Immunohistochemical evidence of lactoferrin in human embryo–fetal bone and cartilage tissues

Lf (lactoferrin) is an 80‐kDa iron‐binding protein, which has been suggested to promote bone growth in murine models. In view of this, we aimed to analyse the immunohistochemical distribution of Lf in human embryonal and fetal bone and cartilaginous tissues at different gestational weeks in order to evaluate whether a role for this protein might be proposed also in human osteogenesis. Bone and cartilaginous specimens were taken at autopsy from 25 fetuses (8–34 weeks of gestation). Ten samples of human adult bone and cartilage were also submitted to the immunohistochemical procedures. Sections, 4‐μm thick, were cut from formalin‐fixed paraffin‐embedded tissue blocks and stained with a monoclonal antibody against human Lf, following antigen retrieval procedures. Lf immunoreactivity was maily localized in the mesenchymal cells forming the periosteum as well as in chondroblasts at the eighth gestational week; a strong Lf immunoexpression in immature osteocytes and osteoblasts was noted up to the 18th gestation week, with a considerable decrease by the 24th week. No Lf expression was found in any bone area after the 30th and up to the 34th week of gestation. Our data seem to suggest an important role for Lf as a bone growth regulator in the early phases of the human endochondral ossification, with an anabolic action similar to that previously reported in cell culture lines and in animal models.     

Inflammation-related proteins in the blood of extremely low gestational age newborns. The contribution of inflammation to the appearance of developmental regulation

BackgroundWe wanted to assess to what extent concentrations of circulating proteins appear to be developmentally regulated, and to what extent such regulation is influenced by intra-uterine inflammation.MethodsWe measured 22 proteins in blood obtained on postnatal days 1, 7, and 14 from 818 children born before the 28th week of gestation for whom we also had information about placenta morphology.ResultsWithin the narrow gestational age range of this sample, some protein concentrations increase in blood with increasing gestational age. More commonly, the concentrations of inflammation-related proteins decrease with increasing gestational age. We observed this inverse pattern both in children whose placenta was and was not inflamed.Conclusions/InferencesRegardless of whether or not the placenta is inflamed, the concentrations of inflammation-related proteins in early blood specimens appear to be developmentally regulated with the most common pattern being a decrease with increasing gestational age.     

Ontogeny of Human Langerhans Islets. A Review of Some Light- and Electron-Microscopical, Immunohistochemical, and Functional Data on Fetal Development of the Endocrine Pancreas (Review)

The parenchymal cells of the islets of Langerhans belong to the extensive human neuroendocrine system. Its messenger substances are biogenic amines and neurohormonal peptides. Like other neuroendocrine cells, the islet cells might have originated from the neural crest. However, in the fetal life, their stem cells are located in the epithelium of the pancreatic ductuli. As early as at the 8th gestational week, these stem cells have been found to contain secretory granules of the neuroendocrine type. Evidences for production of insulin, somatostatin, glucagon, and PP (the pancreatic polypeptide) have been obtained immunohistochemically in the samples from the 10–12th gestational weeks. In the samples from the 14th week, cell clusters have been observed, which are outgrowing from the ductular epithelium and forming primitive Langerhans islets. The insulin cells predominate markedly and are shown to respond functionally to glucose stimulation. By the 16th week, the islets become vascularized, with the primary innervation. The completely formed endocrine pancreas, as it is observed at birth, is revealed at the 26th gestational week. Based on some light-microscopical, ultrastructural, and immunohistochemical characteristics of the islet parenchymal cells and their supply with blood vessels and nerves, three phases of the gland embryonal/fetal development are identified.     

A systematic review to calculate background miscarriage rates using life table analysis

The objectives of the current study were to calculate: (1) the expected rates of miscarriage by gestational week; (2) the cumulative risk of miscarriage; and (3) the remaining risk of miscarriage for gestational weeks five through 20, through a systematic review of the literature. We searched MEDLINE for articles published in English through the end of 2009. References of articles were also searched. Four studies were identified to have the three necessary pieces of information for the proposed calculations: (1) gestational age at study entry, (2) pregnancy outcome; and (3) the gestational age at which the pregnancy outcome occurred. Data were extracted from each study and Life Table Analysis Methods were conducted. Weekly miscarriage rates varied in the early gestational weeks with the highest rate documented at >20 miscarriages per 1000 women-weeks at each week of gestation prior to week 13. By week 14, the rate for all studies became relatively comparable and fell below 10 miscarriages per 1000 woman-weeks at risk and fell even lower through week 20. The cumulative risk of miscarriage for weeks 5 through 20 of gestation ranged from 11 miscarriages per 100 women to 22 miscarriages per 100 women (11-22%). Based on data from comparable study populations, a range of background miscarriage rates by week of gestation for weeks 5 through 20, the cumulative risk of miscarriage, and the remaining risk of miscarriage are presented. Wider variation of miscarriage rates and risks occurred early in gestation (<14 weeks).     

Cerebrovascular response to normal pregnancy: a longitudinal study

We used a longitudinal study design (gestational weeks 8, 15, 22, 29, and 36 and 12 wk postpartum ) to investigate the effect of normal pregnancy on cerebral autoregulation and pressor response. Blood flow velocities in the right internal carotid artery, end-tidal CO2, and mean arterial pressure (MAP) were simultaneously and continuously recorded in 16 healthy pregnant women during standardized hyperventilation and handgrip. Blood flow velocities were recorded using Doppler ultrasound sampled beat by beat using the ECG signal. The results demonstrate that the vasoconstrictor response to hyperventilation is unchanged during pregnancy. During standardized handgrip, MAP showed a statistically significant increase during pregnancy that did not affect cerebral blood flow. A statistically significant reduction in the MAP response to handgrip was seen in week 36. In conclusion, pregnancy has no impact on cerebral autoregulation. There is an impact on the pressor response resulting in a blunted reaction at week 36, probably caused by a fall in the baroreflex set point.     

Intermediate filament typing of the human embryonic and fetal notochord

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.     

Characteristics of human cortical pyramidal neuron development during the second gestational trimester

Prenatal ontogeny of the human neocortex exhibits specific characteristics that make its organization unique. Therefore, experimental data obtained on animal models cannot be extrapolated to human cortex morphogenesis during the middle and late gestational periods. Characteristics of the development of cortical pyramidal neurons of the human brain were studied in the brains of eight fetuses at gestational ages between 16 and 26 weeks. Immunohistochemical labeling of neurons was performed using antibodies against microtubule associated protein 2 (MAP2), a structural protein of microtubules. Expression of this protein marks the beginning of dendrogenesis. MAP2 is mainly located in the neuron body and dendrites, which allowed the neuron morphotype and location in specific cortical layers to be determined. It was shown that MAP2-immunopositive neurons were identifiable in embryonic cortical layer eV as early as the 18th gestational week. By the 25th gestational week, two populations of pyramidal neurons were discernible in the cortical plate, one of them located in layer eV and the other, in layer eIII, which developed later. Since differentiating neurons are known to be more vulnerable than neuroblasts and mature neurons, these results suggest that critical periods for corticofugal and corticocortical populations of pyramidal cells occur at different stages of the second gestational trimester.     

Ontogeny of immunoreactive glicentin in the human gastrointestinal tract and endocrine pancreas

The gestational time of appearance and distribution of immunoreactive glicentin was compared to that of immunoreactive glucagon in the gastrointestinal tract and endocrine pancreas of human fetuses, aged between 5 and 24 weeks, by an indirect immunoperoxidase method. With the glicentin antiserum No. R 64, the first immunoreactive cells were detected at the 10th week of gestation in the oxyntic mucosa and proximal small intestine, at the 8th week in the ileum and at the 12th week in the colon. In the endocrine pancreas, the first immunoreactive cells were observed as early as 8 weeks within the walls of the primitive pancreatic ductules. At a more advanced stage of development (12 weeks), they were found interspersed among the islet cell clusters and still later (16 weeks) inside the recognizable islets of Langerhans. With the glucagon antiserum No. GB 5667, no immunoreactive cells were demonstrated in the gastrointestinal tract whatever the age of the fetuses. In the endocrine pancreas, the first immunoreactive cells were observed at the 8th week of gestation in the pancreatic parenchyma. The distribution of glucagon-containing cells in the pancreas was similar to that of glicentin immunoreactivity throughout ontogenesis. In the pancreatic islets of one 18-week-old human fetus, the study of consecutive semithin sections treated by both antisera showed that the same cells were labelled. The significance of these findings concerning the role of glicentin as a glucagon precursor is discussed.     

Appearance of interstitial cells of Cajal in the human midgut

Several subtypes of the interstitial cells of Cajal (ICC) form networks that play a role in gastrointestinal motor control. ICC express c-kit and depend on signaling via Kit receptors for development and phenotype maintenance. At 7-8 weeks of development, c-kit-immunoreactive (c-kit-IR) cells are present in the human oesophagus, stomach and proximal duodenum wall. In the remaining small and large bowel, c-kit-IR cells appear later. The object of the present study is to determine the timing of the appearance of c-kit-IR ICC in the parts of the digestive tube originating from the midgut (distal duodenum, jejunum, ileum and proximal colon). Specimens were obtained from eight human embryos and 11 fetuses at 7-12 weeks of gestational age. The specimens were exposed to anti-c-kit antibodies to investigate ICC differentiation. The differentiation of enteric neurons and smooth muscle cells was immunohistochemically examined by using anti-PGP9,5 and anti-desmin antibodies, respectively. In the distal duodenum, jejunum and ileum, c-kit-IR cells emerged at week 9 at the level of the myenteric plexus in the form of a thin row of cells encircling the inception of the ganglia. These cells were multipolar or spindle-shaped with two long processes and corresponded to the ICC of the myenteric plexus. In the proximal colon, c-kit-IR cells emerged at week 9-10 in the form of two parallel belts of cells extending at the submucosal plexus and the myenteric plexus levels. We conclude that ICC develop following two different patterns in the human midgut.     

Immunohistochemical localization of prostaglandin endoperoxide synthase in human fetal membranes and decidua

Prostaglandins play an important role during the maintenance of pregnancy and the initiation of parturition. Prostaglandin endoperoxide synthase activity has been demonstrated in human fetal membranes and decidua. Using immunohistochemical techniques, we identified in these tissues the cell types that contain prostaglandin endoperoxide synthase. A total of 33 specimens, ranging from 8 wk to 42 wk gestation, were studied. Decidualized stromal cells stained the most intensely and consistently of all cell types. Cytotrophoblast of the chorion and early placental villi and syncytotrophoblast of all gestational ages demonstrated a lighter, more variable staining pattern. Regardless of gestational age, amnion stained in a heterogeneous fashion, with some cells demonstrating an intense staining and other cells having no staining. There were no observable differences in laboring compared to nonlaboring term specimens. In summary, the specific cell types that contain immunoreactive prostaglandin endoperoxide synthase have been identified in fetal membranes and decidua.     

Impact of maternal undernutrition during the periconceptional period, fetal number, and fetal sex on the development of the hypothalamo-pituitary adrenal axis in sheep during late gestation

Evidence from epidemiologic, clinical, and experimental studies has shown that a suboptimal intrauterine environment during early pregnancy can alter fetal growth and gestation length and is associated with an increased prevalence of adult hypertension and cardiovascular disease. It has been postulated that maternal nutrient restriction may act to reprogram the development of the pituitary-adrenal axis, resulting in excess glucocorticoid exposure and adverse health outcomes in later life. It is unknown, however, whether maternal nutrient restriction during the periconceptional period alters the development of the fetal pituitary-adrenal axis or whether the effects of periconceptional undernutrition can be reversed by the provision of an adequate level of maternal nutrition throughout the remainder of pregnancy. We have investigated the effect of restricted periconceptional nutrition (70% of control feed allowance) from 60 days before until 7 days after mating and the effect of restricted gestational nutrition from Day 8 to 147 of gestation on the development of the fetal hypothalamo-pituitary adrenal (HPA) axis in the sheep. In these studies, we have also investigated the effects of fetal number and sex on the pituitary-adrenal responses to periconceptional and gestational undernutrition. In ewes maintained on a control diet throughout the periconceptional and gestational periods, fetal plasma ACTH concentrations were higher and the prepartum surge in cortisol occurred earlier in singletons compared with twins. Plasma ACTH concentrations were also significantly higher in male compared with female singletons, and in twin fetuses, the prepartum surge in cortisol concentrations occurred earlier in males than in females. Periconceptional undernutrition resulted in higher fetal plasma concentrations of ACTH between 110 and 145 days of gestation and a significantly greater cortisol response to a bolus dose of corticotropin-releasing hormone in twin, but not singleton, fetuses in late gestation. We have therefore demonstrated that fetal number and sex each has an impact on the timing of the prepartum activation of the HPA axis in the sheep. Restriction of the level of maternal nutrition before and in the first week of a twin pregnancy results in stimulation of the fetal pituitary-adrenal axis in late gestation, and this effect is not reversed by the provision of a maintenance control diet from the second week of pregnancy.     

Development of precise maps in visual cortex requires patterned spontaneous activity in the retina

The visual cortex is organized into retinotopic maps that preserve an orderly representation of the visual world, achieved by topographically precise inputs from the lateral geniculate nucleus. We show here that geniculocortical mapping is imprecise when the waves of spontaneous activity in the retina during the first postnatal week are disrupted genetically. This anatomical mapping defect is present by postnatal day 8 and has functional consequences, as revealed by optical imaging and microelectrode recording in adults. Pharmacological disruption of these retinal waves during the first week phenocopies the mapping defect, confirming both the site and the timing of the disruption in neural activity responsible for the defect. Analysis shows that the geniculocortical miswiring is not a trivial or necessary consequence of the retinogeniculate defect. Our findings demonstrate that disrupting early spontaneous activity in the eye alters thalamic connections to the cortex.     

Optical and scanning electron microscopy observation of the mucosa of human fetal tongue

The structural features of the human foetal tongue have been studied in foetuses from 8th to 20th week of pregnancy. The characteristics of the developing papillae as well as of epithelial and mesenchymal layers have been pointed out. An early differentiation of the mesenchymal tissue has been observed, concerning phenomena of cellular condensation and reticular fibers organization both in superficial and deep layers. The hypothesis of the existence of straight interactions between epithelium and mesenchyme also in the developing human tongue mucosa has been suggested. Also the observations at SEM demonstrate that from the 8th to the 20th week the epithelial surface of the tongue reaches a stable structural pattern. From 11th week a characteristic cellular polymorphism occurs: cells with microvilli that diminish progressively, ciliated cells that disappear almost completely at the 20th week and cells whose free surface show microplicae, definitive stage of the tongue cell evolution.     

Alterations in antioxidant enzyme activities in the eyes, aorta and kidneys of diabetic rats relevant to the onset of oxidative stress

Profound changes in antioxidant enzyme activities were observed in a number of vascular tissues during the development of streptozotocin-induced diabetes in rats. In the eyes, there was an increase in superoxide dismutase activity at week 4 of diabetes. However, no difference in superoxide dismutase activity was observed between the control and diabetic animals at week 8. On the other hand, the diabetic state did not seem to affect the catalase activity in the eyes. There was a generalized increase in catalase activity of the eyes from week 4 to week 8 irrespective of the diabetic state. For glutathione peroxidase in the eyes, a decreased activity was observed in the diabetic animals at week 8, but not in week 4. A different pattern of enzyme activity changes was observed in the aorta where an increase in superoxide dismutase activity was observed in the diabetic group at week 4 but not in week 8. On the other hand, an increase in catalase activity was observed only at week 8 but not at week 4. Whereas there was no observed difference between the control and diabetic animals in glutathione peroxidase activity in the aorta, except for a generalized decrease from week 4 to week 8 in both groups of animals. In big contrast to the eyes and aorta where an increase in superoxide dismutase activity was observed at week 4 of diabetes, no change in kidney superoxide dismutase activity was noted at week 4 and a decrease was observed at week 8. A similar pattern of enzyme activity changes was observed for glutathione peroxidase in the kidneys. The catalase activity in the kidneys was not affected at all by the diabetic state at both week 4 and week 8. These results clearly demonstrate the active involvement of these antioxidant enzymes during the development of diabetes, and could be rationalized by the differential response of the tissues towards the different extent of oxidative stress imposed by the diabetic state on the different tissues.     

The natural history of gastroschisis during fetal life: development of the fibrous coating on the bowel loops

Patients with gastroschisis have a paraumbilical defect of the anterior abdominal wall through which bowel loops protrude. These bowel loops are edematous and covered by a fibrous coating. The moment of occurrence of gastroschisis as well as the development of the fibrous coating are unknown. This prompted an investigation of 26 human embryos and fetuses with gastroschisis at various developmental stages (crown-rump length 25-240 mm) and two stillborn fetuses (gestational age 30 weeks) with gastroschisis, as well as resected material from ten newborns (gestational age 33-40 weeks) operated for gastroschisis. Progressive changes of the serosa were only noted after the 30th week of gestation and consisted of an amniotic fluid peritonitis and progressive fibrosis. The changes are in agreement with experimental data and correlate with changes in the composition of amniotic fluid. The findings prove that gastroschisis is a very early occurrence, while the fibrous coating is a late development.     

Development of human pancreas

The developmental sequence of human pancreatic secretory proteins has not previously been studied in detail. We applied immunohistochemistry to study 20 fetal and neonatal pancreas' (8th to 39th gestational weeks) using antisera against the following pancreatic secretory proteins: pancreatic secretory trypsin inhibitor (PSTI), serine proteinases (trypsin, chymotrypsin, and elastase I), and amylase. PSTI was first detected in developing buds of the pancreas during the 8th gestational week, and proteinases were observed in acinar cells during the 14th week of gestation. Immunoreactivity for both PSTI and proteinases was found in most acinar cells soon after their appearance. Immunoreactivity for amylase could not be detected in fetal or neonatal pancreas tissue. PSTI was also found in developing islets during the 14th gestational week, but the number of immunoreactive cells had decreased by term. Cells positive for serine proteinases were occasionally in contact with islets in second-trimester fetuses. In discussing these results, we give particular attention to the nonparallel appearance of secretory products in the fetal pancreas, and the significance of cells immunoreactive for secretory proteins in endocrine islets.     

Stabilizing cumulative incidence estimation of pregnancy outcome with delayed entries

A pregnancy may end up with (at least) three possible events: live birth, spontaneous abortion, or elective termination, yielding a competing risks issue when studying an association between a risk factor and a pregnancy outcome. Cumulative incidences (probabilities to end up with the different outcomes depending on gestational age) can be estimated via the Aalen–Johansen estimate. Another issue is that women are usually not entering such an observational study from the first day of pregnancy, resulting in delayed entries. As in traditional survival analysis, this can be solved by considering “at risk” at a given gestational age only for those women who entered the study before that age. However, the number of women at risk at an early gestational age might be extremely low, such that the estimates of cumulative incidence may increase exaggeratedly at that age because of a single event. One solution to reduce the problem has been recently proposed in the literature, which is to ignore simply those early events, creating a small mean bias but enhancing stability of estimates. In the present paper, we propose an alternative computationally simple approach to tackle this problem that consists to postpone to later gestational ages (rather than to ignore) those early events. The two approaches are compared with respect to bias, stability, and sensitivity on the smoothing parameter via simulations reproducing realistic pregnancy scenarios, and are illustrated with data from a study on the effects of statins on pregnancy outcomes. We also outline that all three approaches are asymptotically equivalent.     

Ultrastructural evidence for adrenergic nerve degeneration in the guinea pig uterus during pregnancy

Summary In the guinea pig myometrium, the adrenergic nerves selectively demonstrated at the ultrastructural level after treatment with 5-OH-DA, show varying degree of degeneration during pregnancy. The changes are more extensive in a late gestational stage (40–45 days) than in an early one (20–25 days), and are particularly evident in the uterus overlying the conceptus as compared to the regions between the fetuses. Scattered degenerative changes were also observed in myometrial specimens from virgin animals, but probably reflect the normal continuous turnover of axons.     

Hormonal changes accompanying cigarette smoke-induced preterm births in a mouse model

Epidemiologic evidence indicates that maternal smoking increases the risk of preterm birth. While a number of plausible mechanisms for early delivery have been offered, the role of gestational hormones in this smoke-induced outcome is uncertain. Thus, a toxicologic study was performed to examine the effects and underlying hormonal mechanisms of mainstream cigarette smoke (MCS) exposure on gestational duration. Pregnant B6C3F1 mice were exposed by inhalation to MCS for 5 days/week (4 hrs/day) from Gestational Day (GD) 4 to parturition. Smoke-induced effects on gestational length, interpubic ligament length, maternal hormone secretion patterns (estradiol-17beta, progesterone, prolactin, and relaxin), body weight gain, postimplantation loss, litter size, and offspring sex ratio were examined. Dams exposed to MCS at a concentration equivalent to smoking less than one pack of cigarettes/day (carbon monoxide = 25 parts per million, total suspended particulates = 16 mg/m3) demonstrated a significant (P < 0.05) shortening of gestational duration (compared with pregnant, air-exposed mice). In addition, MCS-exposed mice sacrificed on GD 18 had significantly (P < 0.05) increased interpubic ligament length, elevated serum estrogen levels, and a reduced progesterone to estradiol-17beta ratio (compared with air-exposed controls); levels of progesterone and prolactin were only modestly decreased and increased, respectively, in the MCS-exposed mice. Smoke exposure had no significant effects on maternal relaxin levels, body weight gain, postimplantation loss, litter size, or sex ratio. Results of this study demonstrate that inhalation exposure of pregnant mice to a low dose of MCS shortens gestation and alters hormone secretory patterns, which are important for maintaining pregnancy and inducing parturition. These findings support the view that pregnant women who smoke (even modestly) may be at increased risk for preterm birth, and that early delivery may be related (at least partly) to MCS-induced.     

Release of human chorionic gonadotropin (hCG) and its alpha-subunit (hCG-alpha) from perifused human placenta

The release of human chorionic gonadotropin (hCG) and its alpha-subunit (hCG-alpha) from the normal human placenta and the effect of some stimulatory agents on their release were studied in vitro using a perfusion system. Each perfusate was assayed for hCG and hCG-alpha in its own homologous radioimmunoassay systems. Both hCG and hCG-alpha were released from the placenta at any stage of gestation in our perfusion system. Much more hCG than hCG-alpha was released from the placenta in early gestation. By comparison, however, hCG-alpha increased gradually with the gestational age. The amount of hCG-alpha released was almost equal to that of hCG in the placenta in the 17th gestational week. After the 22nd gestational week, hCG-alpha was released in larger quantities than hCG, and about 10 times more hCG-alpha than hCG was released from the term placenta. These results were also confirmed by gel filtration of perfusates on a Sephadex G-100 column. hCG-alpha, compared with hCG, was present in excess in gel filtrated perfusates in the last two trimesters. By adding 1 mM dibutyryl cyclic AMP to the perifusion medium, the release of both hCG and hCG-alpha was stimulated significantly. Synthetic luteinizing hormone releasing hormone (LH-RH) at concentrations of 10 ng/ml and 100 ng/ml had no effect, but at a high concentration (1 microgram/ml), LH-RH stimulated the release of them. Moreover, mouse epidermal growth factor (EGF) stimulated not only the release of hCG and hCG-alpha but also their production, because both hCG and hCG-alpha levels rose progressively with the time course in the presence of EGF. The present studies demonstrate that the perifusion system of chorionic tissues is a useful method for investigating the release of hCG and its subunits in vitro.