Abou-Elghait, Amal T.a; Abdel-Aziz, Hoda A.b; Mahmoud, Faten Y.b
The placenta is a complex organ that is essential for fetal growth and development. It is composed of highly specialized cells that have a wide spectrum of functions including transport of maternal fuels to the fetus and the synthesis of various hormones and growth factors . It separates the maternal and the fetal circulation, with which it is in contact through different surfaces, that is, the syncytiotrophoblast exposes the placenta to the maternal circulation and the endothelium is in contact with fetal blood . Because of this unique position, the placenta is exposed to the regulatory influence of hormones and the different substrates present in both circulations . In turn, it can produce molecules that will affect the mother and the fetus independently. In the placental villi, almost the entire maternofetal and fetomaternal exchange takes place . In addition, most metabolic and endocrine activities of the placenta are localized in the villi [3,4]. Placental development is characterized by three distinct periods. At the beginning of gestation, a series of critical proliferation and differentiation processes predominantly of the trophoblasts eventually lead to the formation of villous and extravillous structures. The latter anchor the placenta in the uterus and remodel the uterine spiral arteries into low-resistance vessels. Then the newly formed villi differentiate through various steps of maturation. The end of gestation is associated with the expansion of placental mass, that is, villous growth . During the first half of gestation, the trophoblast is the key tissue that undergoes the most marked alterations, whereas extensive angiogenesis and vascularization occur in the second half of gestation. This period is also accompanied by extensive vascular remodeling and stabilization of the vascular bed [2,6].
Despite the advances made in recent years in the management of diabetes mellitus and, in particular the management of the diabetic pregnancy, diabetes remains a significant threat during pregnancy with an increased risk of malformation and still birth . Many studies suggest that the prevalence of diabetes mellitus among women of childbearing age is increasing because of more sedentary lifestyles, changes in diet, and childhood and adolescent obesity that is currently emerging in many countries . Gestational diabetes mellitus (GDM) is indicated by abnormal glucose tolerance with onset or first recognition during pregnancy, but that was normal before and will usually be normal after pregnancy [9,10]. It is associated with an increased risk of maternal complications and an adverse outcome of the pregnancy, including miscarriage, stillbirth, macrosomia, intrauterine growth retardation, and congenital anomalies [8,11]. The elevated blood glucose level in gestational diabetes is caused by hormones released by the placenta during pregnancy, called the human placental lactogen, also known as human chorionic somatomammotropic. It is similar to growth hormone; thus, it aids the growth of the baby, but it actually modifies the mother's metabolism and the processing of carbohydrates and lipids . The placenta in pregnancy complicated with diabetes is generally larger than normal and has numerous structural abnormalities that are likely to play a role, resulting in disturbances in fetal growth and development [13,14]. In an attempt to explain the discrepancy between fetal and placental changes, it is essential to study the microscopic structure of the placenta.
Aim of the work
In comparison with metabolically normal pregnant mothers, the present study will characterize the histological changes in the placental villi of women with poorly controlled gestational diabetes, that is, pregnant women who did not strictly follow the management of diabetes mellitus during pregnancy and excluding other complicating factors.
Materials and methods
The group studied included 22 pregnant women randomly chosen from the outpatient obstetric care unit of the Obstetrics and Gynecology Clinics, Faculty of Medicine, Assiut University. These mothers ranged in age from 20 to 38 years and the parity ranged from 1 to 5.
Ten women were included in a control group. These women fulfilled the following criteria: normal oral glucose tolerance test (OGTT) with 75g glucose, no endocrine abnormalities, no family history of diabetes, and no hypertension at the end of pregnancy. Twelve gestational diabetic pregnant women (GDM) were included in this study. Gestational diabetic pregnant women were diagnosed according to the WHO criteria, according to which 1-h OGTT value exceeding 8.9mmol/l was considered to indicate gestational diabetes . None of the studied pregnant women with positive OGTT followed the treatment when they attended the antenatal clinic in the late third trimester on initial presentation. These patients who were consistently not following the strict management of GDM were considered poorly controlled GDM patients. Informed consent from all the patients and the control participants was obtained before participation in this study.
Placental tissue samples were immediately collected after delivery from the central part of the maternal surface of the placenta. Specimens 1cm3 in size were processed for light microscopic examination, where they were fixed in 10% neutral-buffered formalin, dehydrated through alcohols, cleared in xylene, and embedded in paraffin wax. Subsequently, 5μm thick sections were stained with H&E, Van Gieson stain, and the PAS reagent . For electron microscopy, thin slices of the placental tissue were fixed in a 2.5% glutaraldehyde solution and then processed to obtain semithin sections (0.5μm) that were stained with toluidine blue. Ultrathin sections (80–90nm) were cut using an ultramicrotome, stained with uranyl acetate and lead citrate, and examined by JEOL transmission electron microscopy at 80 kV in EM unit, Assiut University, Assiut, Egypt.
Serial sections (5μm thick) were obtained from each case and subjected to immunohistochemistry using S100 protein antibodies. The avidin–biotin peroxidase method was used . Adult rat brain served as a positive control and another section without the primary antibody served as a negative control. The tissue was prepared in a manner similar to the placenta.
Light microscopic examination of the normal uncomplicated placenta showed terminal chorionic villi cut in various planes and separated by inter-villous space that contained maternal blood (Fig. 1). Each villous consisted of a mesenchymal core that contained fetal blood capillaries and was surrounded by trophoblasts (Fig. 1). The outer covering of trophoblasts had a multinucleated syncytiotrophoblast and a generative cytotrophoblast. The former appeared as a single layer of syncytial cytoplasm containing an apical acidophilic brush border. Their nuclei were small and hyperchromatic (Fig. 1). Cytotrophoblasts were few and pale on staining. Villous stroma comprised loose mesenchymal tissue containing delicate connective tissue fibers and many kinds of stroma cells (Fig. 1). Fetal blood vessels with clearly evident erythrocytes could be identified inside the placental villi. These vessels had variable sizes and were lined with flattened endothelial cells. Their nuclei were flat and deeply stained (Fig. 1). Van Geison-stained section showed delicate concentric layers of collagen fibers in the connective tissue stroma and around the fetal blood capillaries (Fig. 2a). Periodic acid Schiff-stained sections showed concentrated magenta red staining area at the brush border and around the fetal capillaries (Fig. 2b). Semithin section stained with toluidine blue showed normal chorionic villi, surrounded by trophoblasts and have dilecate amount of connective tissue fibers surrounding mainly the blood vessels (Fig. 3).
The ultrastructural appearance of the normal placenta showed chorionic villi surrounded by a layer of multinucleated syncytiotrophoblasts that had a continuous syncytial layer with multiple hyperchromatic nuclei and numerous apical microvilli (Fig. 4). The syncytial cytoplasm contained numerous binocytotic vesicles, mitochondria, and different cytoplasmic inclusions such as glycogen granules and lipid droplets (Fig. 5). A trophoblastic basement membrane was present between the trophoblastic layer and the stroma. It was continuous, uniform, and thin (Fig. 5). Cytotrophoblasts had a electron-lucent cytoplasm and contained single, large nuclei (Fig. 6). They were scattered beneath the syncytium and had a granular cytoplasm containing mitochondria as well as glycogen granules (Fig. 9). The stroma had a connective tissue core of the chorionic villous separating the trophoblastic basement membrane from the capillary endothelial basal lamina, and contained different kinds of stromal cells and scattered fine collagen fibrils (Fig. 7). It also contained fetal blood vessels, which were lined with flat endothelial cells that contained a few organelles and flat nuclei (Figs 8 and 9).
Under a light microscope, the poorly controlled diabetic placenta showed histological features that were different from those of the normal placenta. It was characterized by an increase in the cut sections of terminal villi and associated capillaries (Fig. 10). A focal area of syncytial thinning and or even degeneration was observed. An increase in syncytial knots was frequently observed (Fig. 10). Villous stroma showed the deposition of a homogeneous acidophilic fibrinoid substance that was sometimes vacuolated (Fig. 10). Fetal blood vessels were markedly dilated and congested (Fig. 11). Van Geison stain showed excessive concentric layers of collagen fibers, mainly around the fetal blood vessels (Fig. 12a). Periodic acid Schiff-stained sections showed a highly concentrated magenta red staining area at the brush border, villous core, and around the fetal capillaries (Fig. 12b).
In the semithin section, a collection of dense irregular syncytial nuclei associated with moderate thickening of the basement membrane of the syncytiotrophoblast was observed (Fig. 13). Hyperplasia of the cytotrophoblast was frequently observed. Concentric lamellae of fibrinoid materials in the connective tissue stroma were also observed (Fig. 13).
The ultrastructural appearance of the poorly controlled diabetic placenta showed patchy focal syncytiotrophoblastic necrosis. The syncytiotrophoblastic cytoplasm had numerous cytoplasmic vacuoles with marked thickening of the basement membrane of the trophoblast (Fig. 14). Syncytial nuclei were highly infolded with chromatin condensation (nuclear pyknosis) (Figs 14 and 15). Syncytial thinning and damage in the form of distorted microvilli that were shortened or completely absent in many areas were also observed (Figs 15 and 16). Cytotrophoblasts had a vacuolated cytoplasm, multiple lipid droplets, and a dilated smooth endoplasmic reticulum (Fig. 17). Villous stroma showed fetal blood vessels with swelling of the endothelium, separation of the basal membranes in some capillaries, dilatation of vessel-lumen, and deposition of filamentous material. Disarrangements of the perivascular space and irregular thickening of the endothelial basal lamina were observed (Figs 18 and 19).
One of the most characteristic features of diabetic placenta was the frequent appearance of Hofbauer cells, which were usually present just underneath the degenerated syncytium. It had a flat irregular nucleus and cytoplasm containing multiple phagocytic vacuoles (Fig. 19). Some of these cells showed abundant cytoplasmic vacuoles and long strands of rough endoplasmic reticulum in the juxtanuclear region (Fig. 20). A group of these cells appeared with a voluminous cytoplasm engorged with numerous phagocytic vacuoles containing parts of degenerated materials (Fig. 21). In fully degenerated villous, the intravillous stroma appeared as a mass of a fibrinoid substance, which showed scanty cores of stroma containing cellular debris (Fig. 22). Also, a group of round-shaped cells, with a central euchromatic nucleus and a prominent nucleolus, was often observed (Fig. 23). In advanced stages, the fibrinoid mass increased in size, whereas the syncytiotrophoblast overlying it underwent atrophy and degeneration.
In comparison with normal placenta, which showed a negative reaction for S100 protein throughout the chorionic villi (Fig. 24), diabetic placenta showed a positive reaction for S100 protein in the trophoblastic layer, connective tissue core, and stromal cells (Fig. 25). The reaction appeared as brown cytoplasmic irregular granules scattered all over the cytoplasm (Fig. 25, inset).
Recently, there has been an interest in the role of placenta in the pathogenesis of variety of pregnancy disorders. Considerable advances have been made in understanding the complex fetomaternal relationship in complicated pregnancies and its influence on the intrauterine development of the fetus . Samples from the central part of placentas were collected in this study to avoid areas of fibrosis or calcification . In the present study, histological examination of a normal full-term placenta showed variations in the shape and apparent size of chorionic villi. Each villus had an outer trophoblastic layer and an inner connective tissue core that was interposed between maternal and fetal circulation. The trophoblastic layer had syncytiotrophoblasts and cytotrophoblasts. Similar observations have been documented previously [20,21]. This was in contrast with the findings of some authors , who did not observe the presence of cytotrophoblastic cells at normal full term. In the present work, marked histological differences were found between the diabetic and the normal groups of placentas; the histological sections of diabetic placenta tended to show smaller and numerous chorionic villi in comparison with the normal placentas. This might be a result of the continuous branching of the chorionic villi in response to prolonged ischemia in an attempt to increase the surface area of villi to insure good fetal nutrition . The terminal villi showed changes in maturation and increased vascularization. A huge number of villi showed fibrinoid necrosis. These observations were in agreement with those of many studies [24–26] that attributed these findings to the hypoxia resulting from diabetes. However, these changes might be considered as a morphological landmark for an immunological reaction in the villous stroma . In the present work, cytotrophoblastic hyperplasia and more syncytial knots were frequently observed in the diabetic group, which was in agreement with many studies, which assumed that these knots might act as cellular bridges to protect the villus capillaries from the effect of sudden changes in the intervillus space pressure during labor . However, the increased syncytial knots in diabetic cases might be an indication of active placental growth. This was based on the hypothesis that hyperinsulinemia is a growth stimulus in diabetic pregnancies  or it might be attributed to the inability of syncytiotrophoblasts to proliferate, therefore depending on the cytotrophoblast population for growth and renewal. The proliferating cytotrophoblasts begin to differentiate, leading to fusion of these cells; a second differentiation process occurs in the outer syncytiotrophoblastic layer, in which the aging nuclei are packed into syncytial knots and extruded . A previous study  has reported that the proliferation of cytotrophoblasts was associated with the formation of syncytial knots as a very common finding in pre-eclampsia because of an impairment in vasculature. In addition, syncytial knots were numerous in cases of low oxygen tension as a slow accommodation to chronic hypoxia; thus, these knots might be increased to compensate the loss of syncytium in areas of infarction . Also, areas of fibrinoid material in the connective tissue stroma of diabetic placenta were observed frequently, especially around the fetal blood capillaries. It was suggested that these stromal changes in diabetic placenta might have resulted from an immunological reaction in the villous stroma . Many researchers have reported that the microfibrils and the amorphous components of the fibrinoid substance appear to be synthesized by the residual cytotrophoblasts. They also added that immunohistochemical evidences support the cytotrophoblastic origin and the secretory nature of fibrinoid material, and the cellular debris certainly derives from degeneration of the syncytiotrophoblast that covers the fibrinoid mass [33,34]. Many authors have concluded that most of the placental abnormalities may be because of some unknown constituent factors of the diabetic state, which is only partially influenced by the diet or insulin . In this study, excessive concentric deposition of collagenous fibers in the stroma of many diabetic villi and around fetal blood capillaries was observed. These findings indicated a placental response to altered glycemia that could have important consequences for the fetus, as this perivascular fibrosis might be the cause of congested fetal blood vessels .
In the present work, the electron microscopic examination of placental villi of diabetic mothers showed widespread and sometimes severe damage of syncytium. Multiple cytoplasmic vacuoles of variable sizes were observed, associated with scarce cell organelles and destroyed surface microvilli. In some areas, very short or completely absent microvilli were observed. These syncytial trophoblastic microvilli were believed to be one of the important structures responsible for transplacental metabolism exchanges [36,37]. Apical microvillar density has been reported to be related to the degree of trophoblastic maturation  and had been considered to be the most involved syncytiotrophoblastic structure under oxygen and nutrient exchange . Decreased microvillar density has been observed in intrauterine growth retardation placentas , pre-eclamptic placentas , and in placenta from pregnancies with other complications, including a decreased metabolic exchange state in diabetic placentas. This might be attributed to exposure to hypoxia . In addition, a recent study has reported that several genes related to transplacental metabolic exchange also showed changes in complicated placenta . The present study showed thickening of the trophoblastic basement membrane, which was one of the most characteristic features that was seen in diabetic placenta. In agreement with our study, many authors observed a marked increase in the thickness of the basement membrane of the syncytiotrophoblast in diabetic placentas. Some authors attributed this change to the pathological immune complex, resulting in the pathological immune complex deposition in the area of the basal membranes of a syncytiotrophoblast and vascular endothelium . Many studies [42,44] have suggested that thickening of the basement membrane might be a result of the hypoxic state of diabetes, as some authors have observed thickening and separation of the basement membrane in placenta derived from women who live at high altitudes , indicating that the hypoxic state could alter placental morphology. In addition to the previous studies, many authors have concluded that the pathologic changes in the placentas of diabetic women such as significant thickening of the basement membrane of trophoblasts, separation of basal membranes in basal capillaries, distention and proliferation of endothelial cells, and disarrangements of the perivascular space are significant factors resulting from placental hypoxia, and they contribute toward fetal anoxia in pregnancy complicated by diabetes mellitus . Other researchers  have concluded that the accumulation of histochemical compounds, for example carbohydrates and fat droplets in the basement membrane leads to structural changes such as a thickened basement membrane . A frequent association between villous fibrinoid masses and thickening of the trophoblastic basement membrane has been observed, as already reported in many studies [46,47]. This association, however, is not fully understood. According to previous researches, both fibrinoid deposition and thickening of the basement membrane might be manifestations of antigen–antibody reactions [47,48].
In the present study, the diabetic placenta showed numerous fetal blood capillaries in comparison with control one; these capillaries were in close association with the trophoblast and consequently came in contact with the thinned-out syncytiotrophoblasts. In recent studies, these results have been explained as follows: in a pregnancy complicated by maternal diabetes, fetal hyperglycemia induces changes in the placental vasculature such as increased growth and angiogenesis. Also, the metabolic effects of insulin result in the stimulation of glycogen synthesis and modulation of angiogenesis on the placental arterial endothelium . In addition to this, many authors have observed that the syncytiotrophoblasts come in contact with some peripherally shifted capillaries with no intervening connective tissue stroma in between, forming the so-called vasculosyncytial membrane, which might represent an attenuated form of the placental barrier [28,49]. Also, in this work, the multilaminal basement membrane that surrounded the capillaries showed a huge variation, possibly because of the variation in capillary age . These structural changes resulted in the thickening of the placental barrier, leading to impairment in transplacental transport, and thus decreased placental blood flow, which is crucial for the transport of water, nutrients, and waste from the maternal to the fetal space to sustain normal placental and fetal development .
In the present study, electron microscopic examination of the stroma of the placental villi in the diabetic group showed numerous Hofbauer cells in comparison with the control group. Recent studies  have reported that Hofbauer cells are placental macrophages that are present in the villus across gestation. Although they were identified more than 100 years ago, their specific role in placental function remains largely unelucidated. Many other studies have concluded that Hofbauer cells are present within the core of the chorionic villous of the placenta, particularly numerous in early pregnancies. They are believed to be a type of macrophage and are most likely involved in preventing the transmission of pathogens from the mother to the fetus . Many authors have reported that Hofbauer cells often lie adjacent to a thick basement membrane, which underlies a layer of cytotrophoblast stem cells that are the progenitors of all the trophoblast lineages. Other researchers have added that they are the major cell type of the human placental villous core and are particularly numerous at the beginning of pregnancy. Other workers have concluded that Hofbauer cells are fetal placental antigen-presenting cells that share a number of features with macrophages but also have some distinct properties [54–58]. A recent study hypothesized that the locations and numbers of Hofbauer cells were significantly correlated with the vascular structures within the placental villi core and therefore might be implicated to play roles in placental vasculogenesis and angiogenesis . In agreement with our result, the presence of Hofbauer cells more frequently in the diabetic group, many studies have proved that these cells are present in immature placenta and the placenta of complicated pregnancies and either disappear or become very scanty after the fourth month of normal gestation [60–62]. Also, there has been wide agreement that these cells are a feature of placenta from cases of rhesus incompatibility , villitis of unknown etiology , in placenta from pregnancies with histological chorioamnionitis , and placenta from diabetic women [52,66,67]. Therefore, we concluded that Hofbauer cells might play a key role in placental pathophysiology, and further studies of their isolation and culture are needed.
Many studies have shown that the vast majority of neurological abnormalities present during childhood have a prenatal or a perinatal origin, especially in complicated pregnancies . Until recently, clinical laboratory assessments were based essentially on biochemical parameters, ultrasound and Doppler patterns, and the determination of blood pH and gases. However, the measurement of brain constituents may provide a direct indication of cell damage in the nervous system. The S100 protein, a calcium-binding protein highly concentrated in the nervous system, appears to be a marker in prenatal and perinatal medicine because of its reproducible, simple, and reliable measurements . In the present work, localization of S100 protein was observed in the trophoblastic and villous stroma cells of diabetic placenta, and was almost absent in the control group. In agreement with our result [68,69], many studies have shown that the concentration of S100 protein increased in amniotic fluid and in the cord blood of fetuses with brain damage, suggesting that these tissues may, at least in part, be responsible for the high level found in the fetal circulation of high-risk pregnancies. Also, they added that the significance of placental S100 protein should be taken into account when this protein is used as a marker of brain injury in the fetus or infant at birth. It was suggested that the direct assessment of neurologically adverse effects in the newborn by means of markers in peripheral blood is of great clinical significance. Also, measurements of brain-specific proteins could be used to monitor the effects of pharmacologic interventions to protect the brain as well as to evaluate the possible adverse or beneficial effects of new interventions . It was concluded that significantly higher values of these proteins were found under conditions associated with adverse neonatal outcome, especially after perinatal asphyxia and preterm labor . In addition to this, a strong positive correlation was found between amniotic fluid and villous tissue S100 protein and the oxidative stress resulting from pregnancies, such as chronic fetal hypoxia . Hence, it was suggested that chronic fetal hypoxia increases the intrauterine release of S100 protein .
In the present work, structural abnormalities in the diabetic placental villi have been observed. These histological abnormalities were not observed in every diabetic placenta but they were not completely absent in any. Also, these findings might support the hypothesis that impaired placental function in gestational diabetes was one of the main reasons for the increased frequency of fetal complications in diabetic pregnancies. According to our results, the presence of S100 protein may serve as a marker of brain damage in diabetic pregnancy.
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Conflicts of interest
There are no conflicts of interest.
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