Small for gestational age (SGA) human newborns occur in as many as 10% of pregnancies. Smallness for gestational age is a main cause of fetal perinatal morbidity and mortality. In the United States, intrauterine growth restriction is linked to an increase of six to ten times in perinatal mortality.1 The pathophysiologic processes that occur at the cellular and molecular levels in intrauterine growth restriction are still largely unknown. Clinical studies have documented that the rate of intrauterine growth restriction varies among ethnic groups, implying that intrauterine growth restriction has genetic components. Many studies have shown that genetic factors influence the fetus weight and length.2,3 Investigators have also shown that intrauterine growth restriction occurs when there is a mismatch between maternal placental perfusion and fetal demand.4 This commonly occurs as a result of failed maternal blood volume expansion.5
Angiotensinogen is the precursor of the vasoactive hormone angiotensin II, which plays an important role in blood pressure regulation, body fluid volume, and vascular remodeling. It has been shown that a common mutation in the angiotensinogen promoter leads to elevated expression of this gene's pleiotropic effects, including abnormal spiral artery remodeling and failed pregnancy volume expansion.6,7 Molecular variants of the angiotensinogen gene, which increase angiotensinogen expression in certain local systems, predispose women to develop specific pregnancy complications.8
This study examines the association of angiotensinogen allele frequencies with intrauterine growth restriction.
MATERIALS AND METHODS
Intrauterine growth restriction patients and normal controls were recruited from women with singleton pregnancies delivered at University of Utah Health Sciences Center in 1998–2000. Written informed consent was obtained from women donating their blood and placenta samples. Approval for this study was obtained from the institutional review board of the University of Utah. Maternal blood samples were obtained at the time of hospital admission, and the fetal blood samples were obtained from the umbilical cord after delivery. Intrauterine growth restriction in this study was defined as the delivery of a structurally normal singleton fetus with a birth weight of less than the fifth percentile using population-specific birth weight charts. Preeclampsia was defined as the development of new-onset hypertension of at least 140/90 with postpartum resolution and either more than 500 mg of protein in 24 hours or a new 3+ dipstick without infection in a pregnant woman with a singleton pregnancy. Because of the known association in our population of angiotensinogen Thr235 polymorphisms with preeclampsia, we distinguished between pregnancies complicated by intrauterine growth restriction alone and those complicated by intrauterine growth restriction plus preeclampsia. Women with chronic hypertension, renal disease, diabetes mellitus, or fetal abnormalities were excluded from the study. We recruited 174 patients with intrauterine growth restriction and 60 patients with both intrauterine growth restriction and preeclampsia as the study groups and 400 consecutive cases of women with term pregnancies and their infants with birth weight between the fifth and 95th percentiles. For the fetal study, we recruited 162 umbilical cord blood samples from intrauterine growth restriction cases and 240 as the control group. No significant differences were found in ethnicity or maternal age between groups of control, intrauterine growth restriction, and preeclampsia/intrauterine growth restriction.
Maternal and fetal genomic deoxyribonucleic acid (DNA) was extracted from whole blood by means of the Puregene system (Gentra System Inc., Research Triangle Park, NC). Deoxyribonucleic acid was genotyped for Thr235/Met235 polymorphism by mutagenically separated polymerase chain reaction (PCR), which was performed using two different allele-specific upstream primers and a common downstream primer (angiotensinogen Int 2: 5′-AGC AGA GTT TGC CTT ACC-3′) from angiotensinogen intron 2. The upstream primers were as follows: 1) angiotensinogen Met235: 5′-GTT CAT GCA GGC TGT GAC AGC TTG GAA GAC TGG CTG CTC CCT CAT-3′, its 3′-nucleotide anneals to the Met235 polymorphic site (ATG), and 2) angiotensinogen Thr235: 5′-GAT GGA AGA CTG GCT GCT CCC AGA C-3′, its 3′-nucleotide anneals to the Thr235 polymorphic site (ACG). The angiotensinogen Met235-specific primer was designed to be 20 nucleotides longer than the angiotensinogen Thr235 primer. Thus, samples that are homozygous Met235 generated a 118–base pair product. Samples that are homozygous Thr235 generated a 98–base pair product and Met235/ Thr235 heterozygous generated both a 118– and a 98–base pair product. Polymerase chain reaction was performed in a total volume of 25 μL containing the following: 300 ng of genomic DNA; 10-mmol/L trishydrochloride, pH 8.4; 40-mmol/L NaCl; 1.5-mmol/L MgCl2, 0.5-μmol/L angiotensinogen Int 2; 0.125-μmol/L angiotensinogen Thr235; 0.25-μmol/L angiotensinogen Met235; 0.25-mmol/L spermidine; 200-mmol/L deoxynucleotide triphosphates; and 1.25 U of Taq polymerase (Perkin Elmer Corp., Norwalk, CT). Reactions were denatured at 94C for 5 minutes, followed by five cycles of 94C (10 seconds), 65C (10 seconds), and 72C (20 seconds), and then 30 cycles of 94C (10 seconds), 61C (10 seconds), and, 72C (20 seconds), at last hold at 4C (Gen′ Amp PCR 9600, Perkin Elmer). The mutagenically separated PCR products were visualized by ethidium bromide staining after electrophoretic fractionation through a 5% 3:1 Nusiere, Seakem agarose gel (FMC Bioproducts, Rockland, ME). Controls were sequenced to confirm genotype, and standard 100–base pair marks were used for observation. The presence of only the Met235- or the Thr235-specific PCR product indicated that the subject was homozygous (MM) for the Met235 allele or homozygous (TT) for the Thr235 allele, whereas when both products were seen, such a subject was heterozygous (MT).
The number of Met235 alleles was compared with that of Thr235 alleles, and the number of homozygous TT women was compared with those of other genotypes. Genotype and allele frequencies in the intrauterine growth restriction or intrauterine growth restriction/preeclampsia group were compared with those in the control group. Fetal birth weight was analyzed on the basis of the angiotensinogen genotype. The differences were statistically analyzed by the χ2 test (df = 1).
Genotypes were determined in 174 intrauterine growth restriction women, 60 intrauterine growth restriction/ preeclampsia women, and 400 normal women. For fetal genotype determination, we recruited 162 intrauterine growth restriction fetuses and 240 normal fetuses. Clinical characteristics and pregnancy outcomes of different groups are shown in Table 1.
The genotype results were divided into three groups: MM (homozygous for the angiotensinogen Met235 allele), TT (homozygous for the angiotensinogen Thr235 allele), and MT (heterozygous) (Figure 1). The results show that the rate of TT homozygosity and the frequency of the T allele were both significantly higher in the intrauterine growth restriction or preeclampsia/intrauterine growth restriction group in both maternal and fetal genotypes than those of the control group (P < .001). In the intrauterine growth restriction or preeclampsia/intrauterine growth restriction group, the maternal MT genotype is higher than MM. The frequency of the TT genotype is higher than that of MT or MM (P < .001, respectively) (Table 2). The frequencies of the angiotensinogen T235 allele are .60 in the intrauterine growth restriction group and .63 in the preeclampsia/ intrauterine growth restriction group, and are significantly higher than that in the control group (.36) (P < .001, respectively) (Table 3).
In fetal genotype, there are more MT and TT in the intrauterine growth restriction than in the control group (P < .001) (Table 2). The angiotensinogen T235 allele in intrauterine growth restriction has a frequency of .59, much higher than in the control group (.38) (P < .001) (Table 3). Fetal birth weight was divided into four grades of less than 1500 g, 1500–2499 g, 2500–3499 g, and 3500 g or more. Its association with the angiotensinogen genotype was shown in Table 4. Analysis based on the different genotype shows that the birth weights in the MM, MT, and TT groups significantly differ (P < .01). There are more MT or TT genotypes in the birth weight range of less than 1500 g and 1500–2499 g (41.8% for MT and 39.2% for TT) than the MM genotype (22.0%). The difference is statistically significant (P < .01 between MT and MM, P < .05 between TT and MM).
Intrauterine growth restriction infants have more cognitive and medical problems, such as asphyxia, neonatal hypoglycemia, meconium aspiration, and persistent fetal circulation. Intrauterine growth restriction is a leading cause of fetal perinatal morbidity and mortality.9–11 Most commonly caused by inadequate maternal-fetal circulation, a decrease in fetal growth defines intrauterine growth restriction.12 The cellular and molecular mechanism of the development of intrauterine growth restriction is largely unknown, but there are many hypotheses regarding the pathophysiology of intrauterine growth restriction.13–16 Epidemiological studies have shown that intrauterine growth restriction is a multifactorial disease dependent on both genetic and epigenetic factors. Failure of either of these mechanisms leads to fetal growth restriction.
Discovery of an underlying major gene could lead to a reanalysis of many of the disputed or contradictory findings in the intrauterine growth restriction literature. Stratifying the results of other studies by angiotensinogen genotype or adjusting data for the angiotensinogen allele frequencies may clarify the role of other pathophysiologic factors.17
Our previous studies have focused on the detection of angiotensinogen polymorphisms and the corresponding genotypes in women with preeclampsia.6 Our results show that the Thr235 allele is associated with uterine spiral artery changes in the first trimester that may be a key factor in the development of preeclampsia. A similar study in Japan found that pregnant women with the Thr235 allele were significantly more likely than normal controls to develop preeclampsia. The women carrying the Thr235 allele had higher concentrations of plasma angiotensinogen, a finding that persisted in the nonpregnant period.18
The adaptation of maternal and fetal circulation(s) plays a key role in fetal growth and development. During pregnancy, the placental circulation undergoes extensive adaptation to fulfill its role in delivery of nutrients, disposal of wastes, and heat exchange. Maternal arterial umbilical blood flow increases from 50 mL per minute early in pregnancy to about 700 mL per minute at term.19 The increase is followed by a gradual decrease in blood through the pregnancy. This physiologic change is due to the maternal spiral artery changes. Disturbance of the normal circulatory adaptation during pregnancy may threaten maternal and fetal well-being. Because pregnancies with increased placental mass (eg, twins) and relative reduction of placental perfusion are at increased risk of intrauterine growth restriction, investigators have postulated that intrauterine growth restriction occurs when there is a mismatch between maternal blood supply and fetal or placental demand.20–24 Pregnant women with intrauterine growth restriction have abnormal spiral artery remodeling, reduced maternal blood volume, and reduced placental blood flow.25,26 All of these abnormalities are seen with increased frequency in pregnancies where the angiotensinogen Thr235 allele is present in either the maternal or the fetal genotype.6,27 This study also demonstrates that the fetal angiotensinogen Thr235 allele is associated with intrauterine growth restriction, there being more in homozygous positive neonates than in heterozygous positive neonates.
The uteroplacental insufficiency is thought to be a failure of trophoblastic invasion by maternal spiral arterioles by 20–22 weeks' gestation. Current studies have demonstrated that the pregnancy-induced physiologic changes of the spiral arteries are different even in the first trimester. This failure causes narrowing and medial degeneration, leading to diminished blood flow to the developing infant. Consequently, these fetuses fail to grow normally.
A number of the common pregnancy disorders appear to be related to similar pathologic changes in the placenta. Intrauterine growth restriction, preeclampsia, spontaneous abortion, and preterm labor have similar placental findings.5,28 Histological studies have demonstrated failure of trophoblastic invasion in both preeclampsia and intrauterine growth restriction.29
This study has demonstrated the association between angiotensinogen Thr235 and intrauterine growth restriction. The results show that angiotensinogen Thr235 is a possible risk factor for fetal growth impairment. The mechanisms by which this occurs and how this allele influences the metabolism of angiotensin and the maternal-placental and fetal-placental circulation are the subject of ongoing investigations.
1. Bernstein I, Gabbe SG. Intrauterine growth restriction. In: Gabbe SG, Niebyl JR, Simpson JL, Annas GJ, eds. Obstetrics: Normal and problem pregnancies, 3rd ed. New York: Churchill-Livingstone, 1996:863–86.
2. Nogami H, Yokose T, Tachibana T. Regulation of growth hormone expression in fetal rat pituitary gland by thyroid or glucocorticoid hormone. Am J Physiol 1995;268:E262–7.
3. Amante A, Borgiani P, Gimelfarb A, Gloria-Bottini F. Interethnic variability in birth weight and genetic background: A study of placental alkaline phosphatase. Am J Phys Anthropol 1996;101:449–53.
4. Khong TY, De Wolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants. Br J Obstet Gynaecol 1986;93:1049–59.
5. Salafia CM, Vogel CA, Bantham KF, Vintzileos AM, Pezzullo J, Silberman L. Preterm delivery: Correlations of fetal growth and placental pathology. Am J Perinatol 1992; 9:190–3.
6. Morgan T, Craven C, Lalouel J-M, Ward K. Angiotensinogen Thr235 variant is associated with abnormal physiologic change of the uterine spiral arteries in first-trimester decidua. Am J Obstet Gynecol 1999;180:95–102.
7. Ward K, Hata A, Jeunemaitre X, Helin C, Nelson L, Namikawa C, et al. A molecular variant of angiotensinogen associated with preeclampsia. Nat Genet 1993;4:59–61.
8. Olson AL, Perlman S, Robillard JE. Developmental regulation of angiotensinogen gene expression in sheep. Pediatr Res 1990;28:183–5.
9. Piper JM, Xenakis EM, McFarland M, Elliott BD, Berkus MD, Langer O. Do growth-retarded premature infants have different rates of perinatal morbidity and mortality than appropriately grown premature infants? Obstet Gynecol 1996;87:169–74.
10. Dashe JS, McIntire DD, Lucas MJ, Leveno KJ. Effects of symmetric and asymmetric fetal growth on pregnancy outcomes. Obstet Gynecol 2000;96:321–7.
11. Savitz DA, Ananth CV, Berkowitz GS, Lapinski R. Concordance among measures of pregnancy outcome based on fetal size and duration of gestation. Am J Epidemiol 2000;151:627–33.
12. Joern H, Rath W. Comparison of Doppler sonographic examinations of the umbilical and uterine arteries in high-risk pregnancies. Fetal Diagn Ther 1998;13:150–3.
13. Gruenwald P. Growth of the human fetus. I. Normal growth and its variation. Am J Obstet Gynecol 1966;94:1112–9.
14. Keirse MJ. Epidemiology and aetiology of the growth retarded baby. Clin Obstet Gynaecol 1984;11:415–36.
15. Casalino M. Intrauterine growth restriction: A neonatologist's approach. J Reprod Med 1975;14:248–50.
16. Meredith HV. Body weight at birth of viable human infants: A worldwide comparative treatise. Hum Biol 1970;42:217–64.
17. Cooper AC, Robinson G, Vinson GP, Cheung WT, Broughton Pipkin F. The localization and expression of the renin-angiotensin system in the human placenta throughout pregnancy. Placenta 1999;20:467–74.
18. Rotimi C, Cooper R, Ogunbiyi O, Morrison L, Ladipo M, Tewksbury D, et al. Hypertension, serum angiotensinogen, and molecular variants of the angiotensinogen gene among Nigerians. Circulation 1997;95:2348–50.
19. Vandenbosche RC, Kirchner JT. Intrauterine growth restriction. Am Fam Physician 1998;58:1384–90,1393–4.
20. Macara L, Kingdom JC, Kaufmann P, Kohnen G, Hair J, More IA, et al. Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms. Placenta 1996;17:37–48.
21. Roberts R. Pregnancy related hypertension. In: Resnik R, ed. Maternal fetal medicine: Principles and practice. Philadelphia: W. B. Saunders, 1989:777–823.
22. Redman CW. Current topics: Pre-eclampsia and the placenta. Placenta 1991;12:301–8.
23. Tenney B. Placenta in toxemia of pregnancy. Am J Obstet Gynecol 1940;39:10000–5.
24. Schweikhart G, Kaufmann P, Beck T. Morphology of placental villi after premature delivery and its clinical relevance. Arch Gynecol 1986;239:101–14.
25. Kingdom JC, Kaufmann P. Oxygen and placental vascular development. Adv Exp Med Biol 1999;474:259–75.
26. Salas SP, Rosso P, Espinoza R, Robert JA, Valdes G, Donoso E. Maternal plasma volume expansion and hormonal changes in women with idiopathic fetal growth restriction. Obstet Gynecol 1993;81:1029–33.
27. Morgan T, Craven C, Nelson L, Lalouel JM, Ward K. Angiotensinogen Thr235 expression is elevated in decidual spiral arteries. J Clin Invest 1997;100:1406–15.
28. Rai R, Regan L. Obstetric complications of antiphospholipid antibodies. Curr Opin Obstet Gynecol 1997;9:387–90.
29. Stallmach T, Hebisch G, Joller-Jemelka HI, Orban P, Schwaller J, Engelmann M. Cytokine production and visualized effects in the feto-maternal unit. Quantitative and topographic data on cytokines during intrauterine disease. Lab Invest 1995;73:384–92.