- Email: [email protected]
Increased fetoplacental angiogenesis during first trimester in anaemic women
EARLY REPORTS
Increased fetoplacental angiogenesis during first trimester in anaemic women Mamed Kadyrov, Georg Kosanke, John Kingdom, Peter Kaufmann
Summary Background Epidemiological studies describe an association between relative size of the placenta at delivery and cardiovascular morbidity and mortality during adult life. Some determinants of placental size, such as maternal anaemia, have been acknowledged, but no plausible mechanism has been advanced to explain the initiation of postnatal disease. Methods Placental villous vascularisation in anaemic women (Hb<90 g/L) was assessed in the first and third trimesters of pregnancy by immunohistochemical identification of villous capillaries and compared with that of gestational age-matched groups of women with normal (Hb>110 g/L; control group) concentrations of haemoglobin, and an intermediate group (Hb 90–110 g/L). Findings Anaemia, especially in the first trimester, was associated with increased numbers of capillaries per villous cross section (mean 11·70 [SE 0·35] vs 4·14 [0·27]) located mainly in the outer third of the stroma beneath the trophoblast (94% [1·15] vs 67% [1·82]) and with increased numbers of villous macrophages and of proliferating MIB-1-positive cells compared with the control group. Interpretation Maternal anaemia in early pregnancy seems to influence the pattern of placental vascularisation. Such changes might alter placental vascular impedance during early fetal life, thereby exerting important effects on cardiovascular development.
Lancet 1998; 352: 1747–49
Introduction Maternal anaemia resulting from iron deficiency is a common complication of pregnancy worldwide,1 particularly in developing countries.2 Screening and treatment are encouraged to prevent unnecessary blood transfusion after childbirth, though the need for routine iron supplementation is debated in well-nourished communities. Maternal anaemia is associated with increased placental weight, placental-weight ratio,3,4 and intrauterine-growth restriction,5 and epidemiological studies have linked these perinatal factors to adult cardiovascular disease.6,7 By term, the gas-exchanging placental villi show adaptive features in response to chronic anaemia, which increase the transfer of oxygen to Department of Anatomy, Medical Faculty, RWTH Aachen, Germany (M Kadyrov MD, G Kosanke PhD, Prof P Kaufmann MD); and Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Canada (J Kingdom MD) Correspondence to: Prof Peter Kaufmann, Department of Anatomy, Technical University of Aachen, Wendligweg 2, D-52057 Aachen, Germany (e-mail: [email protected])
THE LANCET • Vol 352 • November 28, 1998
the fetus,8,9 though the effects of iron-deficiency anaemia on early placental development are unknown. We investigated the hypothesis that angiogenesis in the developing human placenta is altered by pre-existing iron-deficiency anaemia. Our data support the general notion of placental programming in early gestation, which in turn may have effects on fetal development.
Methods Ethics-committee approval was obtained for the study. Placentas were collected consecutively according to their preoperative/predelivery Hb value, after elimination of all those cases which, first, were complicated by illness other than anaemia and which, second, did not meet criteria for gestational age (10–12 and 38–41 postmenstrual weeks, respectively). Placentas were collected from the following groups of otherwise healthy pregnant women: eight anaemic women (Hb<90 g/L, n=4 in each trimester) with a blood film consistent with iron deficiency; eight women with a blood Hb concentration of 90–110 g/L (n=4 in each trimester), with a blood film consistent with iron-deficiency; and a control group (n=8; Hb>110 g/L, n=4 in each trimester). All placentas were collected at the Medical Institute, Aschbad, Turkmenia. The tissues were fixed for 24 h in 4% phosphate-buffered formaldehyde (pH 7·2) after which a random 1 cm3 block was embedded in paraffin (melting point 52°C). 4 m paraffin sections were mounted on glass slides covered with 3-aminopropyl-ethoxy-silan (Sigma, Deisenhofen, Germany) and a standard haematoxylin and eosin stain was used to assess histological structure. Immunohistochemistry was done with three primary antibodies—QB-END/10, directed to the endothelial cell CD34 antigen (Quantum Biosystems, Cambridge, UK; dilution 1/10), anti-CD68, a specific marker of placental macrophages (Hofbauer cells; Dako, Glostrup, Denmark; dilution 1/1000), and MIB 1, a proliferation marker (Dianova, Hamburg, Germany; dilution 1/20). These were omitted in negative-control experiments. We detected primary antibody binding using a streptavidin-biotin sequence with AEC (3-amino-9-ethyl-carbazol; Sigma, Deisenhofen, Germany) as chromogen. For morphometric analysis, one tissue section per specimen was covered with a transparent grid consisting of 121 1 mm2 squares, 40 of which were randomly selected and numbered. We determined the total cross-sectional area of struturally intact villi in these squares by image analysis (MOP Videoplan, Kontron, Germany). The maximum diameter (oriented vertically to the longitudinal axis of each capillary profile) and cross-sectional area of each QB-End/10-positive capillary profile (lumen excluding endothelium) were measured by image analysis, together with its spatial orientation in the villus (defined by the allocation of the most peripheral point of each capillary profile to the outer, middle, or central third of the villous stroma, respectively). Data were pooled in each group (table). We measured capillary density (mean number of capillaries per villus and percentage of villous sectional area occupied by capillaries), together with mean capillary diameter. We assessed the extent of “peripheralisation” of the villous capillaries by calculating the percentage of capillaries allocated to the outer third of the stroma. The density of CD68-positive macrophages and of MIB-1 positive proliferating villous stromal cells per 10 000 mm2 total villous stromal sectional area were assessed in a similar way. We assessed the significance of the quantitative
1747
EARLY REPORTS Haemoglobin concentration in maternal blood (g/L)
Mean (SE) Number of capillaries per villous section
Percentage of villous stromal area occupied by capillaries
Capillary diameter in (m)
Percentage of capillaries in outer third of stroma
Number of macrophages per 10 000 m2
Number of proliferating cells per 10 000 m2
First trimester >110 90–110 <90
4·14 (0·27)* 7·89 (0·53) 11·70 (0·35)
0·53 (0·12) 0·97 (0·13) 0·73 (0·06)
20·42 (4·74) 18·29 (0·91) 16·21 (0·76)
66·5 (1·82)* 91·8 (0·80) 93·7 (1·15)
1·19 (0·26) 2·57 (0·34) 2·37 (0·32)
1·44 (0·23) 2·31 (0·21) 2·52 (0·30)
Third trimester >110 90–110 <90
5·95 (0·36)* 11·62 (0·61) 11·66 (0·50)
3·14 (0·06) 1·82 (0·07) 2·93 (0·41)
12·57 (0·96) 10·83 (0·55) 12·38 (0·56)
87·3 (1·93)* 86·8 (1·33) 86·4 (2·70)
0·98 (0·26)* 3·98 (1·11) 4·09 (0·68)
0·24 (0·01)* 0·54 (0·10) 0·70 (0·09)
Significant differences within each group of three mean values are marked by * (Kruskal-Wallis test; p<0·05). Further assessment of Wilcoxon test for all those groups marked by * revealed significant differences between controls (Hb>110 g/L) and intermediate groups (Hb 90–110 g/L), as well as between controls and those with anaemia (Hb<90 g/L). Increased numbers of capillary profiles indicate excessive branching angiogenesis in response to anaemia. For all groups, n=4.
Morphometric analysis of villous capillarisation, macrophage density, and stromal-cell proliferation in first and third trimester placentas according to haemoglobin concentrations in maternal blood data using the Kruskal-Wallis test. Significant differences were further analysed with the Wilcoxon test. For both methods, p values <0·05 were deemed significant.
Results Data are summarised in the table and representative histological sections are illustrated in the figure. In the first trimester, decreasing concentrations of haemoglobin in maternal blood were associated with a significant increase in the number of capillaries per villus cross section, which were almost exclusively located in the outer zone of the stroma, close to the trophoblast layer, in the anaemic group. These more numerous capillaries were of a smaller diameter than those in the control specimens, such that the increase in percentage of stroma occupied by capillaries was not significant. In the third
trimester, we recorded a similar increase in the number of capillary profiles per villus cross section, though we found no significant differences in the proportion of villous sectional stromal area occupied by capillaries, mean capillary diameter, or degree of peripheralisation. The density of villous macrophages increased with decreasing haemoglobin concentrations in both stages of gestation, as did the percentage of proliferating MIB-1 positive stromal (endothelial and connective tissue) cells, which were mostly integral parts of the developing stromal capillaries.
Discussion This study shows an alteration to the normal pattern of placental villous capillary development during the first trimester of pregnancy in anaemic women. It is
Representative sections of first trimester (A,B) and third trimester (C,D) placentas stained with QB-END/10 to localise villous capillaries Note increased peripheral vascularisation in anaemic group (Hb<90 g/L; B,D) compared with villi of control group (Hb>110 g/L; A,C) which is most pronounced in first trimester. Formation of superficial capillary networks (arrows) points to prevalence of branching angiogenesis in maternal anaemia (magnification ⫻110).
1748
THE LANCET • Vol 352 • November 28, 1998
EARLY REPORTS
characterised by increased numbers of proliferating stromal cells and increased density of fetal capillarisation in the outer part of the villous stroma. Our data suggest that most of the proliferating stromal cells are in fact endothelial cells (and adjacent pericytes). Their proliferation may be an adaptive response to increase oxygen transfer to the early developing fetus. Although some debate exists as to the timing of onset of maternal intervillous blood flow in early pregnancy,10 this circulation is probably established gradually by 12 weeks of postmenstrual age.11 Our data thus suggest that developing villi during the first trimester alter their normal pattern of angiogenesis in response to maternal anaemia. Previous studies have shown alterations in the density and structure of peripheral gas-exchanging villi in placentas from anaemic women at term,12 favouring increased fractional extraction of oxygen by the fetus from the intervillous space, though the timing of onset of these changes was previously unknown. During the first trimester, placental villi are progressively vascularised by a process of branching angiogenesis within the stromal core.13 This process seems to be under the influence of angiogenic growth factors—in particular, vascular endothelial growth factor (VEGF), a potent endothelial mitogen, which is produced by the villous trophoblast and stromal Since VEGF transcription is macrophages.14,15 upregulated in vitro by hypoxia,16,17 the increased villous angiogenesis that occurs in association with maternal anaemia may possibly be mediated through increased paracrine activity of VEGF. Our findings of increased numbers of villous macrophages, potential sources of VEGF, together with increased stromal and endothelial cell proliferation, supports this proposed mechanism of altered angiogenesis. Normal villous development during the second and early third trimesters is characterised by a gradual shift towards non-branching angiogenesis, resulting in the formation of gas-exchanging peripheral villi whose poorly branched capillary loops are located close to the syncytial surface for optimum diffusional exchange.13 Formation of these so-called terminal villi is essential for normal fetal growth. A gradual decline in placental expression of VEGF and its fit-1 receptor, in favour of increased placenta-like growth factor and KDR-receptor expression during pregnancy, may facilitate this switch to nonbranching angiogenesis.18 Persistent branching angiogenesis may be the basis for the typical histological features of the anaemic placenta at term8,9 owing to “preplacental” hypoxia.12 Persistent branching angiogenesis within peripheral villi may therefore account for the epidemiological association between maternal anaemia and a placenta that is large in proportion to the weight of the fetus.4 However, despite an increase in placental size, the functional capacity of the placenta is impaired, since fetal growth may be suboptimum.5 Such observations are of interest outside the perinatal period, since a proportionately large placenta at birth has been linked to cardiovascular morbidity during adult life.6 Interestingly, maternal anaemia throughout gestation was associated with a doubling of fetal heart weight in rats, which suggests an effect of altered early placental development on the fetal heart.19 Therefore, the short-term advantage to the fetus of increased placental angiogenesis in response to maternal anaemia may be at the expense of optimum
THE LANCET • Vol 352 • November 28, 1998
cardiovascular development during postnatal life. Our data thus provide a mechanistic link between maternal anaemia in pregnancy and adverse cardiovascular health in the offspring. This link deserves further inquiry, since the identification and treatment of anaemia in early pregnancy is a fairly straightforward task, even in underdeveloped communities.20 Contributors M Kadyrov collected material, and was responsible for immunohistochemistry and structural assessment. G Kosanke carried out morphometric assessment and statistical analyses. J C P Kingdom interpreted clinical data and prepared the manuscript. P Kaufmann designed the study, interpreted morphological data, and prepared the manuscript.
Acknowledgments We thank Thorsten Reinke (IMIB, Technical University of Aachen) for statistical assistance and Uta Zahn and Rita Ketabchi for technical assistance. M Kadyrov’s stay in Aachen was funded by Deutscher Akademischer Austauschdienst (DAAD) number A/95/14174. The study was supported by Deutsche Forschungsgemeinschaft grant number Gr 902/9-1 to GK and PK.
References 1 2
3 4
5 6 7 8
9
10
11
12 13 14
15
16 17
18
19
20
Allen LH. Pregnancy and iron deficiency: unresolved issues. Nutr Rev 1997; 55: 91–101. Guidozzi F, Patel R, MacPhail AP. A prospective study of iron status in white and black pregnant women in an urban hospital. S Afr Med J 1995; 85: 170–73. Lao TT, Wong WM. Placental ratio: its relationship with maternal anaemia. Placenta 1997; 18: 593–96. Williams LA, Evans SF, Newnham JP. Prospective cohort study of factors influencing the relative weight of the placenta and the newborn infant. BMJ 1997; 314: 1864–68. Singla PN, Tyagi M, Dash D, Shankar R. Fetal growth in maternal anaemia. J Trop Pediatr 1997; 43: 89–92. Barker DJP, Bull AR, Osmond C, Simmonds SJ. Fetal and placental size and risk of hypertension in adult life. BMJ 1990; 301: 259–62. Law CM, de Swiet M, Osmond C, et al. Initiation of hypertension in utero and its amplification throughout life. BMJ 1993; 306: 24–27. Reshetnikova OS, Burton GJ, Teleshova OV. Placental histomorphometry and morphometric diffusing capacity of the villous membrane in pregnancies complicated by maternal iron-deficiency anemia. Am J Obstet Gynecol 1995; 173: 724–27. Burton GJ, Reshetnikova OS, Milovanov AP, Teleshova OV. Stereological evaluation of vascular adaptations in human placental villi to differing forms of hypoxic stress. Placenta 1996; 17: 49–55. Jaffe RJ, Jauniaux E, Hustin J. Maternal circulation in the firsttrimester human placenta—myth or reality? Am J Obstet Gynecol 1997; 176: 695–705. Hustin J, Schaaps JP, Lambotte R. Anatomical studies of the uteroplacental vascularization in the first trimester of pregnancy. Trophoblast Res 1988; 3: 49–60. Kingdom JCP, Kaufmann P. Oxygen and placental villous development: origins of fetal hypoxia. Placenta 1997; 18: 613–21. Benirschka K, Kaufmann P. Pathology of the human placenta. 3rd edn. New York: Springer, 1995. Sharkey AM, Charnock-Jones DS, Boocock CA, Brown KD, Smith SK. Expression of mRNA for vascular endothelial growth factor in human placenta. J Reprod Fertil 1993; 99: 609–15. Ahmed A, Li XF, Whittle MJ, Rushton DI, Rollason T. Colocalisation of vascular endothelial growth factor and its flt-1 receptor in human placenta. Growth Factors 1995; 12: 235–43. Wheeler T, Elcock CL, Anthony FW. Angiogenesis and the placental environment. Placenta 1995; 16: 289–96. Shore VH, Wang TH, Torry RJ, Caudle MR, Torry D. Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast. Placenta 1997; 18: 657–65. Kaufmann P, Kingdom JCP. Development of the vascular system in the placenta. In: Risau W, ed. Morphogenesis of endothelium. Harwood Academic Publishers: Churchill Livingstone, 1998 (in press). Crowe C, Dandekar P, Fox M, Dhingra K, Bennet L, Hanson MA. The effects of anaemia on heart, placenta and body weight, and blood pressure in fetal and neonatal rats. J Physiol (Lond) 1995; 488: 515–19. Suharno D, West CE, Muhilal, Karyadi D, Hautvast JG. Supplementation with vitamin A and iron for nutritional anaemia in pregnant women in West Java, Indonesia. Lancet 1993; 342: 1325–28.
1749
Increased fetoplacental angiogenesis during first trimester in anaemic women Mamed Kadyrov, Georg Kosanke, John Kingdom, Peter Kaufmann
Summary Background Epidemiological studies describe an association between relative size of the placenta at delivery and cardiovascular morbidity and mortality during adult life. Some determinants of placental size, such as maternal anaemia, have been acknowledged, but no plausible mechanism has been advanced to explain the initiation of postnatal disease. Methods Placental villous vascularisation in anaemic women (Hb<90 g/L) was assessed in the first and third trimesters of pregnancy by immunohistochemical identification of villous capillaries and compared with that of gestational age-matched groups of women with normal (Hb>110 g/L; control group) concentrations of haemoglobin, and an intermediate group (Hb 90–110 g/L). Findings Anaemia, especially in the first trimester, was associated with increased numbers of capillaries per villous cross section (mean 11·70 [SE 0·35] vs 4·14 [0·27]) located mainly in the outer third of the stroma beneath the trophoblast (94% [1·15] vs 67% [1·82]) and with increased numbers of villous macrophages and of proliferating MIB-1-positive cells compared with the control group. Interpretation Maternal anaemia in early pregnancy seems to influence the pattern of placental vascularisation. Such changes might alter placental vascular impedance during early fetal life, thereby exerting important effects on cardiovascular development.
Lancet 1998; 352: 1747–49
Introduction Maternal anaemia resulting from iron deficiency is a common complication of pregnancy worldwide,1 particularly in developing countries.2 Screening and treatment are encouraged to prevent unnecessary blood transfusion after childbirth, though the need for routine iron supplementation is debated in well-nourished communities. Maternal anaemia is associated with increased placental weight, placental-weight ratio,3,4 and intrauterine-growth restriction,5 and epidemiological studies have linked these perinatal factors to adult cardiovascular disease.6,7 By term, the gas-exchanging placental villi show adaptive features in response to chronic anaemia, which increase the transfer of oxygen to Department of Anatomy, Medical Faculty, RWTH Aachen, Germany (M Kadyrov MD, G Kosanke PhD, Prof P Kaufmann MD); and Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Canada (J Kingdom MD) Correspondence to: Prof Peter Kaufmann, Department of Anatomy, Technical University of Aachen, Wendligweg 2, D-52057 Aachen, Germany (e-mail: [email protected])
THE LANCET • Vol 352 • November 28, 1998
the fetus,8,9 though the effects of iron-deficiency anaemia on early placental development are unknown. We investigated the hypothesis that angiogenesis in the developing human placenta is altered by pre-existing iron-deficiency anaemia. Our data support the general notion of placental programming in early gestation, which in turn may have effects on fetal development.
Methods Ethics-committee approval was obtained for the study. Placentas were collected consecutively according to their preoperative/predelivery Hb value, after elimination of all those cases which, first, were complicated by illness other than anaemia and which, second, did not meet criteria for gestational age (10–12 and 38–41 postmenstrual weeks, respectively). Placentas were collected from the following groups of otherwise healthy pregnant women: eight anaemic women (Hb<90 g/L, n=4 in each trimester) with a blood film consistent with iron deficiency; eight women with a blood Hb concentration of 90–110 g/L (n=4 in each trimester), with a blood film consistent with iron-deficiency; and a control group (n=8; Hb>110 g/L, n=4 in each trimester). All placentas were collected at the Medical Institute, Aschbad, Turkmenia. The tissues were fixed for 24 h in 4% phosphate-buffered formaldehyde (pH 7·2) after which a random 1 cm3 block was embedded in paraffin (melting point 52°C). 4 m paraffin sections were mounted on glass slides covered with 3-aminopropyl-ethoxy-silan (Sigma, Deisenhofen, Germany) and a standard haematoxylin and eosin stain was used to assess histological structure. Immunohistochemistry was done with three primary antibodies—QB-END/10, directed to the endothelial cell CD34 antigen (Quantum Biosystems, Cambridge, UK; dilution 1/10), anti-CD68, a specific marker of placental macrophages (Hofbauer cells; Dako, Glostrup, Denmark; dilution 1/1000), and MIB 1, a proliferation marker (Dianova, Hamburg, Germany; dilution 1/20). These were omitted in negative-control experiments. We detected primary antibody binding using a streptavidin-biotin sequence with AEC (3-amino-9-ethyl-carbazol; Sigma, Deisenhofen, Germany) as chromogen. For morphometric analysis, one tissue section per specimen was covered with a transparent grid consisting of 121 1 mm2 squares, 40 of which were randomly selected and numbered. We determined the total cross-sectional area of struturally intact villi in these squares by image analysis (MOP Videoplan, Kontron, Germany). The maximum diameter (oriented vertically to the longitudinal axis of each capillary profile) and cross-sectional area of each QB-End/10-positive capillary profile (lumen excluding endothelium) were measured by image analysis, together with its spatial orientation in the villus (defined by the allocation of the most peripheral point of each capillary profile to the outer, middle, or central third of the villous stroma, respectively). Data were pooled in each group (table). We measured capillary density (mean number of capillaries per villus and percentage of villous sectional area occupied by capillaries), together with mean capillary diameter. We assessed the extent of “peripheralisation” of the villous capillaries by calculating the percentage of capillaries allocated to the outer third of the stroma. The density of CD68-positive macrophages and of MIB-1 positive proliferating villous stromal cells per 10 000 mm2 total villous stromal sectional area were assessed in a similar way. We assessed the significance of the quantitative
1747
EARLY REPORTS Haemoglobin concentration in maternal blood (g/L)
Mean (SE) Number of capillaries per villous section
Percentage of villous stromal area occupied by capillaries
Capillary diameter in (m)
Percentage of capillaries in outer third of stroma
Number of macrophages per 10 000 m2
Number of proliferating cells per 10 000 m2
First trimester >110 90–110 <90
4·14 (0·27)* 7·89 (0·53) 11·70 (0·35)
0·53 (0·12) 0·97 (0·13) 0·73 (0·06)
20·42 (4·74) 18·29 (0·91) 16·21 (0·76)
66·5 (1·82)* 91·8 (0·80) 93·7 (1·15)
1·19 (0·26) 2·57 (0·34) 2·37 (0·32)
1·44 (0·23) 2·31 (0·21) 2·52 (0·30)
Third trimester >110 90–110 <90
5·95 (0·36)* 11·62 (0·61) 11·66 (0·50)
3·14 (0·06) 1·82 (0·07) 2·93 (0·41)
12·57 (0·96) 10·83 (0·55) 12·38 (0·56)
87·3 (1·93)* 86·8 (1·33) 86·4 (2·70)
0·98 (0·26)* 3·98 (1·11) 4·09 (0·68)
0·24 (0·01)* 0·54 (0·10) 0·70 (0·09)
Significant differences within each group of three mean values are marked by * (Kruskal-Wallis test; p<0·05). Further assessment of Wilcoxon test for all those groups marked by * revealed significant differences between controls (Hb>110 g/L) and intermediate groups (Hb 90–110 g/L), as well as between controls and those with anaemia (Hb<90 g/L). Increased numbers of capillary profiles indicate excessive branching angiogenesis in response to anaemia. For all groups, n=4.
Morphometric analysis of villous capillarisation, macrophage density, and stromal-cell proliferation in first and third trimester placentas according to haemoglobin concentrations in maternal blood data using the Kruskal-Wallis test. Significant differences were further analysed with the Wilcoxon test. For both methods, p values <0·05 were deemed significant.
Results Data are summarised in the table and representative histological sections are illustrated in the figure. In the first trimester, decreasing concentrations of haemoglobin in maternal blood were associated with a significant increase in the number of capillaries per villus cross section, which were almost exclusively located in the outer zone of the stroma, close to the trophoblast layer, in the anaemic group. These more numerous capillaries were of a smaller diameter than those in the control specimens, such that the increase in percentage of stroma occupied by capillaries was not significant. In the third
trimester, we recorded a similar increase in the number of capillary profiles per villus cross section, though we found no significant differences in the proportion of villous sectional stromal area occupied by capillaries, mean capillary diameter, or degree of peripheralisation. The density of villous macrophages increased with decreasing haemoglobin concentrations in both stages of gestation, as did the percentage of proliferating MIB-1 positive stromal (endothelial and connective tissue) cells, which were mostly integral parts of the developing stromal capillaries.
Discussion This study shows an alteration to the normal pattern of placental villous capillary development during the first trimester of pregnancy in anaemic women. It is
Representative sections of first trimester (A,B) and third trimester (C,D) placentas stained with QB-END/10 to localise villous capillaries Note increased peripheral vascularisation in anaemic group (Hb<90 g/L; B,D) compared with villi of control group (Hb>110 g/L; A,C) which is most pronounced in first trimester. Formation of superficial capillary networks (arrows) points to prevalence of branching angiogenesis in maternal anaemia (magnification ⫻110).
1748
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EARLY REPORTS
characterised by increased numbers of proliferating stromal cells and increased density of fetal capillarisation in the outer part of the villous stroma. Our data suggest that most of the proliferating stromal cells are in fact endothelial cells (and adjacent pericytes). Their proliferation may be an adaptive response to increase oxygen transfer to the early developing fetus. Although some debate exists as to the timing of onset of maternal intervillous blood flow in early pregnancy,10 this circulation is probably established gradually by 12 weeks of postmenstrual age.11 Our data thus suggest that developing villi during the first trimester alter their normal pattern of angiogenesis in response to maternal anaemia. Previous studies have shown alterations in the density and structure of peripheral gas-exchanging villi in placentas from anaemic women at term,12 favouring increased fractional extraction of oxygen by the fetus from the intervillous space, though the timing of onset of these changes was previously unknown. During the first trimester, placental villi are progressively vascularised by a process of branching angiogenesis within the stromal core.13 This process seems to be under the influence of angiogenic growth factors—in particular, vascular endothelial growth factor (VEGF), a potent endothelial mitogen, which is produced by the villous trophoblast and stromal Since VEGF transcription is macrophages.14,15 upregulated in vitro by hypoxia,16,17 the increased villous angiogenesis that occurs in association with maternal anaemia may possibly be mediated through increased paracrine activity of VEGF. Our findings of increased numbers of villous macrophages, potential sources of VEGF, together with increased stromal and endothelial cell proliferation, supports this proposed mechanism of altered angiogenesis. Normal villous development during the second and early third trimesters is characterised by a gradual shift towards non-branching angiogenesis, resulting in the formation of gas-exchanging peripheral villi whose poorly branched capillary loops are located close to the syncytial surface for optimum diffusional exchange.13 Formation of these so-called terminal villi is essential for normal fetal growth. A gradual decline in placental expression of VEGF and its fit-1 receptor, in favour of increased placenta-like growth factor and KDR-receptor expression during pregnancy, may facilitate this switch to nonbranching angiogenesis.18 Persistent branching angiogenesis may be the basis for the typical histological features of the anaemic placenta at term8,9 owing to “preplacental” hypoxia.12 Persistent branching angiogenesis within peripheral villi may therefore account for the epidemiological association between maternal anaemia and a placenta that is large in proportion to the weight of the fetus.4 However, despite an increase in placental size, the functional capacity of the placenta is impaired, since fetal growth may be suboptimum.5 Such observations are of interest outside the perinatal period, since a proportionately large placenta at birth has been linked to cardiovascular morbidity during adult life.6 Interestingly, maternal anaemia throughout gestation was associated with a doubling of fetal heart weight in rats, which suggests an effect of altered early placental development on the fetal heart.19 Therefore, the short-term advantage to the fetus of increased placental angiogenesis in response to maternal anaemia may be at the expense of optimum
THE LANCET • Vol 352 • November 28, 1998
cardiovascular development during postnatal life. Our data thus provide a mechanistic link between maternal anaemia in pregnancy and adverse cardiovascular health in the offspring. This link deserves further inquiry, since the identification and treatment of anaemia in early pregnancy is a fairly straightforward task, even in underdeveloped communities.20 Contributors M Kadyrov collected material, and was responsible for immunohistochemistry and structural assessment. G Kosanke carried out morphometric assessment and statistical analyses. J C P Kingdom interpreted clinical data and prepared the manuscript. P Kaufmann designed the study, interpreted morphological data, and prepared the manuscript.
Acknowledgments We thank Thorsten Reinke (IMIB, Technical University of Aachen) for statistical assistance and Uta Zahn and Rita Ketabchi for technical assistance. M Kadyrov’s stay in Aachen was funded by Deutscher Akademischer Austauschdienst (DAAD) number A/95/14174. The study was supported by Deutsche Forschungsgemeinschaft grant number Gr 902/9-1 to GK and PK.
References 1 2
3 4
5 6 7 8
9
10
11
12 13 14
15
16 17
18
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