Spatial and temporal immunoreaction of nestin, CD44, collagen IX and GFAP in human retinal Müller cells in the developing fetal eye.

The purpose of this study was to investigate Müller cells during the fetal development of the human eye. Müller cells in eyes of 39 human fetuses (11-38 weeks of gestation, WOG) and 6 infants (5 died of abusive head trauma, AHT, aged 1-9 months) were immunohistochemically stained and investigated for spatial and temporal immunoreaction of nestin, CD44, collagen IX and GFAP, which are stem cell markers or markers of intermediate filaments, respectively, in one of the hitherto largest cohorts of fetal eyes. Müller cells could be detected immunohistochemically as early as 12 WOG by immunohistochemical staining with nestin. Nestin was more strongly expressed in Müller cells of the peripheral retina and a centroperipheral gradient of immunoreaction over time was observed. CD44 was predominantly expressed in fetal eyes of the late second and early third trimester between (23 and 27 WOG) and significantly stronger in the infant eyes. Collagen IX labeling in the central retina was significantly stronger than in more peripheral sectors and increased with fetal age. GFAP staining in Müller cells was seen in the eye of a fetus of 38 WOG who died postnatally and in the infant eyes with and without history of AHT. Additionally, GFAP staining was present in the astrocytes of fetal and infant eyes. All examined markers were expressed by Müller cells at different developmental stages highlighting the plasticity of Müller cells during the development of the human eye. GFAP should be cautiously used as a marker for AHT as it was also expressed in fetal astrocytes and Müller cells in eyes without history of AHT.

Placental Invasion into the Small Bowel Intestine Through a Myomectomy Scar: A Case Report With Literature Review.

Although extremely rare, uterine damage after hysteroscopic myomectomy sets the precondition for various life-threatening placental attachment disorders like placenta percreta (PP) or scar pregnancy. Due to vast clinical similarities, these terms are often used interchangeably. We report a case of a 47-yr-old patient at 27 wk + 4 d of gestation who presented with rectal bleeding. Clinical history revealed a previous uterine posterior wall myomectomy. The patient received intensive diagnostic work-up including sonography and magnetic resonance imaging. Under the suspicion of a bleeding Meckel diverticulum, an emergency laparotomy was performed. Intraoperatively it was observed that the placental tissue infiltrated the small bowel intestine at the location of the previous myomectomy. The adjacent intestine and the infiltrating placenta were surgically removed. The placenta could be easily detached from the uterus, which is why no hysterectomy was performed. Retrospectively, no radiologic or clinical hints of PP or scar pregnancy were evident before the surgery. Moreover, the pathologic work-up carried out afterwards proved no histopathologic evidence for PP. Our case underlines several clinical and pathologic difficulties. First, invasive placenta disorders including infiltration of intestinal organs have to be considered even after minor surgical interventions such as myomectomy. Second, clinical presentation is extremely variable and sometimes misleading, depending on the localization and the type of invasion. Our case underlines the importance of histopathologic work-up for distinguishing between various placenta attachment disorders such as PP and scar pregnancy. Given the large overlap in clinical presentation, pathophysiology and definition, we propose that the current definitions for PP and scar pregnancy have to be carefully reevaluated and broadened.

[Intrauterine fetal demise in extensive SARS-CoV-2-associated placental maternal vascular malperfusion in the setting of SARS-CoV-2 placentitis (severe acute respiratory syndrome coronavirus 2)]. / Intrauteriner Fruchttod bei massiver SARS-CoV-2-assoziierter ("severe acute respiratory syndrome coronavirus 2") plazentarer maternaler Malperfusion im Rahmen einer SARS-CoV-2-Plazentitis.

We report a case of a placenta with extensive maternal vascular malperfusion and chronic histiocytic intervillositis corresponding to SARS-CoV­2 placentitis in the context of fetal demise at 31 weeks of gestation. Placental swamp and PCR of the placental parenchyma, umbilical cord and amnion-chorion membrane showed SARS-CoV-2- and B­betacoronavirus-specific RNA. Maternal vascular malperfusion has been described in cases of SARS-CoV­2 infection; however, the manifested severity of this case in the setting of a severe SARS-CoV­2 placentitis is rare. It emphasizes the need of a maternal prophylactic anticoagulation.

Discordancy for a Villous Maturation Defect in a Dizygotic Twin Placenta.

Background: Dizygotic twin pregnancies with discordant manifestation of abnormalities with unclear etiology are of interest because they arise in the same environment. Case report: We present a dizygotic third trimester twin placenta with discordant villous maturation, one placenta lacking developed syncytiocapillary membranes. The twins were eutrophic with no perinatal or postnatal complications. Conclusions: Discordant manifestation of villous maturation in dizygotic twin placentas could be a hint for a genetic rather than an environmental etiology. Villous maturation defect may be underrecognized and has been associated with perinatal morbidity and stillbirth in the late third trimester. Proper recognition is important because of the increased recurrence risk of villous dysmaturity.

[A case of acrania?].

The article reports on the case of a premature and stillborn child. As a first step it had to be clarified whether the child had been alive. The pathological examination of the placenta performed after autopsy could prove a retroplacental hematoma as the cause of death. Furthermore the autopsy revealed a severe skull deformity that would probably have made the survival of the child impossible.

New osseous soft markers for trisomy 13, 18 and 21.

INTRODUCTION: For ultrasonographic diagnosis of a fetal trisomy so-called "soft markers" (=ultrasonographically detectable morphological variants) are used. Detection of a certain number of them increases the diagnostic certainty of a fetal trisomy. Up to now there are very few diagnostically accepted osseous soft markers for trisomy. Hence potential osseous soft markers applicable for first and second trimester ultrasound screening for trisomy 21, 18 or 13 were studied. METHODS: Postmortal fetal X-rays (ap, lateral) of 358 fetuses (trisomy 21: n = 109, trisomy 18: n = 46; trisomy 13: n = 38, control group: n = 165). RESULTS: Not yet described but with trisomy 21 statistically associated soft markers were un-timely os sternale ossification, delayed os sacrum ossification, shortened os maxillare, reduced os maxillare-jaw-corner distance, augmented orbita height, premature os calcaneus ossification, bell-shaped thorax, coronal clefts, trend to wider binocular as well as wider intraocular distances; for trisomy 18: elevated clavicula slope, reduced number of ribs, bell-shaped thorax, coronal clefts, reduced os maxillare-jaw-corner distance, shortened ramus mandibulare, shortened os metacarpale IV and V, augmented ratio between biparietal diameter and (osseus and soft-tissue) shoulder width; for trisomy 13: longer os nasale, elevated clavicula slope, premature sternum, delayed os sacrum ossification, delayed/premature cranium ossification, reduced number of ribs, coronal clefts, reduced os maxillare-jaw-corner distance, shortened ramus mandibulare, augmented orbita height, shortened os metacarpale V and a tendency for a shortened os metacarpale IV. CONCLUSION: We found several not yet published osseous soft markers statistically associated with trisomy 21, 18 and 13, which can help to ensure sonographically these aneuploidy diagnoses.

Expression of AP-2γ in placentas of patients with preeclampsia and of smokers.

INTRODUCTION: There is evidence of a probable key role of the activator protein-2 γ (AP-2γ) in placental development. It is still an open question whether AP-2γ expression may be influenced by preeclampsia, which is a serious pregnancy complication, or by smoking, which has deleterious effects on trophoblastic development. MATERIAL AND METHODS: Thus, the expression of AP-2γ was studied in trophoblastic epithelium and endothelium of placentas from patients with preeclampsia (n = 43) and smokers (n = 45) as well as placentas of healthy pregnant women (control group, n = 26) between gestational ages 23 and 43 weeks. To allow differential expression in primary, secondary and tertiary villi, AP-2γ expression (arbitrary units) was determined immunohistologically. RESULTS: In preeclamptic placentas trophoblastic as well as endothelial cells AP-2γ expression was significantly higher compared to that in control placentas. Endothelial AP-2γ expression in placentas from smokers was similar to that of healthy women while trophoblastic AP-2γ expression in smokers' placenta was insignificantly higher compared to that of control placentas. In all three groups expression rates of AP-2γ did not differ between primary, secondary and tertiary villi. CONCLUSION: A correlation between increased trophoblastic and endothelial AP-2γ expression in patients with preeclampsia and reduced trophoblastic invasion and migration in preeclampsia has to be discussed. Furthermore, increased AP-2γ expression may play a protective role in preeclampsia, protecting from raised blood pressure. The tendency of an enhanced trophoblastic AP-2γ expression in smokers may indicate a compensatory response to the disturbed balance between proliferation and differentiation of villi induced by smoking.

Expression of the lymphatic marker podoplanin (D2-40) in human fetal eyes.

During human ocular development, expression of proteins varies in different maturation stages. This study aims to characterize structures in human fetal eyes stained by the lymphatic marker podoplanin (D2-40) with emphasis on the stage of maturation and the presence of intraocular lymphatic structures. Formalin-fixed paraffin-embedded eyes from 40 human fetuses between 10 and 38 weeks of gestation (WoG) were investigated. Immunohistochemical stains were performed for D2-40, LYVE-1 as a secondary lymphatic marker, and CD34 as a control for endothelial reactivity. A semiquantitative analysis of antigen expression in different segments of the eye was performed by light microscopy. The intensity of antigen expression was graded with a score ranging from 0 to 3. Podoplanin expression was found with a variable intensity in 97.5% of the eyes, in particular in lymphatic vessels of the conjunctiva (n = 26), conjunctival and corneal epithelium (n = 33), corneal endothelium (n = 4), trabecular meshwork (n = 28), and optic nerve sheaths (n = 23). A slight, equivocal staining reaction was noted in the choroid (n = 14). There was a correlation of antigen reactivity and the gestational age for corneal endothelial reactivity in earlier gestational stages (p = 0.003) and trabecular meshwork in older eyes (p = 0.031). D2-40 positive Müller cells were detected in two eyes ≥32 WoG. Thus, aside from conjunctival lymphatic vessels, podoplanin was expressed in several structures of the human fetal eye and the ocular adnexae at different gestational stages. Podoplanin positive structures were also found in the choroid and the chamber angle. However, lymphatic vessels or its progenitors could not be unequivocally identified in intraocular structures during 10-38 weeks of gestation. There is no evidence from our data that transient intraocular lymphactics develop in the fetal eye between 10 and 38 weeks of gestation.

Absence of lymphatic vessels in the developing human sclera.

The adult sclera is free of lymphatic vessels, but contains a net of blood vessels. Whether and when this selectively lymphangiogenic privilege is achieved during embryologic development is not known yet. Therefore, we investigated the developing human sclera for blood- and lymphatic vessels in 34 abortions/stillborns (12-38 weeks of gestation). The probes were subdivided into three groups (group 1: 12-18 weeks of gestation, n = 10; group 2: 19-23 weeks of gestation, n = 13; group 3: 24-38 weeks of gestation, n = 11), and prepared for paraffin sections followed by immunohistochemistry against CD31 to detect blood vessels, and against lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1)/podoplanin to detect lymphatic vessels. We could show, that in the human episclera distinct CD31 + blood vessels are present as early as week of gestation 13. Their amount increased during pregnancy, whereas stromal CD31 + blood vessels were elevated in early pregnancy and regressed with ongoing pregnancy. In the lamina fusca CD31 + blood vessels were absent at any time point investigated. Single LYVE1 + cells were identified primarily in the episclera; their amount decreased significantly with increasing gestational ages (group 1 compared to group 3: p < 0.01). However, LYVE1+/podoplanin + lymphatic vessels were not detectable in the sclera at any gestational ages analyzed. In contrast to the conjunctiva where LYVE1+/podoplanin + lymphatic vessels were detectable as early as week 17, the amount of LYVE1 + cells in the sclera was highest in early pregnancy (group 1), with a significant decrease during continuing pregnancy (p < 0.001). These findings are the first evidence for a fetal lymphangiogenic privilege of the sclera and show, that the fetal human sclera contains CD31 + blood vessels, but is primarily alymphatic. Our findings suggest a strong expression of selectively antilymphangiogenic factors, making the developing sclera a potential model to discern antilymphangiogenic mechanisms.

Lens artifacts in human fetal eyes - the challenge of interpreting the histomorphology of human fetal lenses.

BACKGROUND: Evaluation of the lens, including cataractous changes, is often of paramount importance in the classification of fetal syndromes or forensic questions. On histology, the crystalline lens is - especially in fetal and infant eyes - an organ susceptible to numerous artifacts. Thus, the aim of our study was to study various factors (including fixatives) that might have an impact on lens histomorphology. METHODS: Twenty eyes from ten fetuses (formalin fixation: n = 10, glutaraldehyde fixation: n = 10), matched for gestational age and abortion (spontaneous vs. induced), were investigated macroscopically and by light microscopy. Sections were stained with routine hematoxylin & eosin (H&E), and periodic acid schiff (PAS). The age of the fetal eyes ranged from 15 to 36 weeks of gestation. Lens artifacts were analyzed and compared to fetal and adult lenses with definitive cataractous changes. In addition, 34 eyes from 27 fetuses with trisomy 21 were investigated for lens changes. RESULTS: All lenses showed artifacts of varying extent, in particular globules, vacuoles, clefts, anterior/posterior capsular separation, subcapsular proteinaceous material, fragmentation of the lens capsule/epithelium, and a posterior umbilication. Glutaraldehyde-fixed lenses displayed less artifacts compared to those fixed in formalin. Slight differences in the appearance of artifacts were found dependent on the fixative (formaldehyde vs glutaraldehyde) and the kind of abortion (iatrogenous vs spontaneous). The gestational age did not have a significant influence on the type and extent of lens artifacts. The lenses from fetuses with trisomy 21 displayed similar lens artifacts with no specific findings. CONCLUSIONS: Alterations in fetal lens morphology are extremely frequent and variable. These artifacts have to be carefully taken into account when interpreting post-mortem findings. Thus, the postmortem diagnosis of a fetal cataract should be made with great caution, and should include, in adherence to our proposed diagnostic flow diagram, the macroscopic lens assessment. Reference slides with a proven cataract are recommended for comparison in equivocal cases.

Is the lung floating test a valuable tool or obsolete? A prospective autopsy study.

The lung floating test is still an obligatory measure to distinguish whether a newborn was born dead or alive. In order to verify the reliability of the floating test, a new clinical trial should examine the results of current cases and thus expose, if the test is still contemporary. Following the question, if the test is appropriate for the nowadays birth collective, 208 lungs of newborns were tested with the floating test. The test showed the expected correct result in 204 cases. However, it indicated a false negative result in four cases, in which the lungs sank, although prior life had been reported by medical staff. Overall, the study was able to prove that the results of the floating test are reliable in 98 %. Further, there was not a single false-positive result (lungs of a stillborn swim). Nevertheless, the test demonstrates that a negative test result cannot be taken as proof for a newborn never to have breathed at all.

Vascular endothelial cadherin expression in lung specimens of patients with sepsis-induced acute respiratory distress syndrome and endothelial cell cultures.

AIMS: Vascular endothelial (VE) cadherin is a cell adhesion molecule localized at endothelial cell (EC) junctions. As a major component of endothelial adherens junctions, its main function is the maintenance and regulation of EC integrity. In the acute respiratory distress syndrome (ARDS), increased vascular permeability is a major mechanism in pulmonary edema and lung dysfunction. In this study, VE-cadherin expression was investigated in ARDS lungs and control tissue as well as in an ARDS cell culture model. METHODS: Lung specimens of patients with ARDS due to Gram-negative sepsis (n = 20; control lung tissue: n = 41) and cell cultures of human pulmonary microvascular ECs and human umbilical vein ECs stimulated with LPS, TNF-α and IFN-γ were stained with a VE-cadherin antibody. Staining intensity was semiquantitatively evaluated by conventional light and immunofluorescence microscopy. RESULTS: VE-cadherin expression was statistically significantly reduced in the endothelium of all vessel types in ARDS lungs compared to control tissue. Cell cultures showing disrupted cellular borders confirmed these results. CONCLUSION: Reduced expression of VE-cadherin has to be considered as a major mechanism of increased vessel permeability in ARDS. The previously described vessel-type-specific expression pattern of VE-cadherin in the human lung is not influenced by ARDS.

Hydrocephalus, agenesis of the corpus callosum, and cleft lip/palate represent frequent associations in fetuses with Peters' plus syndrome and B3GALTL mutations. Fetal PPS phenotypes, expanded by Dandy Walker cyst and encephalocele.

OBJECTIVE: Fetal pathology aims to recognize syndromal patterns of anomalies for goal-directed mutation analyses, genetic counseling, and early prenatal diagnosis in consecutive pregnancies. Here, we report on five fetuses with Peters' plus syndrome (PPS) from two distinct families aborted after prenatal ultrasound diagnosis of hydrocephaly. METHOD: We performed fetal autopsies and molecular analyses. RESULTS: Among 44 fetuses with prenatally diagnosed hydrocephaly, four fetuses of 16 to 21 gestational weeks presented with additional cleft lip/palate and/or agenesis of the corpus callosum. Other features were growth retardation, hypertelorism, anomalies of the eyes, in part consistent with Peters' anterior chamber anomalies, mild brachymelia, brachydactyly, and also internal anomalies. Suspected PPS was confirmed by detection of B3GALTL mutation in these four fetuses and in one additional sib fetus, revealing homozygosity for the common c.660 + 1G > A donor splice site mutation in intron 8. CONCLUSIONS: Autosomal-recessive PPS has not yet been diagnosed prenatally. We want to alert ultrasonographers to the diagnosis of this disorder in growth-retarded fetuses with (recurrent) hydrocephaly, agenesis of the corpus callosum, and cleft lip/palate and stress the more severe fetal manifestation, describing a first such case with additional Dandy-Walker cyst and occult meningoencephalocele.

Spatial, temporal and interindividual epigenetic variation of functionally important DNA methylation patterns.

DNA methylation is an epigenetic modification that plays an important role in gene regulation. It can be influenced by stochastic events, environmental factors and developmental programs. However, little is known about the natural variation of gene-specific methylation patterns. In this study, we performed quantitative methylation analyses of six differentially methylated imprinted genes (H19, MEG3, LIT1, NESP55, PEG3 and SNRPN), one hypermethylated pluripotency gene (OCT4) and one hypomethylated tumor suppressor gene (APC) in chorionic villus, fetal and adult cortex, and adult blood samples. Both average methylation level and range of methylation variation depended on the gene locus, tissue type and/or developmental stage. We found considerable variability of functionally important methylation patterns among unrelated healthy individuals and a trend toward more similar methylation levels in monozygotic twins than in dizygotic twins. Imprinted genes showed relatively little methylation changes associated with aging in individuals who are >25 years. The relative differences in methylation among neighboring CpGs in the generally hypomethylated APC promoter may not only reflect stochastic fluctuations but also depend on the tissue type. Our results are consistent with the view that most methylation variation may arise after fertilization, leading to epigenetic mosaicism.

Extreme methylation values of imprinted genes in human abortions and stillbirths.

Imprinted genes play an important role in fetal and placental development. Using quantitative bisulfite pyrosequencing assays, we determined the DNA methylation levels at two paternally methylated (H19 and MEG3) and four maternally methylated (LIT1, NESP55, PEG3, and SNRPN) imprinted regions in fetal muscle samples from abortions and stillbirths. Two of 55 (4%) spontaneous abortions and 10 of 57 (18%) stillbirths displayed hypermethylation in multiple genes. Interestingly, none of 34 induced abortions had extreme methylation values in multiple genes. All but two abortions/stillbirths with multiple methylation abnormalities were male, indicating that the male embryo may be more susceptible to excess methylation. Hypermethylation of multiple imprinted genes is consistent with stochastic failures of the mechanism, which normally protects the hypomethylated allele from de novo methylation after fertilization. Two of six informative abortions/stillbirths with H19 hypermethylation revealed significant biallelic expression of the autocrine growth factor IGF2. In two other cases hypermethylation of MEG3 was associated with transcriptional down-regulation. We propose that primary epimutations resulting in inappropriate methylation and expression patterns of imprinted genes may contribute to both normal human variation and disease, in particular spontaneous pregnancy loss.

Immunohistochemical Detection of Cancer-Testis Antigen PRAME.

Cancer-testis (CT) antigens were identified by their ability to elicit T- or B-cell immune responses in the autologous host. They are typically expressed in a wide variety of neoplasms and in normal adult tissues are restricted to testicular germ cells. PReferentially expressed Antigen of Melanoma (PRAME) is a member of the family of nonclassical CT antigens being expressed in a few other normal tissues besides testis. Interestingly, knowledge about the protein expression of many CT antigens is still incomplete due to the limited availability of reagents for their immunohistochemical detection. Here, we tested several commercially available serological reagents and identified a monoclonal antibody suitable for the immunohistochemical detection of PRAME in formalin-fixed paraffin-embedded specimens. We also tested a wide array of normal and neoplastic tissues. PRAME protein expression in normal tissues is congruent with original molecular data being present in the testis, and at low levels in the endometrium, adrenal cortex, and adult as well as fetal ovary. In tumors, there is diffuse PRAME immunoreactivity in most metastatic melanomas, myxoid liposarcomas, and synovial sarcomas. Other neoplasms such as seminomas and carcinomas of various origins including endometrial, serous ovarian, mammary ductal, lung, and renal showed an intermediate proportion of cases and variable extent of tumor cells positive for PRAME protein expression. As seen with other CT antigens, hepatocellular and colorectal carcinoma, Leydig cell tumors, mesothelioma, and leiomyosarcoma are poor expressers of PRAME.