Morphological Features of Fetal Appendages in Fetal Distress Induced by Placental Pathology
I.E. Pleşea^{(1)}, I. Ivănuş^{(2)}, O.T. Pop^{(1)}, Mirela Ghiluşi^{(3)}, Claudia Valentina Georgescu^{(3)}, Carmen Popescu^{(3)}, V. Gheorman^{(4)}
^{(1)}Department of Pathology, Faculty of Medicine, Craiova University of Medicine and Pharmacy; ^{(2)}Corabia Hospital, Olt County; ^{(3} Department of Pathology and Cytology, Emergency County Hospital, Craiova; ^{(4)}Department of Obstetrics and Ginecology, Faculty of Medicine, Craiova University of Medicine and Pharmacy
Abstract
Fetal growth is depending on integrity of fetal appendages. The aim of the study was reveal if morphological changes of fetal appendages could be related with the presence of fetal distress (either acute or chronic). Several quantitative parameters, such as placental weight, type of placental pathological process; vascular densities in the placental villi, thickness of umbilical cord vessels’ wall and amniotic membrane thickness were assessed on 20 pacients whose new borns had fetal distress. The results of statistic correlations between those parameters allow us to conclude that umbilical vein wall thickness can affect the vascular density in central placental areas and consequently fetalplacental exchange, resulting in fetal distress.
Keywords: Fetal distress; Fetal appendages
Introduction
Fetal growth depends on maternal and placental genetic factors. It is assumed that the fetus has an inherited growth potential which, under normal conditions, results in a healthy infant, with appropriate morphological parameters. Of all fetal appendages, placental investigation may provide many useful diagnostic tools for newborn care.
Such tools allow improved diagnosis or even establishing diagnoses that were not taken into account during the investigation and nursing of the newborn [Peleg şi colab. 1998; Gilber – Barness 2002].
Many have tried, over time, to define the role of the placenta. Perhaps the most comprehensive definition belongs to H.W. Mossman: „an apposition between parent (usually maternal) and fetal tissue in order to establish physiological the exchange” [Mossman, 1937]. However, the functional aspects of the role of the placenta are better emphasized by the definition proposed by D.H. Steven (1975): „a device consisting of one or more transport epithelia located between fetal and maternal blood supply”.
Maternalplacentalfetal unit act harmoniously to meet the needs of the fetus while supporting the mother's physiological changes. Distribution of fetal and maternal blood supply in the placenta is important for an efficient exchange of oxygen and nutrients and thus for a normal fetal growth [Pardi şi colab. 2002; Peleg şi colab. 1998].
Based on these assumptions, the present study was designed as an attempt to implement the latest acquisitions in morphological investigation techniques to identify the possible involvement of morphological changes of the fetal appendages in inducing fetal distress and, even more, because we found no similar approach in the literature.
Materials and Methods
Materials
The basis of this study was a group made of 20 patients with 21 fetuses (in one case it was a twin pregnacy) in which fetal distress was related to a diagnosed pathological process in the placenta, which was selected in turn from a larger group of 116 cases in which fetal distress was related to a pathological process located in the fetal appendages. The patients were divided into two subgroups, depending on the type of fetal distress identified in newborns: Acute Fetal Distress (AFD)  13 cases and intrauterine growth restriction (IUGR)  8 cases.
Data sources from which the material was selected for the study were: medical records of all the cases in the study (documentation of surgery and delivery protocols) and histopathological samples.
The materials for the study were the selected data from the above mentioned medical records as well as macroscopic samples and and fragments of adnexal tissue (placenta, umbilical cord and amniotic membranes) that were histologically processed.
Methods
„Database”type files were created for data collection in which morphological parameters that were to be studied were gathered.
Analysis of data obtained was divided in two studies: the study of morphological data and the study of correlations between morphological parameters.
The morphological parameters analyzed were: placental weight, the type of placental pathological process; vascular densities in the placental villi (with four components: vascular densities in type I and II placental villi from central and peripheral placenta and capillary densities in type III placental villi from central and peripheral placenta), average thickness of the wall of umbilical cord vessels (with two components: umbilical artery wall thickness and umbilical vein wall thickness) and amniotic membrane thickness.
In order to assess the weight of placenta 8 groups of weight were established (Table 1).
Teble 1: Placental weight groups
Weight group 
Weight 
WG1 
W < 200 g 
WG2 
200 g < W < 300 g 
WG3 
300 g < W < 400 g 
WG4 
400 g < W < 500 g 
WG5 
500 g < W < 600 g 
WG6 
600 g < W < 700 g 
WG7 
700 g < W < 800 g 
WG8 
800 g < W < 900 g 
Tissue samples from fetal adnexa (placenta, umbilical cord) underwent conventional histological processing techniques (fixation and paraffin embedding) and then serial sections were performed from each block. The first section was stained with the usual classical hematoxylin and eosin method for all types of tissue (placenta, umbilical cord, amniotic membrane). The second section was stained using an immunohistochemical technique only for fragments of placental tissue in order to mark the vascular structures. CD34 monoclonal antibody clone QBEnd 10 (Dako) was used (1:50 dilution).
Histopathological aspects were selected using an Olympus CX31 microscope with an X4 magnification eyepiece. Microscopic images were taken with an Olympus DP12 digital camera and downloaded into the computer using the analySIS Pro 3.2 software. The images of placental parenchyma were obtained at X20 magnification for type I and II placental villi and at X40 magnification for type III placental villi. The optically corrected planapo X4 objective was used to obtain the imges of umbilical cord. The optically corrected planapo X40 objective was used to obtain the imges of amniotic membrane.
Quantification of morphometric parameters was performed using the analySIS Pro 3.2 computerized image analysis software, after prior calibration of images acquired for each type of magnification used.
The algorithm for determining vascular densities in placental villi was as follows: 5 fields from the sections stained with antiCD34 antibody from central and peripheral fragments were randomly selected for each case.
The number of vascular structures in each villus, total area of the villi within the field and vascular density/mm^{2} were determined for each field. The mean vascular density/mm2 was calculated for each case.
To assess vascular density in type I and II villi 11 groups were defined for the classification of all the values obtained, with a growth factor of 100 vessels/mm^{2} (Table 2).
Table 2
Group 
Vascular Density (vessels/mm^{2}) 
VD1 
VD < 200/mm^{2} 
VD2 
200/mm^{2} < VD < 299/mm^{2} 
VD3 
300/mm^{2} < VD < 399/mm^{2} 
VD4 
400/mm^{2} < VD < 499/mm^{2} 
VD5 
500/mm^{2} < VD < 599/mm^{2} 
VD6 
600/mm^{2} < VD < 699/mm^{2} 
VD7 
700/mm^{2} < VD < 799/mm^{2} 
VD8 
800/mm^{2} < VD < 899/mm^{2} 
VD9 
900/mm^{2} < VD < 999/mm^{2} 
VD10 
1000/mm^{2} < VD < 1099/mm^{2} 
VD11 
1100/mm^{2} < VD < 1199/mm^{2} 
For the evaluation of capillary density in type III placental villi 5 groups were defined for the classification of all the values obtained, with a growth factor of 500 capillaries/mm^{2} (Table 3).
Table 3
Group 
Vascular Density (capillaries/mm^{2}) 
CD 1 
CD < 1500/mm^{2} 
CD 2 
1500/mm^{2} < CD < 1999/mm^{2} 
CD 3 
2000/mm^{2} < CD < 2499/mm^{2} 
CD 4 
2500/mm^{2} < CD < 2999/mm^{2} 
CD 5 
3000/mm^{2} < CD < 3499/mm^{2} 
The algorithm for determining the wall thickness of umbilical vessels was as follows: 10 determinations of vascular wall thickness for each of the umbilical arteries and the umbilical vein were randomly performed for each case on hematoxylin and eosin stained sections. For each case we calculated: the mean thickness of the first and second umbilical artery as well as the mean thickness of the umbilical vein.
To assess the mean thickness of the vascular wall, 9 groups were defined for the classification of all the values obtained, with a growth factor of 50 μm (Table 4).
Table 4
Group 
Thickness (µm) 
Th 1 
Th < 400 µm 
Th 2 
400 µm < Th < 449 µm 
Th 3 
450 µm < Th < 499 µm 
Th 4 
500 µm < Th < 549 µm 
Th 5 
550 µm < Th < 599 µm 
Th 6 
600 µm < Th < 649 µm 
Th 7 
650 µm < Th < 699 µm 
Th 8 
700 µm < Th < 749 µm 
Th 9 
750 µm < Th < 799 µm 
The algorithm for determining the mean thickness of the amnios was as follows: For each case 5 fields were randomly selected on hematoxylin and eosin stained sections. For each field, 4 random measurements of membrane thickness were performed. Mean thickness of the amnios was calculated for each case.
For the evaluation of mean thickness of the amnios, 7 groups were defined for the classification of all the values, with a growth factor of 5 μm (Table 5).
Table 5
Group 
Thickness (µm) 
AT 1 
AT < 30 µm 
AT 2 
30 µm < AT < 34,99 µm 
AT 3 
35 µm < AT < 39,99 µm 
AT 4 
40 µm < AT < 44,99 µm 
AT 5 
45 µm < AT < 49,99 µm 
AT 6 
50 µm < AT < 54,99 µm 
AT 7 
55 µm < AT < 59,99 µm 
Data obtained from measurements were processed using Microsoft Excel module of the Microsoft Office 2003 Professional software package. The smallest and highest values, the mean value, standard deviation, variation coefficient and confidence interval were obtained for each parameter.
To assess the correlation between two numerically expressed parameters Pearson's correlation coefficient (considered to be the most synthetic indicator of correlation) was used. Values > +0.40 were considered significant for the sample size used, with the tStudent test of statistical significance having value superior to the 1.96 significance threshold considered to be the significant threshold of 95%. The correlation is a direct one, meaning that the two variables compared evolve to the same effect, to increase or to decrease in value. Values < 0.40 were also considered significant but the correlation is of the inverse type, meaning that the increasing trend of one variable is associated with the decreasing trend of the other. Values between 0.40 and +0.40 were considered insignificant as tStudent statistics in this case were lower than the 1.96 threshold.
Morphological Study
Placental Weight
Most placentas in the studied batch (80%) had weights between 300 and 500 g. Weight was dispersed in a narrower range, namely between 270 and 650 g. The mean weight was 409 g, lower than the mean placental weight of term births without fetal distress. Concentration range of most cases was also narrow, highlighted by the standard deviation of only 102.69.
In the subgroup of fetuses with AFD, the weight was dispersed in a narrower range than in the whole group, ie between 300 and 650 g. The mean weight was 424.6 g, higher than that group. The concentration range of most cases was also narrower, highlighted by the standard deviation of only 95.27, also lower than the one in the whole group.
Table 6
Statistical parameter 
AFD 
IUGR 
Wmin 
300 
270 
WMax 
650 
650 
MW 
424,62 
385 
Std. Dev. 
95,27 
116 
V.C. (%) 
22,44 
30,13 
C.I. (95%) 
372,83 – 476,41 
304,61 – 465,39 
Wmin = Minimum weight; WMax = Maximum weight; MW = Mean Weight; Std. Dev. = Standard deviation; V. C. = Variation coefficient; AFD = Acute Fetal Distress; IUGR = Intrauterine Growth Restriction; C.I. = Confidence Interval 
In the subgroup of fetuses with IUGR, weight was dispersed in a range identical to that of the entire group, ie between 270 and 650 g. However, the mean weight was lower than that of the whole group, which is supported by a higher standard deviation, ie 116. Also, the comparison between the two subgroups shows that the dispersion range of the weight was slightly smaller in the subgroup with IUGR. The mean weight was also lower than that in the subgroup with the AFD. However, the concentration range of most cases was higher, which was also supported by a standard deviation of only 116 compared with 95.27.
Placental Pathology
In the studied batch, unspecified antepartum fetal pathology prevailed (15/20 cases). Distribution and prevalence of types of pathological processes diagnosed antepartum revealed a greater number of premature detachments of the normal inserted placentas, followed by placenta praevia (3 out of 6 cases and 2 aut of 6 cases respectively). The percentage represented by an unspecified antepartum fetal pathology was found to be higher in the subgroup of fetuses with IUGR (Table 7).
Table 7
Subgroup 
Type of Pathology 
Total 

Not mentioned 
Mentioned 

AFD 
8 (61,5%) 
5 (38,5%) 
13 
IUGR 
7 (87,5%) 
1 (12,5%) 
8 
Vascular Density in Placental Villi
Since most cases of placental pathology were considered antepartum as placental insufficiency syndrome, it was assumed that poor functioning of the placenta could be determined by a quantitative change of vascular structures within the placenta. Therefore, morphometric determinations of vascular density within the placenta were made taking into account two criteria: the type of the villi and the placental area.
Table 8
Statistical Parameter 
CP 
PP 
VDmin 
261,9 
341,2 
VDMax 
1170 
1219 
MVD 
661,56 
783,37 
Std. Dev. 
237,17 
309,37 
V.C. (%) 
35,85 
39,49 
C.I. (95%) 
547,7 – 775,45 
634,85 – 931,89 
VDmin = Minimum Vascular Density; VDMax = Maximum Vascular Density; MVD = Mean Vascular Density; Std. Dev. = Standard Deviation; V.C. = Variation Coefficient; CP = Central Placenta; PP = Peripheral Placenta; C.I. = Confidence Interval 
Vascular Density in Placental Villi Types I and II. The dispersion range of vascular density values (from the lowest to the highest value determined) had about the same spread in central and peripheral placenta, slightly wider in the central one (908.1 vs. 877.8), but the of central placenta was moved slightly to the left, towards lower densities. Mean vascular density was higher in type I and II villi of the peripheral placenta as compared to the central one. Mean density values were placed within the confidence intervals where most of the determinations are concentrated, intervals that also had the same position as the dispersion ranges (Table 8 and Figure 1).
Figure 1: Vessel counting and determination of villosity areas
Evaluation of the development trend of vascular densities in type I and II villi in the two parts of the placenta confirmed the diverging trends, namely, while vascular density in the central area tends to have lower values, the vascular density of the peripheral area tends to have higher values (Diagram 1).
Diagram 1
Vascular Density in Placental Villi Type III. Dispersion intervals for values of capillary density (from the lowest to the highest value determined) in type III placental villi have different limits and spans. Thus, the lower limit of the interval for capillary densities in central placenta was lower than in peripheral placenta and the upper limit was higher, which led to a wider range of dispersion of capillary density in type III placental villi in the central placenta. However, the mean capillary density in type III placental villi was higher in the central placenta and with a smaller concentration range, which was highlighted by the lower standard deviation. Data were also confirmed by determining the evolution trend of capillary densities in type III placental villi in the two parts of the placenta (Table 9 and Figure 2).
Table 9
Statistical Parameter 
CP 
PP 
VDmin 
1258 
1553 
VDMax 
3100 
2849 
MVD 
2087.09 
2136.51 
Std. Dev. 
495.62 
416.66 
V.C. (%) 
23.75 
19.50 
C.I. (95%) 
1849,15 – 2325,03 
1936,48 – 2336,54 
VDmin = Minimum Vascular Density; VDMax = Maximum Vascular Density; MVD = Mean Vascular Density; Std. Dev. = Standard Deviation; V.C. = Variation Coefficient; CP = Central Placenta; PP = Peripheral Placenta; C.I. = Confidence Interval 
Figure 2: Determination of umbilical arterial wall (Left) and venous wall (Right) thickness
Data were also confirmed by determining the evolution trend of capillary densities in type III placental villi in the two parts of the placenta (Diagram 2).
Diagram 2
Umbilical Cord
Another quantitative parameter that was discussed and evaluated is the thickness of the umbilical cord vessel walls. Statistical analysis of data resulted from determining the mean thickness of each of the three vessels of the umbilical cord has shown that in general, mean wall thickness of the umbilical artery is greater than that of the umbilical vein (Table 10 and Figure 3).
Table 10
Statistical Parameter 
UA 1 
UA 2 
UV 
Tmin 
541,5 
423,8 
374,7 
TMax 
733,5 
796,5 
764 
MT 
659.34 
589.47 
502.70 
Std. Dev. 
53.66 
109.03 
115.79 
V.C. (%) 
8.14 
18.50 
23.03 
C.I. (95%) 
633,58 – 685,10 
537,12 – 641,81 
447,12 – 558,29 
Tmin = Minimum thickness; TMax = Maximum thickness; MT = Mean thickness; Std. Dev. = Standard Deviation; V.C. = Variation Coefficient; C.I. = Confidence Interval 
Figure 3: Determination of amnion thickness
It was seen that one of the umbilical arteries has a thicker wall than the other and that the umbilical vein wall is very thick, its thickness representing between 76% and 85% of that of the thickness of arterial walls.
Determination of the evolution trend of the average thickness of the umbilical vessels confirmed the observations resulting from statistical analysis (Diagram 3).
Diagram 3
Amniotic Membrane
Finally, another quantitative parameter that has been discussed and evaluated was the mean thickness of the amniotic membrane. Mean thickness of the amniotic membrane varied between 25.5 and 58.9 μm. However it should be noted that 60% of cases had a higher amniotic membrane average thickness than that determined for the entire group (Diagram 4).
Diagram 4
The determined value for the mean thickness was around 40 micrometres, being placed within the confidence interval that focused 95% of the tests carried out (Table 11 and Figure 4).
Table 11
Statistical Parameter 
AM 
Minimum Thickness 
25,2 
Maximum Thickness 
58,9 
Mean Thickness 
39,70 
Std. Dev. 
10,80 
V.C. (%) 
27,21 
C.I. (95%) 
34,52 – 44,89 
Std. Dev. = Standard Deviation; V.C. = Variation Coefficient; C.I. = Confidence Interval 
Correlations Between Morphological Parameters
Finally, possible correlations between the parameterstaken into consideration, both clinical and morphological, were assessed using statistical comparison tools. It should be emphasized that the only correlations that were analized were between numerically expressed parameters.
The numerically expressed morphological parameters between which we was attempted to establish statistically significant correlations were: placenta weight, vascular densities in type I and II placental villi in central and peripheral placentas, capillary densities in type III placental villi in central and peripheral placentas, the mean wall thickness of the three umbilical vessels and the mean thickness of the amniotic membrane (Table 12).
Placental weight, the first morphological parameter considered, was directly correlated with capillary and vascular densities of villi in the central area, but not in the periheral one. In other words, the bigger the placenta, the higher the probability for the vascular tree in the center of the placenta to be more dense. Besides this, there were no other correlations between placental weight and any of the other morphological parameters that were analyzed.
Vascular densities in central and peripheral placentas. Variations in the values of all vascular densities calculated from both central and peripheral placenta and in both large and small villi were directlu correlated. Vascular densities increase or decrease in parallel in central and peripheral placenta, in type I and II villi as well as in type III oanes, while the central and peripheral, in vilozităţile Grade I and II and the Grade III, or, in other words, the growth or regression of the placental vascular tree is evenly balanced.
Table 12

FW 
VD Vill. I, II CP 
CD Vill. III CP 
VD Vill. I, II PP 
CD Vill. III PP 
PW 





VD Vill. I, IICP 
0.48 




CD Vill. IIICP 
0.58 
0.97 



VD Vill. I, IIPP 
NS 
0.55 
0.62 


CD Vill. IIIPP 
NS 
0.70 
0.70 
0.78 

UA1 T 
NS 
NS 
0.42 
NS 
NS 
UA2 T 
NS 
NS 
NS 
NS 
0.50 
UV T 
NS 
 0.61 
 0.52 
NS 
NS 
Amn T 
NS 
NS 
NS 
 0.55 
NS 
PW = Placental Weight; VD = Vascular Density; CD = Capillary Density; Vill. = Placental Villi; CP = Central Placenta; PP = Peripheral Placenta; T = Thickness; UA1 = Umbilical Artery 1; UA2 = Umbilical Artery 2; UV = Umbilical Vein; Amn = Amnios; NS = Statistically not significant 
Umbilical arteries.
Umbilical artery wall thickness variations were generally not associated with direct or inverse changes of other morphological parameters. However two direct correlations were observed: the one between the variation of umbilical artery "1" thickness and the variation of capillary density in type III villi of the central placenta, and the one between the variation of the thickness of umbilical artery "2" and the variation of capillary density in type III placental villi of the peripheral placenta. It could be hypothesised that umbilical artery wall thickness is dependent on the density of capillaries in type III placental villi, in other words, thicker the walls of the umbilical arteries.
Umbilical vein. Variations in umbilical vein wall thickness showed two interesting reverse correlations with vascular and capillary density in central areas of the placenta but not with those in peripheral ones. In other words, the greater the thickness of the umbilical vein, the smaller the density of vascular structures within central placenta.
Amniotic membrane. Amniotic membrane thickness variations were not directly or inversely associated with changes in other morphological parameters, with one exception, namely the changes in vascular density in type I and II placental villi of the peripheral placenta. With this, amniotic membrane thickness showed an inverse correlation, in other words, the greater the amniotic membrane thickness, the lower the vascular density in type I and II placental villi of the peripheral placenta, and vice versa.
Conclusions
Placental tissue mass, morphologically quantified by measuring the weight, may be one of the determinants for the onset of FD. Thus, lightweight placentae generated IUGR. Therefore, given that modern investigative currently permit an antepartum morphological evaluation of the placenta, the postpartum diagnosis of IUGR should not be the only option.
In general, but particularly in cases with IUGR, the type of antepartum placental pathology leading to the installation of FD was very hard to specify. When the antepartum diagnosis was possible, premature detachment of the normal inserted placenta emerged.
Microscopic morphometric measurements also highlighted the fact that vascular densities increase or decrease in parallel in the central and peripheral placenta, in type I and II placental villi as well as in type III placental villi, in other words, the growth or regression of the placental vascular tree is evenly balanced.
Analysis of correlation between umbilical artery wall thickness and capillary density in type III placental villi led to the observation that the greater the umbilical artery wall thickness, the greater the density of capillaries in type III villi, suggesting the conclusion that the wall thickness of the umbilical artery depends on the density of capillaries in type III villi in bot central and peripheral placenta.
Another interesting observation resulting from the analysis of correlations between microscopic morphological determinations of structures belonging to the fetal appendages was that umbilical vein wall thickness is even greater as the density of vascular structures in central placenta is lower and vice versa.
Vascular density in villi of the central areas of the placenta was all the greater as the thickness of the umbilical artery wall was higher and as the thickness of the umbilical vein wall smaller.
Finally, the greater the amniotic membrane thickness, the lower the vascular density in type I and II villi of the peripheral placenta and vice versa.
Linking observations on the relationship between umbilical vein wall thickness and vascular density in villi of the central placenta may lead to the conclusion that the umbilical vein wall thickness affects the vascular bed density in the central area of the placenta and, subsequently, the early postnatal adaptation. The hypothesis can be further developed in that the thickness of the umbilical vein wall affects fetalplacental exchange and may thus represent one of the causes of placental insufficiency syndrome.
References
1. Gilber  Barness E.: The significance of the placenta in assessment of the newborn. Crit Rev Clin Lab Sci.; 39 (2): 13992, 2002
1. Mossman, H.W. Comparative morphogenesis of the fetal membranes and accessory uterine structures. Carnegie Institute Contributions to Embryology. 26: 129246, 1937
2. Pardi G., Marconi A.M., Cetin
3. Peleg D. Kennedy C. M, Hunter S.K.: Intrauterine Growth Restriction: Identification and Management. American Family Physician, 50 (1516): 453470, 1998
4. Steven D.H. (Ed.): Comparative placentation. Academic Press, 1975
Corresponding author: Prof. Emil Pleşea, Department of Pathology, Faculty of Medicine, University of Medicine and Pharmacy, Petru Rareş Street 2, Craiova, 200349; Telephone/Fax: +40251 306110; Email pie1956@yahoo.com