Introduction
Umbilical cord is a connecting channel between the uterine vasculature and the fetal vasculature. Umbilical cord shows a specific gross morphology of a vein and two arteries surrounded by Wharton’s jelly. Umbilical cord lacks vasavasorum and depends on the blood in the umbilical vessels and Wharton’s jelly for its nutrition and hence vulnerable to hypoxic injuries easily due to changes in hemodynamic condition [1]. Maternal diabetes significantly influences the expression of genes in the umbilical cord and alters the umbilical vessel phenotype, with possible long term consequences for the neonate [2].
Gestational diabetes mellitus (GDM) is the commonest endocrine disorder of pregnancy presenting after 20th week of gestation and cause various structural and functional changes within umbilical cord blood vessels affecting the normal hemodynamics [3]. Pathological alteration of blood flow would be associated with mal development and reduction in number of terminal villi and may account for reduced feto-placental blood flow leading to hypoxia [4]. Chronic hypoxia of the growing fetus might be responsible for pathological contractions in the vascular bed of cotyledons leading to an increased placental resistance [5]. The pressure inside the blood vessels increases to overcome the increased resistance offered by the placenta and the blood vessels are distended due to its normal compliance. Blood vessels with a higher compliance deform easier than lower compliance blood vessels under the same pressure and volume conditions. Later when there is a continuous increase in pressure the compliance is reduced as the elastic lamellae in the vessel wall are reduced due to defective synthesis of elastin on exposure to fetal hypoxia and placental insufficiency [6]. A further rise in pressure inside the umbilical arteries are compensated by stimulating the increase in contractile unit that is smooth muscle fibers which in turn decrease the luminal radius and increases the wall thickness (WT) of umbilical arteries. On the other hand, umbilical vein withstands the increasing pressure only up to a certain limit and then becomes dilated and thin walled [7]. Endothelial dysfunction of the vessels is also responsible for reduced compliance especially in the smaller arteries. Reduced arterial compliance is often seen in diabetes [8]. These observations can be clinically confirmed through of Doppler flow velocity waveform analysis. Vascular changes at term may lead to early onset of vascular diseases like systemic arterial hypertension. Fetal programming is now studied as a determinant of adult diseases which has an increasing interdisciplinary research scope and major impact on public health [9]. Impaired elastin synthesis in fetal life may be associated with long-lasting increased vessel stiffness and therefore, may constitute a risk factor for the development of hypertension in adulthood [10].
Arterial wall enlargement may be due to an increase in the thickness of both tunica intima and media. The thickening of tunica intima has been attributed to migration of smooth muscle cells towards the endothelium, accompanied by splitting of internal elastic lamina. This migration is supposed to be due to augmentation of sulphated glycosaminoglycans in umbilical arteries and reduced expression of elastin. Thickness can be added by interstitial edema indicated by widening of intercellular spaces [7, 11].
Usually, both umbilical arteries are of similar diameter and sum of them roughly correlate with the vein diameter. However, discordance in the artery diameters was associated with placental abnormalities, variation of the umbilical cord insertion and clinical conditions, such as GDM [12]. The presence of discordant umbilical artery is not only evident by different diameters, but also as a sign of different umbilical artery blood flow indices [13]. Smaller artery has higher resistance to flow than larger arteries and can cause macroscopic and microscopic changes in placental lobe supplied by it [12, 14].
The present study was conducted to compare the effects of GDM and its management on the microscopic structure of umbilical cord and its vessels with normal pregnant female.
Materials and Methods
The study was approved by the Institutional Review Board of PK Das Institute of Medical Sciences (IEC/12-2/2015) and was thereafter conducted with umbilical cords of 50 normal pregnant females and 56 individuals with GDM (23 GDM mothers managed by diet [GDM-Diet] and 33 GDM mothers managed by drugs [GDM-Drug]) from December 2016 to December 2019.
The collected specimens were tagged and washed thoroughly to remove blood and mucus. Approximately 1×1 cm tissue samples were collected from three different regions fetal, maternal and central part of umbilical cord according to College of American Pathologists protocol. Further specimen preparation and staining techniques were done as per the standard protocol [15]. The tissues were fixed in 10% neutral buffered formalin for 48 hours and were sent to Department of Pathology, PK Das Institute of Medical Sciences where further tissue processing was done to make five blocks from each umbilical cord and systematic random samples of umbilical cord were taken for further study. Serial sections of 3 microns size were generated from the selected block and two sections of umbilical cord were fixed on each glass slide moistened with egg albumin. These slides were then stained with hematoxylin and eosin and Verhoeff’s Van Geison staining.
The stained slides were studied under Olympus Cx 2li microscope (Olympus Life Science) for assessing the histological appearance and measuring various histomorphometric parameters. Digital images were captured from a randomly selected 4×, low power (10×) and high power (40×) fields per slide using Olympus DP-20 colour video camera (Olympus Life Science) interfaced with an Olympus Cx 2li microscope. The images were scanned at a resolution of 1,600×1,200 pixels and images were captured using Olympus DP-20 BSW software (version 2.2; Olympus Life Science). Captured images were stored as raw data to analyze the histological and histomorphometric features using the image analysis software Image J (version 1.37) after calibration.
Hoboken nodules (HN), smooth muscle disintegration (SMD) and smooth muscle migration (SMM), myofibroblasts and empty spaces in Wharton’s jelly and number of HN of the arteries were examined under 10× magnification. The empty spaces and number of myofibroblasts in the Wharton’s jelly, and the disintegration and migration of smooth muscles to tunica intima were examined under high power field (40×).
Micrometry of umbilical vessels
Lumen diameter (LD) was considered as an average of three longest and three shortest diameter under 10×. Total WT was measured from the lumen to the outer margin of tunica media. The tunica media thickness (TMT) was measured from internal elastic lamina to the outer margin of tunica media. Thickness of the tunica intima was obtained by reducing the TMT from the total WT. The measurements of the two umbilical arteries of the umbilical cord were separately measured for all the parameters. The average of both was considered as the arterial measurement.
Elastin fibers
Verhoeff’s Van Geison stained slides were observed under high power field and elastic lamellae were counted from three different fields from the wall of each umbilical artery and vein and an average of the three fields were noted and recorded. Mean arterial elastic fibers were calculated by taking the average of elastic fibers of both the arteries of each umbilical cord. Mean arterial elastic fibers and venous elastic fibers were compared among normal and GDM groups.
Statistical analysis
Statistical analysis and plotting of graphs were carried out using Saigma Plot 13.0 (Syatat Software). All the continuous data were presented as mean±standard error of mean and categorical data as percentage and frequencies. The mean was analyzed by one-way analysis of variance with multiple comparison tests of Student-Newman-Keuls test. Chi-square test was used to compare the frequencies to see associations. P<0.05 is considered as statistically significant.
Results
Hoboken nodules, number of myofibroblasts and empty spaces in Wharton’s jelly
In the current study, a reduced number of HN was observed in the umbilical cord of GDM-Diet patients compared to the control group (Fig. 1).
The mean and standard error of number of HN and number of myofibroblasts are shown in Table 1. The mean number of HN of normal, GDM-Diet and GDM-Drug are. 2.57, 1.07, and 2.17, respectively. The mean of GDM-Diet (1.07) was found to be statistically significant when compared to the normal and GDM-Drug. In GDM-Diet it was found to be significantly reduced (P=0.002). The number of HN did not show any significant difference between the normal and GDM-Drug (P=0.286).
Increased empty spaces and reduced myofibroblasts were observed in Wharton’s jelly of GDM (G) compared to normal (N) umbilical cord (Fig. 2).
The mean number of myofibroblasts in normal, GDM-Diet and GDM-Drug are 29.02, 22.87, and 20.21, respectively. The mean 22.87 and 20.21 was found to be significant. When compared to the normal, GDM-Diet and GDM-Drug showed decrease in number of myofibroblasts in Wharton’s jelly. It was found to be statistically significant (P<0.001). The number of myofibroblasts did not show any significant difference between GDM-Diet and GDM-Drug (P=0.357) (Table 1).
Association of empty spaces in the Wharton’s jelly with GDM of different groups in umbilical cord is given in Table 2. Analysis showed absence of empty spaces in normal (96%) and presence of empty spaces in GDM-Diet (27.78%) and GDM-Drug (42.42%). It was found to be statistically significant (P≤0.001).
The impact of GDM on the number of HN in the umbilical artery and the number of myofibroblasts in Wharton’s jelly is shown in the graph for normal pregnancy, GDM-Diet, and GDM-Drug in pregnant female (Fig. 3).
Smooth muscle disintegration and migration
Increased SMD and SMM to the intima were observed in the umbilical artery and vein of GDM (G) compared to normal (N) (Figs. 4, 5).
The details of the association of SMD and SMM with GDM in different groups in umbilical artery and vein are given in Table 2.
The SMD in umbilical artery of GDM-Diet and GDM-Drug group was 52.17% and 63.63% respectively and it was statistically significant (P<0.001). The arterial SMM to intima was present in 43.48% cases of GDM-Diet and 48.49% cases of GDM-Drug group respectively and was statistically significant (P=0.003).
The SMD in umbilical veins of GDM-Diet and GDM-Drug group was 86.95% and 69.69% respectively and it was statistically significant (P<0.001). The venous SMM to intima was 86.96% and 69.68% respectively in GDM-Diet GDM-Drug group and too was statistically significant (P<0.001) (Table 2).
Histomorphometry of umbilical artery
Increased total WT and TMT of umbilical artery were observed in the GDM-Drug group compared to normal (N) (Fig. 4).
The mean and standard error of umbilical artery WT, thickness of tunica media, thickness of tunica intima and LD are shown in Table 3. The mean value of WT of normal, GDM-Diet and GDM-Drug groups are 0.92 mm, 0.85 mm, and 1.06 mm, respectively. The mean of GDM-Drug group (1.06 mm) was found to be statistically significant (P=0.002). Compared to normal and GDM-Diet, GDM-Drug group showed increase in WT. The mean value of TMT of normal, GDM-Diet and GDM-Drug group are 0.59 mm, 0.58 mm, and 0.7 mm, respectively. The mean of GDM-Drug (0.7 mm) was found to be statistically significant (P=0.005) (Fig. 4). Compared to normal and GDM-Diet, GDM-Drug group showed increase in TMT GDM-Diet and normal did not show significance in the media thickness (P=0.806). The mean value of tunica intima thickness (TIT) of normal, GDM-Diet and GDM-Drug are 0.35 mm, 0.30 mm, and 0.36 mm, respectively. It was not found to be significant (P=0.148). The mean value of LD of normal, GDM-Diet and GDM-Drug group are 0.77 mm, 1.11 mm, and 0.84 mm, respectively. It was not found to be significant (P=0.092).
The graph displays the effect of GDM on the umbilical artery, specifically focusing on the WT, TMT, TIT, and LD. The study compares these measurements in the umbilical arteries of normal pregnant female, those with GDM-Diet, and those with GDM-Drug (Fig. 6).
Histomorphometry of umbilical vein
Fig. 7 showed an increased LD in the umbilical vein of individuals with GDM compared to those with normal (N) conditions.
The mean and standard error of umbilical vein WT, TMT, TIT, and LD are shown in Table 4. The mean value of WT of normal, GDM-Diet and GDM-Drug are 0.62 mm, 0.47 mm, and 0.60 mm, respectively. It was not found to be significant (P=0.078). The mean value of TMT of normal, GDM-Diet and GDM-Drug are 0.39 mm, 0.30 mm, and 0.38 mm, respectively. It was not found to be significant (P=0.129). The mean value of TIT of normal, GDM-Diet and GDM-Drug are 0.23 mm, 0.23 mm, and 0.23 mm, respectively. It was not found to be significant (P=0.985). The mean value of LD of normal, GDM-Diet and GDM-Drug are 2.09 mm, 2.94 mm, and 2.95 mm, respectively. The mean of GDM-Diet and GDM-Drug was found to be statistically significant (P=0.001). Compared to normal, GDM-Diet and GDM-Drug showed increase in LD (Fig. 7). It was statistically significant. GDM-Diet and GDM-Drug did not show significance in the LD (P=0.970).
The graph shows how GDM affects the thickness of the umbilical vein wall, including measurements of the WT, tunica media, tunica intima, and LD. The data is gathered from normal pregnancies, GDM-Diet, and GDM-Drug in pregnant female (Fig. 8).
Elastic fibers
In individuals with GDM, there was a significant decrease in elastic fibers in both the umbilical artery and vein compared to individuals with a normal umbilical artery and vein (Fig. 9).
The mean and standard error of number of elastic fibers in umbilical arterial wall and venous wall in normal. GDM-Diet and GDM-Drug are shown in Table 5. The mean value of arterial wall elastic fibers (AWE) of normal, GDM-Diet and GDM-Drug are 17.43, 6.44, and 7.8 per high power field respectively. The mean value of venous wall elastic fibers of normal, GDM-Diet and GDM-Drug group are 6.2, 2.0, and 3.0 per high power field respectively. The mean of GDM-Diet and GDM-Drug groups for both artery and vein was found to be statistically significant (P<0.001). Compared to normal, GDM-Diet and GDM-Drug groups showed decrease in elastic fibers. It was statistically significant. However, a statistically significant difference was not observed between GDM-Diet and GDM-Drug groups (P=0.505).
A graph depicting the effect of GDM on the number of elastic fibers in the wall of the umbilical artery and vein: AWE and vein wall elastic fibers of normal, GDM-Diet, and GDM-Drug in pregnant female (Fig. 10).
Discussion
The intrauterine development of the fetus depends on the placental and umbilical vascular system. Inward remodeling is a common finding in smaller resistance vessels and may present with or without increase in the cross-sectional area of the vessels [16]. Umbilical artery blood flow resistance is an indication of ischemic changes in the placenta due to direct effect on vascularization and this emphasizes the importance of the placental-umbilical cord unit in pathologic pregnancies [17]. The structure of the umbilical vessels changes from the placental end to the fetal end. These changes were more predominant in the vein than in the artery. The vein’s wall-luminal ratio increased from the placental to the fetal end suggesting that the fetal end of the umbilical vein has a more refined role in the regulation of blood flow to the fetus [18]. Literature review showed lack of studies on the histomorphometrical changes of umbilical cord in GDM. The present study concentrated on 50 normal and 56 GDM (23 GDM-Diet and 33 GDM-Drug) umbilical cords for their histological and histomorphometric changes.
There are not many studies conducted on histomorphometry of umbilical cord and its vessels in GDM. Total WT and TMT of umbilical artery and umbilical vein lumen was found significantly increased in GDM which is in consistent with the other studies [19-23]. In contrast to the current study, Lateef [22] reported a thinner arterial wall in GDM with reduction in both tunica media and intima. However, number of HN were reported lesser in GDM umbilical arteries which is consistent with the present study. Earlier studies have reported SMD and SMM to intima and empty spaces in Wharton’s jelly in GDM similar to present study [19, 20]. Histomorphometry of umbilical cord and its vessels has been studied in detail in pre-eclampcia (PE) rather than GDM. Thicker as well as thinner vessel walls have been reported in PE [1, 7, 24-26].
The elastin fibers were significantly found reduced in the tunics of umbilical arteries (P<0.001) and veins (P<0.001) in GDM in the present study. A similar finding was observed in a study conducted in Egypt among GDM umbilical cords [27] and in other complicated pregnancy like PE [7]. A reduction of elastin associated with increased SMD and SMM of smooth muscle cells to intima was observed in PE that was attributed to exposure to hypoxia. Compensatory increase in the WT of umbilical arteries were observed as a functional adaptation to improve the altered hemodynamic conditions in PE [1, 7, 18, 28-30]. It has been reported in the literature that in GDM there would be deficiency of vasculo-syncytial membrane in tertiary placental villous leading to reduced oxygen diffusion and fetal hypoxia [31]. Exposure to hypoxia in GDM babies hampers the elastin synthesis as observed in the present study [6]. Elastin content of the vessel wall determines the elastic properties of the vessels. Blood vessels with a higher compliance deform easier than lower compliance blood vessels under the same pressure and volume conditions [7].
Hence, in the present study, exposure to hypoxia might have resulted in a reduction of elastic fibres. This might have further led to the SMD and SMM to intima and the dilated umbilical veins and other compensatory adaptations like thickness of tunica media in umbilical arteries. A reduction of elastic fibres in umbilial arteries could also result in the reduction of HN. Ischemic vascular changes in the placenta leading to the increased blood flow resistance in umbilical artery is a late pathognomic sign of GDM [23]. Exposure of umbilical arteries to increased blood flow resistance in treatment group might have led to the increased proliferation of smooth muscle cells and increased wall and media thickness of umbilical arteries.
Wharton’s jelly was found to have spaces with in it in many of the previous studies conducted in GDM and was supposed to be due to the degeneration [19-22]. In consistent to the previous studies, the present study also had a significant increase in the empty spaces and reduction in the number of myofibroblasts in both GDM groups. This could be due to the leaky endothelium in GDM [19, 23]. Such structural changes in the umbilical vessels and Wharton’s jelly were also reported in pathologic conditions with high pressure blood flow such as preeclampsia. Hence, it is proposed that apart from hypoxia, high pressure blood flow may also play a role in the histomorphometric changes of umbilical vessels in GDM.
In conclusion, GDM causes structural changes in the umbilical vessel wall. Elastic fibers were significantly reduced in umbilical vessel walls in GDM. A significant reduction in the number of myofibroblast and increase in the empty spaces in Wharton’s jelly was seen in GDM. Venous LD was significantly increased in GDM. There was no significant difference between any of the parameters in GDM-Diet and GDM-Drug group other than WT, which was significantly higher in umbilical arteries of GDM-Drug group.