Vascularization in first-trimester chorionic villi in complicated and uncomplicated pregnancies




Objective


The purpose of this study was to investigate possible altered 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 hypertensive 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.


After 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 placentas of uncomplicated pregnancies. 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 Kaufmann 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 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, 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 antibody against the CD34 antigen to visualize vascular elements and subsequently were analyzed with a video-image analysis system.


Patients and Methods


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-μm 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 criteria : 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 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. 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% H 2 O 2 (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. 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 antibody labeling was carried out with peroxidase-conjugated rabbit anti-mouse immunoglobulin (RAM po ; DAKO A/S, Glostrup, Denmark) and peroxidase conjugated goat anti-rabbit immunoglobulin (GAR po ; 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 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.




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 .


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.




Results


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 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 .



TABLE 1

Clinical data of study population





































Variable a Hypertensive disorder b Intrauterine growth restriction
Cases (n = 26) Control subjects (n = 37) Cases (n = 10) c Control subjects (n = 18)
Maternal age, y 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)
Birthweight, g 3090 (1400–4325) 3586 (2525–4620) 2555 (2110–2840) 3840 (3280–4590)

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Jul 8, 2017 | Posted by in GYNECOLOGY | Comments Off on Vascularization in first-trimester chorionic villi in complicated and uncomplicated pregnancies

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