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Vascularization in first-trimester chorionic villi in complicated and uncomplicated pregnancies
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Vascularization in first-trimester chorionic villi in complicated and uncomplicated pregnancies Monique A. Huisman, MD; Bert Timmer, PhD; Janet Stegehuis, MD; Bert Swart, MD; Jan G. Aarnoudse, PhD; JanJaap H. M. Erwich, PhD OBJECTIVE: The purpose of this study was to investigate possible al-
tered chorionic vascularization patterns that are seen already in the first trimester of pregnancies that are complicated by hypertensive disorders or intrauterine growth restriction (IUGR) in the third trimester of pregnancy. STUDY DESIGN: After chorionic villous sampling, surplus of villi were
stored, and a selection was made of pregnancies that were complicated further by hypertensive disorders (n ⫽ 26), normotensive IUGR (n ⫽ 13), and matched control subjects (n ⫽ 60). Vascular parameters of these villi were analyzed with a video-image-analysis system. RESULTS: In pregnancies that are complicated by early-onset hyper-
tensive disorders and IUGR, the mean distance of the peripheral vessels
to the intervillous space and the total of the distances (central and peripheral) are significantly smaller, compared with control subjects (9.3% and 13.8% for hypertensive disorders and 12.2% and 16.1% for IUGR, respectively). CONCLUSION: Differences in vascularization patterns in the placenta
already in the first trimester of pregnancies that are complicated later by hypertensive disorders or IUGR confirm the hypothesis of early changes by means of more vessels and more peripheral vessels that are located in chorionic villi. Key words: chorionic villi, intrauterine growth restriction, preeclampsia, vascularization
Cite this article as: Huisman MA, Timmer B, Stegehuis J, et al. Vascularization in first-trimester chorionic villi in complicated and uncomplicated pregnancies. Am J Obstet Gynecol 2010;202:88.e1-7.
A
fter delivery, placentas of pregnancies that were complicated by preeclampsia or intrauterine growth restriction (IUGR) show morphologically different vascularization patterns in the chorionic villi, compared with those pla-
From the Departments of Obstetrics and Gynecology (Drs Huisman, Stegehuis, Swart, Aarnoudse, and Erwich) and Pathology and Laboratory Medicine (Dr Timmer), University Medical Center Groningen, The Netherlands. Presented at the 52nd Annual Meeting of the Society for Gynecologic Investigation, Los Angeles, CA, March 23-26, 2005. Received March 19, 2009; revised June 14, 2009; accepted Aug. 20, 3009. Reprints: M.A. Huisman, MD, Department of Obstetrics and Gynaecology, Deventer Hospital, Nico Bolkesteinlaan 75,7416 SE Deventer, The Netherlands. [email protected]. Authorship and contribution to the article is limited to the 6 authors indicated. There was no outside funding or technical assistance with the production of this article. 0002-9378/$36.00 © 2010 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2009.08.036
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centas of uncomplicated pregnancies.1,2 The cause of the described differences is not completely known, and differences in early vascularization patterns between early and late onset preeclampsia are not well described. Kingdom and Kaufmann3 proposed a common model to explore the origins of fetal hypoxia with different vascularization patterns. In their model, they distinguish preplacental, uteroplacental, and postplacental (fetal) hypoxia, with each specific morphologic or physiologic antecedents. In the case of uteroplacental hypoxia that is associated with hypertension, failed extravillous cytotrophoblast invasion of the maternal spiral arteries causes a restriction for the normally oxygenated maternal blood to enter the intervillous space. Branching angiogenesis increases, which results in a greater amount of highly vascularized villi and a dilation of the villous capillaries, and causes an increased capillary volume fraction and an increased fetal syncytial surface area for diffusional exchange. In postplacental hypoxia that is associated with IUGR, angiogenesis is nonbranching, and few slender and fibrotic chorionic villi are
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found with reduced numbers of capillaries. In the current study, we investigated whether the morphologically altered vascularization patterns can be found already in first-trimester chorionic villi of ongoing pregnancies that were complicated with early- or late-onset hypertensive disorders such as preeclampsia and/or IUGR in the third trimester of pregnancy, compared with the vascularization patterns of further uncomplicated pregnancies. In accordance to the model of Kingdom and Kaufman,3 we hypothesized to find more and larger and perhaps more peripherally situated vessels in pregnancies that were complicated by hypertensive disorders to allow optimal maternal-fetal exchange of oxygen and nutrition between the intervillous maternal blood and the villi. Subsequently, concerning IUGR, fewer and smaller vessels were expected to be found in pregnancies that were complicated with IUGR, because at term, these pregnancies seemed to lack the adaptive response that was seen in pregnancies that were complicated with preeclampsia. Villi were stained with a monoclonal an-
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FIGURE
Analysis of CD34-stained human chorionic villi
A, Random selection of structurally intact parts of villi; B, contours of immature intermediate villi were traced and drawn on screen (green); C centrally located vascular elements (green) drawn on screen, followed by D, the peripherally located vessels; E, measurement of the distance from the luminal borders of a vascular element to the intervillous space (arrow). Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
tibody against the CD34 antigen to visualize vascular elements and subsequently were analyzed with a video-image analysis system.
P ATIENTS AND M ETHODS Patients Over the years, ⬎2000 woman have undergone vaginal chorionic villous sampling between 10 and 12 weeks of gestation, mainly for maternal age or serum screening that was related risk of aneuploidy. Gestational age was calculated according to the last menstrual period and subsequently confirmed by ultrasound evaluation (crown-rump length). Tissue was obtained with a biopsy catheter (K-CMA-5000; Cook Medical Inc, Bloomington, IN). Surplus material, which was not needed for karyotyping,
was obtained, and decidua was mechanically separated from the villi. The villi were placed in formaldehyde immediately and stored until further processing. Villi were embedded in paraffin, subsequently cut into 4-m sections and fixed on 3-aminopropyl-triethoxy-silane– coated (A3648; Sigma Chemical Company, St. Louis, MO) slides. Follow-up evaluation of the pregnancies was available in ⬎85% of cases and consisted of a questionnaire that was returned by the patient after delivery; additional data were extracted from the clinical notes. A selection was made of pregnancies that were complicated by hypertensive disorders (n ⫽ 26), such as preeclampsia and pregnancy-induced hypertension, and pregnancies that were complicated by normotensive IUGR
(n ⫽ 13). Pregnancy-induced hypertension, preeclampsia, and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome were defined according to the International Society for the Study of Hypertension in Pregnancy criteria4: pregnancy-induced hypertension was defined as blood pressure ⬎140/90 mm Hg after 20 weeks of gestation on 2 occasions at least 6 hours apart; preeclampsia was defined as an increase in blood pressure to at least 140/90 mm Hg after week 20 of gestation in a previously normotensive woman, combined with proteinuria (protein excretion at least 0.3 g per 24 hours, spot urine protein/creatinine ratio of 530 mg/mmol or at least 2⫹ protein by dipstick); HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome was defined
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TABLE 1
Clinical data of study population Hypertensive disorderb
Intrauterine growth restriction
Cases (n ⴝ 26)
Control subjects (n ⴝ 37)
Cases (n ⴝ 10)c
Control subjects (n ⴝ 18)
38 (36–42)
39 (35–43)
38 (36–43)
37 (36–40)
Chorionic villous sampling, d
76 (71–83)
77 (71–85)
76 (72–80)
75 (69–81)
Delivery, wk
39 (32–42)
39 (35–43)
38 (36–43)
38 (36–40)
Variablea Maternal age, y
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
Birthweight, g
3090 (1400–4325)
3586 (2525–4620)
2555 (2110–2840)
3840 (3280–4590)
................................................................................................................................................................................................................................................................................................................................................................................ a
b
Data are given as mean (range); Consisted of 13 cases of pregnancy-induced hypertension, 10 cases of preeclampsia, and 3 cases of HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, according to the criteria of the International Society for the Study of Hypertension in Pregnancy; c All intrauterine growth restriction cases were normotensive.
Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
as hemolysis, lactic dehydrogenase ⬎600 U/L, aspartate aminotransferase ⬎70 U/L, alanine aminotransferase ⬎90 U/L, and platelet-count ⬍100 g/L. For IUGR, birthweight was corrected for gestational age, and percentiles were computed. IUGR was defined as a corrected birthweight below the 5th percentile, according to Kloosterman.5 All the included cases in the IUGR group concerned normotensive pregnancies. For each case, control subjects were selected and matched for maternal age, parity, and gestational age at time of sampling (n ⫽ 60). Patients with concurrent morbidity (eg, preexisting hypertension or diabetes mellitus) who were smoking or receiving medication were excluded. Fetal karyotyping showed no chromosomal abnormalities. Patients were informed that surplus material could be used for research, according to the “Code for proper use of human tissue,” version 2002, of the Federation of Dutch Medical Scientific Societies and the Local Ethics Committee of the University Medical
Center Groningen. Patients who were included gave informed consent; after the outcome of the pregnancy was known, data were made anonymous.
Immunohistochemistry After the slides were deparaffinized with a series of xylene and alcohol and washed in phosphate-buffered saline solution (PBS; pH 7.4) 3 times for 5 minutes, the nonspecific peroxidase activity was blocked with 1% H2O2 (Merck, Darmstadt, Germany) in PBS for 30 minutes. Antigen retrieval was performed by incubation in Tris-HCL (buffer) for 30 minutes by 94°C in a microwave. Subsequently the slides were incubated with the mouse monoclonal primary antibody against the CD34 antigen (Clone QBEND10; Immunotech, Marseille, France) for 1 hour.3 The CD34 antibody was used at 1:100 dilution in PBS, in which 1% bovine serum albumin and 1% normal human AB serum were included, both to minimize nonspecific reactivity. The second and third step of an-
tibody labeling was carried out with peroxidase-conjugated rabbit anti-mouse immunoglobulin (RAMpo; DAKO A/S, Glostrup, Denmark) and peroxidase conjugated goat anti-rabbit immunoglobulin (GARpo; DAKO), respectively, both 1:50 in PBS/1% bovine serum albumin/1% AB serum for 30 minutes. All incubation steps were followed by 3 washes in PBS for 5 minutes. Finally, the peroxidase reaction was visualized with diaminobenzidine (Sigma Chemical Company). Subsequently the slides were counterstained with hematoxylin, dehydrated, and mounted with mounting medium (International Medical Products, Zutphen, The Netherlands).
Video Image Analysis To quantify vascular development in the chorionic villi, a video-image analysis system was used (Qwin 2.4; Leica, Cambridge, England). Microscope images were recorded with a color video camera (DC 300 V2.0; Leica) that was mounted on a light microscope (Axioskop 130 VA Type
TABLE 2
Data of pregnancies with early-onset hypertensive disorders Case
Disorder
Neonatal weight, ga
Week of delivery
1
Pregnancy-induced hypertension
1940 (25-50)
34
2
HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome
1610 (⬍5)
36
3
HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome
1400 (25)
32
4
Preeclampsia
1885 (⬍10)
36
5
Preeclampsia
2110 (25)
35
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
HELLP, hemolysis, elevated liver enzymes, and low platelet count. a
Percentile given in parentheses.
Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
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TABLE 3
Vascular parameters of the pregnancies complicated with intrauterine growth restriction, compared with control subjects
Parameter
Intrauterine growth restriction (n ⴝ 10)
Control subjects (n ⴝ 18)
Mean
Mean
SD
SD
P value
Vascular surface, %
.......................................................................................................................................................................................................................................................................................................................................................................
Central
1.77
0.86
1.94
1.13
NS
Peripheral
1.61
0.49
1.60
0.64
NS
Total
3.38
1.11
3.53
1.38
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Vessel no., mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
40.28
17.64
47.21
17.15
NS
Peripheral
137.87
37.78
124.03
28.81
NS
Total
178.15
42.16
171.24
39.23
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Perimeter, mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
3697
1152
4200
1365
NS
Peripheral
6863
1587
6776
1931
NS
10831
2328
10,976
2749
NS
....................................................................................................................................................................................................................................................................................................................................................................... .......................................................................................................................................................................................................................................................................................................................................................................
Total
................................................................................................................................................................................................................................................................................................................................................................................
Distance luminal border to intervillous space, m
.......................................................................................................................................................................................................................................................................................................................................................................
Central
58.09
11.53
60.49
10.35
NS
Peripheral
24.91
3.22
28.38
3.04
.012
Total
37.27
9.11
44.43
6.33
.045
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................
NS, not significant. Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
B; Carl Zeiss, Goettingen, Germany) with a ⫻20 objective. For each case, 5-15 images with (parts of) structurally intact immature intermediate villi were recorded randomly and evaluated. Random fields were chosen by means of knight’s move selection. Once recorded, no adjustments were done to provide structurally intact villi to prevent selection bias. First, from the recorded chorionic villi (Figure, A), the contours were traced manually on the computer monitor and drawn on screen with a mouse-controlled cursor, after which the villous area could be measured by the analysis system (Figure, B). Subsequently, every single central vascular element, which was made visible by the CD34-stained endothelial cells, was traced manually and drawn on screen, and the vascular area was measured (Figure, C). Any brown-stained endothelial cell or endothelial cell cluster (hemangioblastic cord), with or without a vessel lumen, that was clearly separate from adjacent
vessels was considered to be a single countable vessel. The same was done for every single peripheral vascular element (Figure, D). Division of the vascular elements into central or peripheral was done by the judgment of the investigator regarding the position of the vascular element to the syncytio-/cytotrophoblast. The total vascular area, the total vascular perimeter, and the number of vascular elements per square millimeter were measured for both central and peripheral vascular elements. Furthermore, the total villous area and perimeter per vascular element were measured. Finally, the shortest distance from the luminal border of each central and peripheral vascular element to the intervillous space was drawn on screen and subsequently measured (Figure, E). The investigator was blinded for pregnancy outcome.
Statistics Statistical comparisons were performed with the Student t test for independent
samples to compare groups with their matched control subjects. Kruskal-Wallis test was used where appropriate. A 2-sided probability value of ⬍ .05 was considered to be statistically significant. Statistical analysis was performed with SPSS software (version 11.0 for Windows; Microsoft Corporation, Redmond, WA). Data are presented as mean ⫾ SD, unless otherwise stated.
R ESULTS Clinical data From the selected 99 samples, 26 cases of pregnancies that had been complicated by hypertensive disorders and 37 control subjects could be included. In the IUGR group, 10 cases with 18 control subjects were included. Nine samples were not representative because of very small amounts or damaged tissue. The clinical data from the cases and control subjects are listed in Table 1. There were no significant differences between the cases
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TABLE 4
Mean vascular parameters of the 5 pregnancies that were complicated with early-onset hypertensive disorders (n ⴝ 5), compared with the control subjects (n ⴝ 37) Hypertensive disorder Parameter
Mean
Control subjects SD
Mean
SD
P value
Vascular surface, %
.......................................................................................................................................................................................................................................................................................................................................................................
Central
2.01
0.85
1.99
1.02
NS
Peripheral
1.52
0.36
1.38
0.55
NS
Total
3.54
0.87
3.38
1.31
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Vessel no., mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
51.26
11.49
50.08
17.81
NS
Peripheral
134.70
45.73
112.88
27.67
NS
Total
185.96
50.13
162.96
36.27
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Perimeter, mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
5061.09
1388.45
4248.25
1548.20
NS
Peripheral
6715.56
1973.22
5891.04
1836.81
NS
11776.65
1659.54
10,139.30
2901.54
NS
....................................................................................................................................................................................................................................................................................................................................................................... .......................................................................................................................................................................................................................................................................................................................................................................
Total
................................................................................................................................................................................................................................................................................................................................................................................
Distance luminal border to intervillous space, m
.......................................................................................................................................................................................................................................................................................................................................................................
Central
57.38
11.08
60.41
11.95
Peripheral
24.91
1.52
27.46
3.17
.014
NS
Total
37.89
4.04
43.93
6.81
.023
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................
NS, not significant. Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
and control subjects regarding maternal age, gestational age at chorion villous sampling, or week of delivery, except for birthweight (P ⬍ .005 for both hypertensive and IUGR cases compared with their control subjects). In the hypertensive group, there were 5 cases with early onset of complications at ⬍36 weeks of gestation. Specifications of these pregnancies are listed in Table 2.
Villous vascular measurements. In the IUGR group, the distance of the peripheral vessels to the intervillous space was significantly smaller, compared with the control subjects (24.91 vs 28.38 m; P ⬍ .02). The same was found for the sum of the distances of the central and peripheral vessels to the intervillous space (37.27 vs 44.43 m; P ⬍ .05; Table 3). In the early hypertensive group, we also observed a significantly smaller distance of the peripheral vessels to the intervillous space (24.91 vs 27.46 m; P ⬍ .02) and that the sum of the distances 88.e5
(central and peripheral) was also smaller, compared with the control subjects (37.89 vs 43.93 m; P ⬍ .03). Details of these measurements are listed in Table 4. Regarding the late-onset hypertensive group, no significant differences were found for any of the vascular variables that were measured, compared with the control group (Table 5).
C OMMENT The results of our study show that the altered vascularization patterns, which were seen in chorionic villi at term from pregnancies that were complicated with either hypertensive disorders or IUGR of the fetus, can be seen already in the first trimester of pregnancy. Almost all studies on human chorionic villous vasculature of complicated pregnancies are performed on third-trimester placental tissue. A study from Roberts et al6 that compared trisomic and chromosomally normal pregnancies demonstrated that
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detailed histomorphologic analysis is possible with the use of chorionic villi from ongoing pregnancies and has allowed normal values for some morphometric parameters. These 2-dimensional parameters may not give the more extensive morphometric data that stereologic (3-dimensional) parameters would, as described by Mayhew et al1 in the third trimester, but they do provide the possibility to compare data from first-trimester villi from normal and later complicated pregnancies. Exploring the results of our study, we found significantly more peripherally located vessels in the pregnancies with early-onset hypertensive disorders and a trend for an increase in the number of vascular elements per square millimeter, especially peripheral vessels. This is in accordance with our hypothesis and the “uteroplacental hypoxia” model of Kingdom and Kaufmann.3 In this model, the maternal blood is normally oxygenated but is partly restricted to en-
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TABLE 5
Vascular parameters of pregnancies complicated with hypertensive disorders (n ⴝ 21) compared with controls (n ⴝ 37) Hypertensive disorders Parameter
Mean
Control subjects SD
Mean
SD
P value
Vascular surface, %
.......................................................................................................................................................................................................................................................................................................................................................................
Central
2.11
1.48
1.99
1.03
NS
Peripheral
1.46
0.76
1.38
0.56
NS
Total
3.21
0.90
3.38
1.32
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Vessel no., mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
45.22
12.84
50.08
17.82
NS
Peripheral
131.63
67.31
112.88
27.67
NS
Total
176.85
66.34
162.96
36.27
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Perimeter, mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
4031
Peripheral
6547
10,579
723.81
4248
1548
NS
3242
5891
1837
NS
3367
10,139
2902
NS
....................................................................................................................................................................................................................................................................................................................................................................... .......................................................................................................................................................................................................................................................................................................................................................................
Total
................................................................................................................................................................................................................................................................................................................................................................................
Distance luminal border to intervillous space, m
.......................................................................................................................................................................................................................................................................................................................................................................
Central
58.25
11.42
60.41
11.96
NS
Peripheral
25.75
3.17
27.46
3.18
NS
Total
39.99
9.69
43.93
6.82
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................
NS, not significant. Hypertensive disorders: pregnancy-induced hypertension (n ⫽ 12), preeclampsia (n ⫽ 8) and hemolysis, elevated liver enzymes, and low platelet count. Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
ter the intervillous space because of failed remodeling of spiral arteries by extravillous cytotrophoblast. As a result, the milieu in the placental parenchyma is hypoxic, and villous branching angiogenesis is increased.3,7 Vis et al8 observed a tendency for larger birthweights in relation to larger and more vascularized villi of first-trimester chorionic villous samples; Kadyrov et al9 found altered vascularization patterns in the first trimester of pregnancy of anemic women. In the 5 cases of early hypertensive disorders, there was a tendency toward lower birthweights for gestational age, with 2 of 5 pregnancies resulting in birthweights at ⬍10th percentile. The pregnancies with late-onset hypertensive disorders showed a normal (p10 to p90) birthweight in all but 1 case. In 1 case, the birthweight percentile was below p5 (percentiles according to Kloosterman5). Egbor et al10,11 showed that isolated early-onset preeclampsia was associated with abnormal placental mor-
phologic condition, but placentas from late-onset preeclampsia were morphologically similar to placentas from gestational-age–matched control subjects. This is in accordance with our results, in which we also found a significant difference in vascularization patterns in the early-onset group, but not in the late-onset group. Concerning vascularization, on the contrary, fewer and smaller vessels are expected to be found in pregnancies that are complicated with IUGR. These pregnancies seem to lack the adaptive response that is seen in pregnancies that are complicated with preeclampsia. Third-trimester studies revealed chorionic villi with impaired vascularization patterns in pregnancies that were complicated with IUGR.12,13 In the cases of IUGR and the models of postplacental hypoxia of Kingdom and Kaufmann,3,7 our results not only show some kind of adaptive response but also seem to show a decrease in
centrally located vessels. In the model of Kingdom and Kaufmann, postplacental hypoxia seems to be associated with early onset IUGR with absent end diastolic flow velocity in the umbilical arteries. Normally, oxygenated blood enters the intervillous space, but a failure of the fetoplacental circulation to extract oxygen from the intervillous space places the fetus at risk for hypoxia, and the relatively high oxygen levels in the intervillous space will now stimulate nonbranching angiogenesis, with a reduced number of elongated villi with few unbranched and uncoiled capillaries.3,8,12,14 We found more and more peripherally situated vessels not only in IUGR but also in hypertensive disorders. In the IUGR group, however, the adaptive response seems to be ineffective, considering the lower birthweight, which is contrary to the cases with hypertensive disorders in which the neonatal birthweight is less compromised.
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Subsequently, Mayhew et al1,13,15 found no morphologic differences between preeclampsia and control cases but do describe an impoverished villous development in the IUGR group. In an earlier study, they concluded that it was possible that branching angiogenesis occurred, although overall growth of capillaries and villi was impaired. Ness and Sibai16 and Sibai et al17 stated that IUGR and preeclampsia are pregnancy-specific disorders that have in common abnormal placental implantation. They propose a shared complex placental pathophysiologic condition, but different interaction with the metabolic syndrome. However, both IUGR and preeclampsia seem to arise from a maternal predisposition to endothelial dysfunction. Shibata et al18 describe a placental system of amino acid transport, which is reduced in pregnancies with small-forgestational-age infants but not in preeclampsia with small-for-gestationalage infants. They suggest that growth reduction in the 2 settings may be substantially different and that several of the metabolic changes of preeclampsia (like insulin resistance19,20) are consistent with an adaptive change in preeclampsia to increase nutrient availability that is not present in IUGR. Our findings are in line with the early onset of complications in human pregnancy and may widen the window for therapeutic interventions that are already in early pregnancy, thereby reducing maternal and fetal morbidity. f
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REFERENCES 1. Mayhew TM, Charnock-Jones DS, Kaufmann P. Aspects of human fetoplacental vasculogenesis and angiogenesis: III, changes in complicated pregnancies. Placenta 2004;25: 127-39. 2. Kingdom JC, Huppertz B, Seaward G, Kaufmann P. Development of the placental villous tree and its consequences for fetal growth. Eur J Obstet Gynecol Reprod Biol 2000;92:35-43. 3. Kingdom JC, Kaufmann P. Oxygen and placental villous development: origins of fetal hypoxia. Placenta 1997;18:613-21. 4. Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP) (review). Hypertens Pregn 2001;20: IX-XIV. 5. Kloosterman GJ. Over intra-uteriene groei en de intra-uteriene groeicurve. [Intrauterine growth and intrauterine growth curves]. Ned Tijdschr Verloskd Gynaecol 1969;69:349-65. 6. Roberts L, Sebire NJ, Fowler D, Nicolaides KH. Histomorphological features of chorionic villi at 10-14 weeks of gestation in trisomic and chromosomally normal pregnancies. Placenta 2000;21:678-83. 7. Kingdom JC. Adriana and Luisa Castellucci Award Lecture 1997: placental pathology in obstetrics: adaptation or failure of the villous tree? Placenta 1998;19:347-51. 8. Vis C, Everhardt E, te Velde J, Exalto N. Microscopic investigation of villi from chorionic villous sampling. Hum Reprod 1998;13:2954-7. 9. Kadyrov M, Kosanke G, Kingdom J, Kaufmann P. Increased fetoplacental angiogenesis during first trimester in anaemic women. Lancet 1998;352:1747-9. 10. Egbor M, Ansari T, Morris N, Green CJ, Sibbons PD. Morphometric placental villous and vascular abnormalities in early- and late-onset pre-eclampsia with and without fetal growth restriction. BJOG 2006;113:580-9.
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www.AJOG.org 11. Egbor M, Ansari T, Morris N, Green CJ, Sibbons PD. Pre-eclampsia and fetal growth restriction: how morphometrically different is the placenta? Placenta 2006;27:727-34. 12. Krebs C, Macara LM, Leiser R, Bowman AW, Greer IA, Kingdom JC. Intrauterine growth restriction with absent end-diastolic flow velocity in the umbilical artery is associated with maldevelopment of the placental terminal villous tree. Am J Obstet Gynecol 1996;175:1534-42. 13. Mayhew TM, Wijesekara J, Baker PN, Ong SS. Morphometric evidence that villous development and fetoplacental angiogenesis are compromised by intrauterine growth restriction but not by pre-eclampsia. Placenta 2004; 25:829-33. 14. Macara L, Kingdom JC, Kaufmann P, et al. Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms. Placenta 1996;17:37-48. 15. Mayhew TM, Ohadike C, Baker PN, Crocker IP, Mitchell C, Ong SS. Stereological investigation of placental morphology in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction. Placenta 2003;24:219-26. 16. Ness RB, Sibai BM. Shared and disparate components of the pathophysiologies of fetal growth restriction and preeclampsia. Am J Obstet Gynecol 2006;195:40-9. 17. Sibai BM, Dekker G, Kupferminc M. Preeclampsia. Lancet 2005;365:785-99. 18. Shibata E, Hubel CA, Powers RW, et al. Placental system A amino acid transport is reduced in pregnancies with small for gestational age (SGA) infants but not in preeclampsia with SGA infants. Placenta 2008;29:879-82. 19. Hubel CA. Dyslipidemia, iron and oxidative stress in preeclampsia: assessment of maternal and feto-placental interactions. Semin Reprod Endocrinol 1998;16:75-92. 20. Rodie VA, Freeman DJ, Sattar N, Greer IA. Pre-eclampsia and cardiovascular disease: metabolic syndrome of pregnancy? Atherosclerosis 2004;175:189-202.
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Vascularization in first-trimester chorionic villi in complicated and uncomplicated pregnancies Monique A. Huisman, MD; Bert Timmer, PhD; Janet Stegehuis, MD; Bert Swart, MD; Jan G. Aarnoudse, PhD; JanJaap H. M. Erwich, PhD OBJECTIVE: The purpose of this study was to investigate possible al-
tered chorionic vascularization patterns that are seen already in the first trimester of pregnancies that are complicated by hypertensive disorders or intrauterine growth restriction (IUGR) in the third trimester of pregnancy. STUDY DESIGN: After chorionic villous sampling, surplus of villi were
stored, and a selection was made of pregnancies that were complicated further by hypertensive disorders (n ⫽ 26), normotensive IUGR (n ⫽ 13), and matched control subjects (n ⫽ 60). Vascular parameters of these villi were analyzed with a video-image-analysis system. RESULTS: In pregnancies that are complicated by early-onset hyper-
tensive disorders and IUGR, the mean distance of the peripheral vessels
to the intervillous space and the total of the distances (central and peripheral) are significantly smaller, compared with control subjects (9.3% and 13.8% for hypertensive disorders and 12.2% and 16.1% for IUGR, respectively). CONCLUSION: Differences in vascularization patterns in the placenta
already in the first trimester of pregnancies that are complicated later by hypertensive disorders or IUGR confirm the hypothesis of early changes by means of more vessels and more peripheral vessels that are located in chorionic villi. Key words: chorionic villi, intrauterine growth restriction, preeclampsia, vascularization
Cite this article as: Huisman MA, Timmer B, Stegehuis J, et al. Vascularization in first-trimester chorionic villi in complicated and uncomplicated pregnancies. Am J Obstet Gynecol 2010;202:88.e1-7.
A
fter delivery, placentas of pregnancies that were complicated by preeclampsia or intrauterine growth restriction (IUGR) show morphologically different vascularization patterns in the chorionic villi, compared with those pla-
From the Departments of Obstetrics and Gynecology (Drs Huisman, Stegehuis, Swart, Aarnoudse, and Erwich) and Pathology and Laboratory Medicine (Dr Timmer), University Medical Center Groningen, The Netherlands. Presented at the 52nd Annual Meeting of the Society for Gynecologic Investigation, Los Angeles, CA, March 23-26, 2005. Received March 19, 2009; revised June 14, 2009; accepted Aug. 20, 3009. Reprints: M.A. Huisman, MD, Department of Obstetrics and Gynaecology, Deventer Hospital, Nico Bolkesteinlaan 75,7416 SE Deventer, The Netherlands. [email protected]. Authorship and contribution to the article is limited to the 6 authors indicated. There was no outside funding or technical assistance with the production of this article. 0002-9378/$36.00 © 2010 Mosby, Inc. All rights reserved. doi: 10.1016/j.ajog.2009.08.036
88.e1
centas of uncomplicated pregnancies.1,2 The cause of the described differences is not completely known, and differences in early vascularization patterns between early and late onset preeclampsia are not well described. Kingdom and Kaufmann3 proposed a common model to explore the origins of fetal hypoxia with different vascularization patterns. In their model, they distinguish preplacental, uteroplacental, and postplacental (fetal) hypoxia, with each specific morphologic or physiologic antecedents. In the case of uteroplacental hypoxia that is associated with hypertension, failed extravillous cytotrophoblast invasion of the maternal spiral arteries causes a restriction for the normally oxygenated maternal blood to enter the intervillous space. Branching angiogenesis increases, which results in a greater amount of highly vascularized villi and a dilation of the villous capillaries, and causes an increased capillary volume fraction and an increased fetal syncytial surface area for diffusional exchange. In postplacental hypoxia that is associated with IUGR, angiogenesis is nonbranching, and few slender and fibrotic chorionic villi are
American Journal of Obstetrics & Gynecology JANUARY 2010
found with reduced numbers of capillaries. In the current study, we investigated whether the morphologically altered vascularization patterns can be found already in first-trimester chorionic villi of ongoing pregnancies that were complicated with early- or late-onset hypertensive disorders such as preeclampsia and/or IUGR in the third trimester of pregnancy, compared with the vascularization patterns of further uncomplicated pregnancies. In accordance to the model of Kingdom and Kaufman,3 we hypothesized to find more and larger and perhaps more peripherally situated vessels in pregnancies that were complicated by hypertensive disorders to allow optimal maternal-fetal exchange of oxygen and nutrition between the intervillous maternal blood and the villi. Subsequently, concerning IUGR, fewer and smaller vessels were expected to be found in pregnancies that were complicated with IUGR, because at term, these pregnancies seemed to lack the adaptive response that was seen in pregnancies that were complicated with preeclampsia. Villi were stained with a monoclonal an-
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FIGURE
Analysis of CD34-stained human chorionic villi
A, Random selection of structurally intact parts of villi; B, contours of immature intermediate villi were traced and drawn on screen (green); C centrally located vascular elements (green) drawn on screen, followed by D, the peripherally located vessels; E, measurement of the distance from the luminal borders of a vascular element to the intervillous space (arrow). Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
tibody against the CD34 antigen to visualize vascular elements and subsequently were analyzed with a video-image analysis system.
P ATIENTS AND M ETHODS Patients Over the years, ⬎2000 woman have undergone vaginal chorionic villous sampling between 10 and 12 weeks of gestation, mainly for maternal age or serum screening that was related risk of aneuploidy. Gestational age was calculated according to the last menstrual period and subsequently confirmed by ultrasound evaluation (crown-rump length). Tissue was obtained with a biopsy catheter (K-CMA-5000; Cook Medical Inc, Bloomington, IN). Surplus material, which was not needed for karyotyping,
was obtained, and decidua was mechanically separated from the villi. The villi were placed in formaldehyde immediately and stored until further processing. Villi were embedded in paraffin, subsequently cut into 4-m sections and fixed on 3-aminopropyl-triethoxy-silane– coated (A3648; Sigma Chemical Company, St. Louis, MO) slides. Follow-up evaluation of the pregnancies was available in ⬎85% of cases and consisted of a questionnaire that was returned by the patient after delivery; additional data were extracted from the clinical notes. A selection was made of pregnancies that were complicated by hypertensive disorders (n ⫽ 26), such as preeclampsia and pregnancy-induced hypertension, and pregnancies that were complicated by normotensive IUGR
(n ⫽ 13). Pregnancy-induced hypertension, preeclampsia, and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome were defined according to the International Society for the Study of Hypertension in Pregnancy criteria4: pregnancy-induced hypertension was defined as blood pressure ⬎140/90 mm Hg after 20 weeks of gestation on 2 occasions at least 6 hours apart; preeclampsia was defined as an increase in blood pressure to at least 140/90 mm Hg after week 20 of gestation in a previously normotensive woman, combined with proteinuria (protein excretion at least 0.3 g per 24 hours, spot urine protein/creatinine ratio of 530 mg/mmol or at least 2⫹ protein by dipstick); HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome was defined
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TABLE 1
Clinical data of study population Hypertensive disorderb
Intrauterine growth restriction
Cases (n ⴝ 26)
Control subjects (n ⴝ 37)
Cases (n ⴝ 10)c
Control subjects (n ⴝ 18)
38 (36–42)
39 (35–43)
38 (36–43)
37 (36–40)
Chorionic villous sampling, d
76 (71–83)
77 (71–85)
76 (72–80)
75 (69–81)
Delivery, wk
39 (32–42)
39 (35–43)
38 (36–43)
38 (36–40)
Variablea Maternal age, y
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
Birthweight, g
3090 (1400–4325)
3586 (2525–4620)
2555 (2110–2840)
3840 (3280–4590)
................................................................................................................................................................................................................................................................................................................................................................................ a
b
Data are given as mean (range); Consisted of 13 cases of pregnancy-induced hypertension, 10 cases of preeclampsia, and 3 cases of HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, according to the criteria of the International Society for the Study of Hypertension in Pregnancy; c All intrauterine growth restriction cases were normotensive.
Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
as hemolysis, lactic dehydrogenase ⬎600 U/L, aspartate aminotransferase ⬎70 U/L, alanine aminotransferase ⬎90 U/L, and platelet-count ⬍100 g/L. For IUGR, birthweight was corrected for gestational age, and percentiles were computed. IUGR was defined as a corrected birthweight below the 5th percentile, according to Kloosterman.5 All the included cases in the IUGR group concerned normotensive pregnancies. For each case, control subjects were selected and matched for maternal age, parity, and gestational age at time of sampling (n ⫽ 60). Patients with concurrent morbidity (eg, preexisting hypertension or diabetes mellitus) who were smoking or receiving medication were excluded. Fetal karyotyping showed no chromosomal abnormalities. Patients were informed that surplus material could be used for research, according to the “Code for proper use of human tissue,” version 2002, of the Federation of Dutch Medical Scientific Societies and the Local Ethics Committee of the University Medical
Center Groningen. Patients who were included gave informed consent; after the outcome of the pregnancy was known, data were made anonymous.
Immunohistochemistry After the slides were deparaffinized with a series of xylene and alcohol and washed in phosphate-buffered saline solution (PBS; pH 7.4) 3 times for 5 minutes, the nonspecific peroxidase activity was blocked with 1% H2O2 (Merck, Darmstadt, Germany) in PBS for 30 minutes. Antigen retrieval was performed by incubation in Tris-HCL (buffer) for 30 minutes by 94°C in a microwave. Subsequently the slides were incubated with the mouse monoclonal primary antibody against the CD34 antigen (Clone QBEND10; Immunotech, Marseille, France) for 1 hour.3 The CD34 antibody was used at 1:100 dilution in PBS, in which 1% bovine serum albumin and 1% normal human AB serum were included, both to minimize nonspecific reactivity. The second and third step of an-
tibody labeling was carried out with peroxidase-conjugated rabbit anti-mouse immunoglobulin (RAMpo; DAKO A/S, Glostrup, Denmark) and peroxidase conjugated goat anti-rabbit immunoglobulin (GARpo; DAKO), respectively, both 1:50 in PBS/1% bovine serum albumin/1% AB serum for 30 minutes. All incubation steps were followed by 3 washes in PBS for 5 minutes. Finally, the peroxidase reaction was visualized with diaminobenzidine (Sigma Chemical Company). Subsequently the slides were counterstained with hematoxylin, dehydrated, and mounted with mounting medium (International Medical Products, Zutphen, The Netherlands).
Video Image Analysis To quantify vascular development in the chorionic villi, a video-image analysis system was used (Qwin 2.4; Leica, Cambridge, England). Microscope images were recorded with a color video camera (DC 300 V2.0; Leica) that was mounted on a light microscope (Axioskop 130 VA Type
TABLE 2
Data of pregnancies with early-onset hypertensive disorders Case
Disorder
Neonatal weight, ga
Week of delivery
1
Pregnancy-induced hypertension
1940 (25-50)
34
2
HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome
1610 (⬍5)
36
3
HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome
1400 (25)
32
4
Preeclampsia
1885 (⬍10)
36
5
Preeclampsia
2110 (25)
35
................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................
HELLP, hemolysis, elevated liver enzymes, and low platelet count. a
Percentile given in parentheses.
Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
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TABLE 3
Vascular parameters of the pregnancies complicated with intrauterine growth restriction, compared with control subjects
Parameter
Intrauterine growth restriction (n ⴝ 10)
Control subjects (n ⴝ 18)
Mean
Mean
SD
SD
P value
Vascular surface, %
.......................................................................................................................................................................................................................................................................................................................................................................
Central
1.77
0.86
1.94
1.13
NS
Peripheral
1.61
0.49
1.60
0.64
NS
Total
3.38
1.11
3.53
1.38
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Vessel no., mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
40.28
17.64
47.21
17.15
NS
Peripheral
137.87
37.78
124.03
28.81
NS
Total
178.15
42.16
171.24
39.23
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Perimeter, mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
3697
1152
4200
1365
NS
Peripheral
6863
1587
6776
1931
NS
10831
2328
10,976
2749
NS
....................................................................................................................................................................................................................................................................................................................................................................... .......................................................................................................................................................................................................................................................................................................................................................................
Total
................................................................................................................................................................................................................................................................................................................................................................................
Distance luminal border to intervillous space, m
.......................................................................................................................................................................................................................................................................................................................................................................
Central
58.09
11.53
60.49
10.35
NS
Peripheral
24.91
3.22
28.38
3.04
.012
Total
37.27
9.11
44.43
6.33
.045
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................
NS, not significant. Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
B; Carl Zeiss, Goettingen, Germany) with a ⫻20 objective. For each case, 5-15 images with (parts of) structurally intact immature intermediate villi were recorded randomly and evaluated. Random fields were chosen by means of knight’s move selection. Once recorded, no adjustments were done to provide structurally intact villi to prevent selection bias. First, from the recorded chorionic villi (Figure, A), the contours were traced manually on the computer monitor and drawn on screen with a mouse-controlled cursor, after which the villous area could be measured by the analysis system (Figure, B). Subsequently, every single central vascular element, which was made visible by the CD34-stained endothelial cells, was traced manually and drawn on screen, and the vascular area was measured (Figure, C). Any brown-stained endothelial cell or endothelial cell cluster (hemangioblastic cord), with or without a vessel lumen, that was clearly separate from adjacent
vessels was considered to be a single countable vessel. The same was done for every single peripheral vascular element (Figure, D). Division of the vascular elements into central or peripheral was done by the judgment of the investigator regarding the position of the vascular element to the syncytio-/cytotrophoblast. The total vascular area, the total vascular perimeter, and the number of vascular elements per square millimeter were measured for both central and peripheral vascular elements. Furthermore, the total villous area and perimeter per vascular element were measured. Finally, the shortest distance from the luminal border of each central and peripheral vascular element to the intervillous space was drawn on screen and subsequently measured (Figure, E). The investigator was blinded for pregnancy outcome.
Statistics Statistical comparisons were performed with the Student t test for independent
samples to compare groups with their matched control subjects. Kruskal-Wallis test was used where appropriate. A 2-sided probability value of ⬍ .05 was considered to be statistically significant. Statistical analysis was performed with SPSS software (version 11.0 for Windows; Microsoft Corporation, Redmond, WA). Data are presented as mean ⫾ SD, unless otherwise stated.
R ESULTS Clinical data From the selected 99 samples, 26 cases of pregnancies that had been complicated by hypertensive disorders and 37 control subjects could be included. In the IUGR group, 10 cases with 18 control subjects were included. Nine samples were not representative because of very small amounts or damaged tissue. The clinical data from the cases and control subjects are listed in Table 1. There were no significant differences between the cases
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TABLE 4
Mean vascular parameters of the 5 pregnancies that were complicated with early-onset hypertensive disorders (n ⴝ 5), compared with the control subjects (n ⴝ 37) Hypertensive disorder Parameter
Mean
Control subjects SD
Mean
SD
P value
Vascular surface, %
.......................................................................................................................................................................................................................................................................................................................................................................
Central
2.01
0.85
1.99
1.02
NS
Peripheral
1.52
0.36
1.38
0.55
NS
Total
3.54
0.87
3.38
1.31
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Vessel no., mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
51.26
11.49
50.08
17.81
NS
Peripheral
134.70
45.73
112.88
27.67
NS
Total
185.96
50.13
162.96
36.27
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Perimeter, mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
5061.09
1388.45
4248.25
1548.20
NS
Peripheral
6715.56
1973.22
5891.04
1836.81
NS
11776.65
1659.54
10,139.30
2901.54
NS
....................................................................................................................................................................................................................................................................................................................................................................... .......................................................................................................................................................................................................................................................................................................................................................................
Total
................................................................................................................................................................................................................................................................................................................................................................................
Distance luminal border to intervillous space, m
.......................................................................................................................................................................................................................................................................................................................................................................
Central
57.38
11.08
60.41
11.95
Peripheral
24.91
1.52
27.46
3.17
.014
NS
Total
37.89
4.04
43.93
6.81
.023
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................
NS, not significant. Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
and control subjects regarding maternal age, gestational age at chorion villous sampling, or week of delivery, except for birthweight (P ⬍ .005 for both hypertensive and IUGR cases compared with their control subjects). In the hypertensive group, there were 5 cases with early onset of complications at ⬍36 weeks of gestation. Specifications of these pregnancies are listed in Table 2.
Villous vascular measurements. In the IUGR group, the distance of the peripheral vessels to the intervillous space was significantly smaller, compared with the control subjects (24.91 vs 28.38 m; P ⬍ .02). The same was found for the sum of the distances of the central and peripheral vessels to the intervillous space (37.27 vs 44.43 m; P ⬍ .05; Table 3). In the early hypertensive group, we also observed a significantly smaller distance of the peripheral vessels to the intervillous space (24.91 vs 27.46 m; P ⬍ .02) and that the sum of the distances 88.e5
(central and peripheral) was also smaller, compared with the control subjects (37.89 vs 43.93 m; P ⬍ .03). Details of these measurements are listed in Table 4. Regarding the late-onset hypertensive group, no significant differences were found for any of the vascular variables that were measured, compared with the control group (Table 5).
C OMMENT The results of our study show that the altered vascularization patterns, which were seen in chorionic villi at term from pregnancies that were complicated with either hypertensive disorders or IUGR of the fetus, can be seen already in the first trimester of pregnancy. Almost all studies on human chorionic villous vasculature of complicated pregnancies are performed on third-trimester placental tissue. A study from Roberts et al6 that compared trisomic and chromosomally normal pregnancies demonstrated that
American Journal of Obstetrics & Gynecology JANUARY 2010
detailed histomorphologic analysis is possible with the use of chorionic villi from ongoing pregnancies and has allowed normal values for some morphometric parameters. These 2-dimensional parameters may not give the more extensive morphometric data that stereologic (3-dimensional) parameters would, as described by Mayhew et al1 in the third trimester, but they do provide the possibility to compare data from first-trimester villi from normal and later complicated pregnancies. Exploring the results of our study, we found significantly more peripherally located vessels in the pregnancies with early-onset hypertensive disorders and a trend for an increase in the number of vascular elements per square millimeter, especially peripheral vessels. This is in accordance with our hypothesis and the “uteroplacental hypoxia” model of Kingdom and Kaufmann.3 In this model, the maternal blood is normally oxygenated but is partly restricted to en-
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TABLE 5
Vascular parameters of pregnancies complicated with hypertensive disorders (n ⴝ 21) compared with controls (n ⴝ 37) Hypertensive disorders Parameter
Mean
Control subjects SD
Mean
SD
P value
Vascular surface, %
.......................................................................................................................................................................................................................................................................................................................................................................
Central
2.11
1.48
1.99
1.03
NS
Peripheral
1.46
0.76
1.38
0.56
NS
Total
3.21
0.90
3.38
1.32
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Vessel no., mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
45.22
12.84
50.08
17.82
NS
Peripheral
131.63
67.31
112.88
27.67
NS
Total
176.85
66.34
162.96
36.27
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................ 2
Perimeter, mm
.......................................................................................................................................................................................................................................................................................................................................................................
Central
4031
Peripheral
6547
10,579
723.81
4248
1548
NS
3242
5891
1837
NS
3367
10,139
2902
NS
....................................................................................................................................................................................................................................................................................................................................................................... .......................................................................................................................................................................................................................................................................................................................................................................
Total
................................................................................................................................................................................................................................................................................................................................................................................
Distance luminal border to intervillous space, m
.......................................................................................................................................................................................................................................................................................................................................................................
Central
58.25
11.42
60.41
11.96
NS
Peripheral
25.75
3.17
27.46
3.18
NS
Total
39.99
9.69
43.93
6.82
NS
....................................................................................................................................................................................................................................................................................................................................................................... ....................................................................................................................................................................................................................................................................................................................................................................... ................................................................................................................................................................................................................................................................................................................................................................................
NS, not significant. Hypertensive disorders: pregnancy-induced hypertension (n ⫽ 12), preeclampsia (n ⫽ 8) and hemolysis, elevated liver enzymes, and low platelet count. Huisman. Vascularization in first-trimester chorionic villi. Am J Obstet Gynecol 2010.
ter the intervillous space because of failed remodeling of spiral arteries by extravillous cytotrophoblast. As a result, the milieu in the placental parenchyma is hypoxic, and villous branching angiogenesis is increased.3,7 Vis et al8 observed a tendency for larger birthweights in relation to larger and more vascularized villi of first-trimester chorionic villous samples; Kadyrov et al9 found altered vascularization patterns in the first trimester of pregnancy of anemic women. In the 5 cases of early hypertensive disorders, there was a tendency toward lower birthweights for gestational age, with 2 of 5 pregnancies resulting in birthweights at ⬍10th percentile. The pregnancies with late-onset hypertensive disorders showed a normal (p10 to p90) birthweight in all but 1 case. In 1 case, the birthweight percentile was below p5 (percentiles according to Kloosterman5). Egbor et al10,11 showed that isolated early-onset preeclampsia was associated with abnormal placental mor-
phologic condition, but placentas from late-onset preeclampsia were morphologically similar to placentas from gestational-age–matched control subjects. This is in accordance with our results, in which we also found a significant difference in vascularization patterns in the early-onset group, but not in the late-onset group. Concerning vascularization, on the contrary, fewer and smaller vessels are expected to be found in pregnancies that are complicated with IUGR. These pregnancies seem to lack the adaptive response that is seen in pregnancies that are complicated with preeclampsia. Third-trimester studies revealed chorionic villi with impaired vascularization patterns in pregnancies that were complicated with IUGR.12,13 In the cases of IUGR and the models of postplacental hypoxia of Kingdom and Kaufmann,3,7 our results not only show some kind of adaptive response but also seem to show a decrease in
centrally located vessels. In the model of Kingdom and Kaufmann, postplacental hypoxia seems to be associated with early onset IUGR with absent end diastolic flow velocity in the umbilical arteries. Normally, oxygenated blood enters the intervillous space, but a failure of the fetoplacental circulation to extract oxygen from the intervillous space places the fetus at risk for hypoxia, and the relatively high oxygen levels in the intervillous space will now stimulate nonbranching angiogenesis, with a reduced number of elongated villi with few unbranched and uncoiled capillaries.3,8,12,14 We found more and more peripherally situated vessels not only in IUGR but also in hypertensive disorders. In the IUGR group, however, the adaptive response seems to be ineffective, considering the lower birthweight, which is contrary to the cases with hypertensive disorders in which the neonatal birthweight is less compromised.
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Research
Basic Science: Obstetrics
Subsequently, Mayhew et al1,13,15 found no morphologic differences between preeclampsia and control cases but do describe an impoverished villous development in the IUGR group. In an earlier study, they concluded that it was possible that branching angiogenesis occurred, although overall growth of capillaries and villi was impaired. Ness and Sibai16 and Sibai et al17 stated that IUGR and preeclampsia are pregnancy-specific disorders that have in common abnormal placental implantation. They propose a shared complex placental pathophysiologic condition, but different interaction with the metabolic syndrome. However, both IUGR and preeclampsia seem to arise from a maternal predisposition to endothelial dysfunction. Shibata et al18 describe a placental system of amino acid transport, which is reduced in pregnancies with small-forgestational-age infants but not in preeclampsia with small-for-gestationalage infants. They suggest that growth reduction in the 2 settings may be substantially different and that several of the metabolic changes of preeclampsia (like insulin resistance19,20) are consistent with an adaptive change in preeclampsia to increase nutrient availability that is not present in IUGR. Our findings are in line with the early onset of complications in human pregnancy and may widen the window for therapeutic interventions that are already in early pregnancy, thereby reducing maternal and fetal morbidity. f
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REFERENCES 1. Mayhew TM, Charnock-Jones DS, Kaufmann P. Aspects of human fetoplacental vasculogenesis and angiogenesis: III, changes in complicated pregnancies. Placenta 2004;25: 127-39. 2. Kingdom JC, Huppertz B, Seaward G, Kaufmann P. Development of the placental villous tree and its consequences for fetal growth. Eur J Obstet Gynecol Reprod Biol 2000;92:35-43. 3. Kingdom JC, Kaufmann P. Oxygen and placental villous development: origins of fetal hypoxia. Placenta 1997;18:613-21. 4. Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP) (review). Hypertens Pregn 2001;20: IX-XIV. 5. Kloosterman GJ. Over intra-uteriene groei en de intra-uteriene groeicurve. [Intrauterine growth and intrauterine growth curves]. Ned Tijdschr Verloskd Gynaecol 1969;69:349-65. 6. Roberts L, Sebire NJ, Fowler D, Nicolaides KH. Histomorphological features of chorionic villi at 10-14 weeks of gestation in trisomic and chromosomally normal pregnancies. Placenta 2000;21:678-83. 7. Kingdom JC. Adriana and Luisa Castellucci Award Lecture 1997: placental pathology in obstetrics: adaptation or failure of the villous tree? Placenta 1998;19:347-51. 8. Vis C, Everhardt E, te Velde J, Exalto N. Microscopic investigation of villi from chorionic villous sampling. Hum Reprod 1998;13:2954-7. 9. Kadyrov M, Kosanke G, Kingdom J, Kaufmann P. Increased fetoplacental angiogenesis during first trimester in anaemic women. Lancet 1998;352:1747-9. 10. Egbor M, Ansari T, Morris N, Green CJ, Sibbons PD. Morphometric placental villous and vascular abnormalities in early- and late-onset pre-eclampsia with and without fetal growth restriction. BJOG 2006;113:580-9.
American Journal of Obstetrics & Gynecology JANUARY 2010
www.AJOG.org 11. Egbor M, Ansari T, Morris N, Green CJ, Sibbons PD. Pre-eclampsia and fetal growth restriction: how morphometrically different is the placenta? Placenta 2006;27:727-34. 12. Krebs C, Macara LM, Leiser R, Bowman AW, Greer IA, Kingdom JC. Intrauterine growth restriction with absent end-diastolic flow velocity in the umbilical artery is associated with maldevelopment of the placental terminal villous tree. Am J Obstet Gynecol 1996;175:1534-42. 13. Mayhew TM, Wijesekara J, Baker PN, Ong SS. Morphometric evidence that villous development and fetoplacental angiogenesis are compromised by intrauterine growth restriction but not by pre-eclampsia. Placenta 2004; 25:829-33. 14. Macara L, Kingdom JC, Kaufmann P, et al. Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms. Placenta 1996;17:37-48. 15. Mayhew TM, Ohadike C, Baker PN, Crocker IP, Mitchell C, Ong SS. Stereological investigation of placental morphology in pregnancies complicated by pre-eclampsia with and without intrauterine growth restriction. Placenta 2003;24:219-26. 16. Ness RB, Sibai BM. Shared and disparate components of the pathophysiologies of fetal growth restriction and preeclampsia. Am J Obstet Gynecol 2006;195:40-9. 17. Sibai BM, Dekker G, Kupferminc M. Preeclampsia. Lancet 2005;365:785-99. 18. Shibata E, Hubel CA, Powers RW, et al. Placental system A amino acid transport is reduced in pregnancies with small for gestational age (SGA) infants but not in preeclampsia with SGA infants. Placenta 2008;29:879-82. 19. Hubel CA. Dyslipidemia, iron and oxidative stress in preeclampsia: assessment of maternal and feto-placental interactions. Semin Reprod Endocrinol 1998;16:75-92. 20. Rodie VA, Freeman DJ, Sattar N, Greer IA. Pre-eclampsia and cardiovascular disease: metabolic syndrome of pregnancy? Atherosclerosis 2004;175:189-202.