings (such as repetitive variable or late decelerations, fetal bradycardia, or tachycardia and no short-term variability), those who required emergency deliveries, and those with delivery complications such as shoulder dystocia.

Maternal smoking status and obstetric histories were assessed by patient questionnaire. Responses were verified by antenatal records. We prospectively recorded maternal age, gravidity, parity, and birth outcome, including gestational age, birth weight, and Apgar scores at 1 and 5 minutes.

Immediately after cord clamping, umbilical artery (UA) samples were collected in heparinized syringes, put on ice, and transported to the laboratory for pH and base excess determination within 10 minutes of collection. Umbilical venous (UV) samples were collected in syringes, transferred to Vacutainer vials (Becton Dickinson, Franklin Lakes, NJ), transported to the laboratory, allowed to clot, and centrifuged for 10 minutes at 3000g, and the serum was stored at -70C for later analysis.

Samples for erythropoietin determination were batch-analyzed in duplicate with the use of a commercially available enzyme-linked immunosorbent assay (ELISA) (RD & S Systems, Minneapolis, MN). Intraassay coefficient of variation was less than 5%, and interassay coefficient was less than 10%. Cotinine levels also were batch-analyzed in duplicate by using the STC Technologies Cotinine, Serum Micro-plate enzyme immunoassay [(STC) Technologies Inc., Bethlehem, PA 18018/1799]. When used for quantitative analysis, this assay has a linearity of 0–50 µg/L, intraassay precision of 6.2% at 50 µg/L, and interassay precision of 9.6% at 50 µg/L. All samples with results above the linearity of the assay were reanalyzed after specimen dilution with a cotinine-free serum pool.

Maternal demographics and birth outcomes were compared by using a t test, or for ordinal variables, a median test. Fetal erythropoietin and cotinine concentrations were compared with the use of a Kruskal-Wallis test. To investigate a possible relationship between erythropoietin and cotinine, and between cigarette consumption and cotinine, a Spearman rank correlation coefficient was calculated. Statistical significance was P < .05. This study was approved by the Ottawa Hospital General Campus Research Ethics Board, and written informed consents were obtained from subjects.


   Results
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Abstract
 Materials and Methods
 Results
Discussion
 References
 
Sixty-one samples were analyzed, 35 from nonsmokers and 26 from smokers. There was no difference between groups in gravidity or proportion of women with labor. Maternal age was statistically greater in the nonsmoker group; however, that difference was not clinically relevant.

Gestational ages were similar, and there was no difference in proportion of fetuses delivered after 40 weeks. Birth weights were significantly lower in fetuses of women who smoked. Commonly used clinical markers of acute hypoxia such as UA pH and Apgar scores at 1 and 5 minutes were similar between groups, and none of the fetuses had a pH of less than 7.1. There was a slight but statistically significant difference in base excess between groups, which we believed to be of little clinical significance (Table 1Go).


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  		Table 1. Maternal Demographics and Birth Outcome
 
To evaluate the effect of maternal smoking on fetal erythropoietin concentration, we compared fetuses of nonsmokers, those exposed to one to five cigarettes per day, six to 15 cigarettes per day, and more than 15 cigarettes per day. The mean and median erythropoietin and cotinine concentrations of those groups, shown in Table 2Go, showed a significant increase in fetal erythropoietin (P = .03) and cotinine (P = .001) with increasing maternal cigarette consumption. The increase was especially marked in fetuses exposed to more than 15 cigarettes per day. In the group of fetuses exposed to smoking, there was a positive correlation between the number of cigarettes smoked per day and the cotinine concentrations (r = .53, P = .005). There was also a positive correlation between fetal erythropoietin and cotinine concentrations (r = .41, P = .04). There was no correlation between birth weight and fetal erythropoietin concentration or birth weight and cotinine concentration in this small sample.


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  		Table 2. Fetal Erythropoietin and Cotinine Concentrations
 

   Discussion
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Abstract
 Materials and Methods
 Results
 Discussion
References
 
We found that in otherwise healthy, term singleton pregnancies, increased maternal cigarette consumption, ranging from none to more than 15 per day is associated with elevated fetal erythropoietin concentrations. Also we found a positive correlation between fetal erythropoietin and cotinine, which suggests a dose-response relationship. Our data indicated that fetuses exposed to maternal smoking are at greater risk of subacute hypoxia and that risk might be more important in mothers with heavier smoking habits. In a previous study that examined influence of smoking on fetal erythropoietin levels, results barely reached statistical significance (P = .05) to show greater erythropoietin concentrations in fetuses of women who smoked.15 Only one fifth of the fetuses had increased erythropoietin levels. Inclusion of preterm and growth-restricted fetuses in that study might have confounded the data. A similar study by Bili et al16 showed elevated erythropoietin concentrations in fetuses of smokers but also did not exclude growth restriction. Dose-response relationship between number of cigarettes per day and fetal erythropoietin also could not be determined in either of those studies, possibly owing to inherent inaccuracies when relying on patient-reported cigarette consumption instead of using objective biomarkers such as cotinine. Some inaccuracies were compounded further by patients’ tendencies to report cigarette consumption in fractions of packs per day, by variations in inhalation techniques and nicotine content of different cigarette brands, and by potential effects of passive smoking which may explain some of the difficulties examining erythropoietin in fetuses of smokers and the need for biological markers of smoking. Difficulty quantifying cigarette consumption was reported as a weakness in another study on smoking and fetal erythropoietin.17 Those authors carefully evaluated growth-restricted fetuses separately and confirmed that in both populations of fetuses (normal and growth-restricted), erythropoietin was elevated when pregnancies were complicated by maternal smoking.

We examined cotinine because there is a linear relationship between maternal and fetal levels18 and there is no placental or fetal sequestration.18 Cotinine also has a half-life of 16.3 hours in the fetus,18 allowing its detection even after a certain period of abstinence such as seen in labor. Using fetal cotinine concentration as a marker for maternal smoking allowed us to identify correctly smokers and helped quantify their consumption, providing us with evidence of dose-dependent association between fetal erythropoietin and maternal smoking.

Several mechanisms might contribute to erythropoietin elevations in fetuses of smokers, including increased fetal carboxyhemoglobin concentrations, vasoconstriction either from nicotine or catecholamine release, and decreased uteroplacental blood flow. We believe that relative fetal hypoxia is caused by those mechanisms combined, thereby stimulating erythropoietin synthesis. That belief is supported by a recent report of a positive correlation between number of cigarettes per day and absolute nucleated red cell counts, another marker of chronic hypoxia,19 which could also explain higher hemoglobin concentrations reported in fetuses of smokers.15 Suboptimal fetal oxygenation could be the basis of several perinatal complications in fetuses of women who smoke. Our sample might have been too small to show correlations between birth weight and erythropoietin; however, fetal cotinine correlated inversely with birth weight in much larger populations,18 so we believe that erythropoietin might have the same positive correlation with fetal cotinine.

Estimating the real risk of subacute hypoxia in fetuses exposed to smoking is difficult, but our data suggest that possibly 50% of fetuses are at such risk because half our fetuses exposed to smoking had erythropoietin concentrations greater than 25 µg/L, a commonly reported value for low-risk control populations.20,21 It appears smoking more than 15 cigarettes per day might be important because that group had a more marked increase in erythropoietin concentrations.


   Footnotes
 
PII S0029-7844(99)00622-5

Received June 16, 1999. Received in revised form October 1, 1999. Accepted October 15, 1999.


   References
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Abstract
 Materials and Methods
 Results
 Discussion
 References
 
1. Eckardt K. The ontogeny of the biological role and production of erythropoietin. J Perinat Med 1995;23:19–29.[Medline]

2. Curo J, Erslev AJ, Silver R, Miller O, Birgigard G. Erythropoietin production in response to anemia or hypoxia in the newborn rat. Blood 1982;60:984–8.[Abstract/Free Full Text]

3. Moritz KM, Cooper E, Wintour EM. The effect of haemorrhage on erythropoietin concentration in the mature ovine fetus. J Dev Physiol 1992;17:157–61.[Medline]

4. Costa-Giomi P, Caro J, Weinmann R. Enhancement by hypoxia of human erythropoietin gene transcription in vitro. J Biol Chem 1990;265:10185–8.[Abstract/Free Full Text]

5. Schneider H, Malek A. Lack of permeability of the human placenta for erythropoietin. J Perinat Med 1995;23:71–6.[Medline]

6. Mamopoulos M, Bili H, Tsantali C, Assimakopoulos E, Manta-llenakis S, Farmakides G. Erythropoietin umbilical serum levels during labor in women with preeclampsia, diabetes, and preterm labor. Am J Perinatol 1994;11:427–9.[Medline]

7. Halmesmaki E, Teramo KA, Widness JA, Clemens JK, Ylikorkala O. Maternal alcohol abuse is associated with elevated fetal erythropoietin levels. Obstet Gynecol 1990;76:219–22.[Abstract/Free Full Text]

8. Jazayeri A, Tsibris JC, Spellacy WN. Elevated umbilical cord plasma erythropoietin levels in prolonged pregnancies. Obstet Gynecol 1998;92:61–3.[Abstract]

9. Snijders RJ, Abbas A, Melby O, Ireland RM, Nicolaides KH. Fetal plasma erythropoietin concentration in severe growth retardation. Am J Obstet Gynecol 1993;168:615–9.[Medline]

10. Moya FR, Grannum PA, Widness J, Clemons GK, Copel JA, Hobbins JC. Erythropoietin in human fetuses with immune hemolytic anemia and hydrops fetalis. Obstet Gynecol 1993;82:353–8.[Abstract/Free Full Text]

11. Bureau MA, Monette J, Shapcott D, Pare C, Mathieu JL, Lippe J, et al. Carboxyhemoglobin concentration in fetal cord blood and in blood of mothers who smoked during labor. Pediatrics 1982;69: 371–3.[Abstract/Free Full Text]

12. Monheit AG, Van Vunakis H, Key TC, Resnick R. Maternal and fetal cardiovascular effects of nicotine infusion in pregnant sheep. Am J Obstet Gynecol 1983;145:290–6.[Medline]

13. Suzuki K, Minei LJ, Johnson EE. Effect of nicotine upon uterine blood flow in the pregnant rhesus monkey. Am J Obstet Gynecol 1980;136:1009–13.[Medline]

14. Widness JA, Peuschel SM, Pezzullo JC, Clemens GK. Elevated erythropoietin levels in cord blood of newborns with Down’s Syndrome. Biol Neonate 1994;66:50–5.[Medline]

15. Varvarigou A, Beratis NG, Makri M, Vagenakis AG. Increased levels and positive correlation between erythropoietin and hemoglobin concentrations in newborn children of mothers who are smokers. J Pediatr 1994;124:480–2.[Medline]

16. Bili H, Mamopoulos M, Tsantali C, Tsevelekis P, Malaka K, Mantalenakis S, et al. Elevated umbilical erythropoietin levels during labor in newborns of smoking mothers. Am J Perinatol 1996;13:85–7.[Medline]

17. Jazayeri A, Tsibris JCM, Spellacy WN. Umbilical cord plasma erythropoietin levels in pregnancies complicated by maternal smoking. Am J Obstet Gynecol 1998;178:433–5.[Medline]

18. Perkins SL, Belcher JM, Livesey JF. A Canadian tertiary care centre study of maternal and umbilical cord cotinine levels as markers of smoking during pregnancy: Relationship to neonatal effects. Can J Public Health 1997;88:232–7.[Medline]

19. Yeruchimovich M, Dollberg S, Green D, Mimouni FB. Nucleated red blood cells in infants of smoking mothers. Obstet Gynecol 1999;93:403–6.[Abstract/Free Full Text]

20. Teramo KA, Widness JA, Clemons GK, Voutilainen P, McKinlay S, Schwartz R. Amniotic fluid erythropoietin correlates with umbilical plasma erythropoietin in normal and abnormal pregnancy. Obstet Gynecol 1987;69:710–6.[Medline]

21. Eckardt KU, Hartman W, Vetter U, Pohlandt F, Burghardt R, Kurtz A. Serum immunoreactive erythropoietin of children in health and disease. Eur J Pediatr 1990;149:459–64.[Medline]





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