Objective
To compare venous cord plasma concentrations of 4 vasoactive peptide precursors: carboxy-terminal proarginine vasopressin, CT-prondothelin (ET)-1, midregional proadrenomedullin, and MR-proatrial natriuretic peptide, between fetuses with intrauterine growth restriction and appropriate for gestational age controls.
Study Design
Matched-pair analysis of 12 fetuses with significant intrauterine growth restriction and 42 healthy appropriate for gestational age control fetuses. All infants were singletons, delivered by elective section after 34 weeks and without chromosomal abnormalities.
Results
Umbilical cord plasma copeptin levels (median [range]) were 4-fold higher in intrauterine growth restriction infants than in matched appropriate for gestational age controls: 23.2 (6.7–449) vs 5.1 (2.5–53) pmol/L ( P < .001). Multivariate regression analysis revealed an association between copeptin and umbilical artery resistance index z-score ( P = .034). The 3 other precursor peptides showed no changes.
Conclusion
High copeptin concentrations in the cord blood of intrauterine growth restriction newborns reflect a fetal stress response and support the fetal programming hypothesis.
Fetuses failing to reach their growth potential attract a diagnosis of intrauterine growth restriction (IUGR), associated with serious short- and long-term sequelae. It is, therefore, important to distinguish them from those merely small for gestational age (SGA). Detection of a small fetus strongly depends on accurate estimation of gestational age (GA) with ultrasound in the first trimester. Umbilical artery Doppler impedance is the reference clinical measure of compromised placental function differentiating IUGR from SGA. The additional presence of oligohydramnios is believed to be a chronic parameter of placental insufficiency.
IUGR fetuses display signs of chronic malnutrition and hypoxia that alter fetal cardiovascular dynamics and endocrine status. Important hormonal regulators of cardiovascular dynamics are the vasoconstrictor peptides arginine vasopressin (AVP) and endothelin-1 (ET-1) and the vasodilator peptides adrenomedullin (ADM) and atrial natriuretic peptide (ANP). Their instability and short half-lives make direct measurement unfeasible in the clinical setting. However, all derive from larger precursor peptides and distinct by-products have been shown to serve as robust surrogate markers, copeptin for AVP, carboxy-terminal proET-1 (CT-proET-1) for ET-1, midregional proADM (MR-proADM) for ADM, and midregional proANP (MR-proANP) for ANP.
We wished to test our hypothesis that umbilical cord blood concentrations of the vasoactive peptide precursors copeptin, CT-proET-1, MR-proADM, and MR-proANP in growth-restricted infants differ from those in matched appropriate for gestational age (AGA) controls.
Materials and Methods
Enrollment in this case-control study was performed between June 2005 and December 2006 and July and September 2009. In the first study period, 12 IUGR infants and 12 matching AGA infants (GA ± 10 days) were recruited at delivery as previously described. In the second period an additional 30 matching healthy AGA infants were identified among a larger study cohort of healthy newborns. To allow matching with healthy AGA controls we included only IUGR infants delivered after 34 weeks. Additional inclusion criteria were singleton pregnancies and elective section without preceding contractions or rupture of the membranes.
IUGR was defined by an estimated fetal weight below the fifth percentile of the reference chart and by pathologic blood flow patterns in the umbilical arteries in combination with oligohydramnios, defined as a 4-quadrant amniotic fluid index ≤6 cm. Fetal weight was calculated applying Hadlock’s formula (biparietal diameter, abdominal circumference, femur length). AGA infant weight was defined as >10th and <90th percentile of the estimated fetal weight on the reference chart.
We recorded umbilical artery blood flow patterns and calculated resistance indices (RI: maximal systolic velocity − maximal diastolic velocity/maximal systolic velocity) as described by Kurmanavicius et al. An increased umbilical artery RI outside the normal range of our hospital reference chart signified a pathologic flow pattern. We excluded pregnancies with chromosomal aberrations, fetal malformation, infection or substance abuse, and mothers with hypertension, preeclampsia, diabetes type I or II, autoimmune disease (antiphospholipid syndrome, lupus erythematosus), arteriosclerosis, or renal disease. No mother in either group had received nonsteroidal anti-inflammatory drugs or vasoactive drugs during pregnancy. The institutional review board approved the study protocol and all mothers provided written informed consent.
Venous cord blood samples were collected immediately after delivery into EDTA tubes, centrifuged and the plasma was stored at −80°C. Propeptides were each measured in a single batch using BRAHMS KRYPTOR automated immunofluorescent assays for CT-proET1, MR-proADM, and MR-proANP, as previously described, and using a research sandwich immunoluminometric assay (B·R·A·H·M·S C-terminal proAVP luminescence immunoassay; B·R·A·H·M·S AG, Hennigsdorf, Germany), as described by Morgenthaler et al, except that we replaced the capture antibody by a murine monoclonal antibody directed against the 137-144 amino acid residue (GRAGAL) of proAVP.
We performed all statistical analyses using STATA 10.1 statistics/data analysis software (StataCorp, College Station, TX). We used the Mann-Whitney test and χ 2 test then appropriate to compare groups at a 95% significance level, the Spearman rank correlation to test for associations between 2 variables, and stepwise multiple regression to test for putative influences on copeptin variation such as GA at delivery, birthweight percentile and umbilical artery RI z-scores.
Results
The study enrolled 54 pregnant mothers (IUGR: n = 12; AGA: n = 42). Indications for elective section in the IUGR group were a nonreassuring fetal heart rate pattern before the occurrence of any contractions and absence of fetal growth on serial ultrasound evaluation; no infant died or had severe morbidity after delivery. Indications for elective section in the AGA group were previous section, breech presentation, placenta previa, and section on demand.
Median maternal age, maternal parity and sex were similar in IUGR and AGA infants ( Table 1 ). Median IUGR birthweight was 2000 g, corresponding to the 0.4 th weight percentile on our reference chart. Median AGA birthweight was 3250 g, corresponding to the 55 th percentile. GA at delivery was significantly lower in the IUGR group, the GA ranges were similar in both groups, and therefore this difference was interpreted as less relevant ( Table 1 ).
| Characteristic | IUGR (n = 12) | AGA (n = 42) | P value |
|---|---|---|---|
| Maternal age, y | 31 (24–42) | 33 (22–45) | .235 |
| Multiparity, % | 58 | 62 | .823 |
| Gestational age, d | 257.5 (244–276) | 267.5 (247–278) | .001 |
| Birthweight, g | 2000 (1070–2560) | 3205 (2230–4000) | < .001 |
| Birthweight percentile | 0.4 (0–11.7) | 55 (4.2–99) | < .001 |
| Infant sex, male n, % | 8 (67) | 23 (55) | .462 |
| Umbilical artery pH | 7.30 (7.21–7.35) | 7.32 (7.20–7.38) | .300 |
| 5 min Apgar | 9 (9–9) | 9 (7–9) | .134 |
| Umbilical artery resistance index (AGA n = 16) | 0.77 (0.63–0.87) | 0.64 (0.52–0.73) | < .001 |
Blood flow was pathologic, defined by an increased umbilical artery RI, in all IUGR infants as opposed to normal, in terms of our reference chart, in all 16 AGA infants in which it was measured ( P < .001). End-diastolic flow in the umbilical arteries was absent in 2 IUGR infants.
Umbilical plasma concentrations of the 4 propeptides in the 12 IUGR and 12 AGA infants matched 1:1 in the 2005-2006 period were as follows (median [range]): copeptin 23.2 (6.7–449) vs 6.3 (4–9) pmol/L ( P < .001); CT-proET-1 132 (74–187) vs 118 (56–175) pmol/L (nonsignificant [NS]); MR-proADM 0.9 (0.69–2.8) vs 0.7 (0.57–0.89) nmol/L ( P = .019); and MR-proANP 307 (142–623) vs 238 (169–663) pmol/L (NS).
To compare the IUGR infants with a larger AGA group we matched an additional 30 AGA infants using the same criteria. Again, copeptin concentrations were significantly higher in the IUGR group ( P < .001, Figure ). Other plasma peptide concentrations did not differ between the 2 groups ( Table 2 ). Stepwise multiple regression revealed significant influence by the umbilical artery RI z-score ( P = .008) but none by either birthweight percentile ( P = .602) or GA at delivery ( P = .335).
| Peptide precursor | IUGR | AGA | P value a |
|---|---|---|---|
| Copeptin, pmol/L | 23.15 (6.7–449) | 5.14 (2.47–53.0) | < .01 |
| CT-proET-1, ng/mL | 132.0 (74.0–187.0) | 134.1 (56.0–202.6) | .49 |
| MR-proADM, pg/mL | 0.90 (0.69–2.8) | 0.93 (0.49–1.89) | .42 |
| MR-proANP, pg/mL | 307.0 (142.0–1146.0) | 241.2 (139.5–1590.3) | .60 |
Results
The study enrolled 54 pregnant mothers (IUGR: n = 12; AGA: n = 42). Indications for elective section in the IUGR group were a nonreassuring fetal heart rate pattern before the occurrence of any contractions and absence of fetal growth on serial ultrasound evaluation; no infant died or had severe morbidity after delivery. Indications for elective section in the AGA group were previous section, breech presentation, placenta previa, and section on demand.
Median maternal age, maternal parity and sex were similar in IUGR and AGA infants ( Table 1 ). Median IUGR birthweight was 2000 g, corresponding to the 0.4 th weight percentile on our reference chart. Median AGA birthweight was 3250 g, corresponding to the 55 th percentile. GA at delivery was significantly lower in the IUGR group, the GA ranges were similar in both groups, and therefore this difference was interpreted as less relevant ( Table 1 ).
