Does COVID-19 infection acquired in different pregnancy trimester influence placental pathology?

OBJECTIVES: To determine the morphological characteristics of the placentas from COVID-19 positive mothers in regard to the trimester of COVID-19 infection onset and low weight molecular heparin (LMWH) treatment. METHODS: Placentas were collected in the period April 1st till September 1st 2021 after delivery at Department of Obstetrics and Gynecology University Hospital Split, Croatia, and sent for pathological examination. Medical history and pathology reports were used to collect the data. Pregnant women were divided based on the onset of COVID-19 infection and stratified into low molecular weight heparin (LMWH)+ or LMWH-. Depending on the data distribution, the following test were used: chi-squared test. Student's t-test, Mann-Whitney U test, ANOVA and Kruskal-Wallis test. RESULTS: In 38% of patients the onset of COVID-19 infection was the 1st trimester of pregnancy, in 27% in the 2nd and 35% of women were infected in the 3rd trimester The fetal vascular malperfusion (FVM) occurrence was statistically significantly higher in the LMWH- group and if the onset of infection was in the 2nd trimester, while the perivillous fibrin deposition was most likely to happen if the COVID-19 infection that occured in the 1st trimester of pregnancy. CONCLUSIONS: The onset of COVID-19 infection has the influence on trophoblast damage and subsequent morphological appearance of the placenta. LMWH use in COVID positive pregnant women decreases the rate of the FVM in examined placentas.

The Effect of GLUT1 and HIF-1α Expressions on Glucose Uptake and Patient Survival in Non-Small-Cell Lung Carcinoma.

Lung cancer is the second-most-common cancer while being the leading cause of cancer deaths worldwide. It has been found that glucose transporter 1 (GLUT1) and hypoxia-inducible factor 1α (HIF-1α) are overexpressed in various malignancies and that they correlate with the maximum standard uptake values (SUVmax) on 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG PET/CT) and poor prognosis. In this study, we aim to evaluate the relationship between the SUVmax, GLUT1, and HIF-1α expression with primary tumor size, histological type, lymph node metastases, and patient survival. Of the 48 patients with non-small-cell lung cancer, those with squamous cell carcinomas (SCCs) had significantly higher GLUT1 and HIF-1α immunohistochemical expressions in comparison to adenocarcinomas (ACs), while there was no statistically significant difference in FDG accumulation between them. No significant correlation was noted between either GLUT1 or HIF-1α protein expression and FDG uptake and overall survival. However, an analysis of tumor transcriptomics showed a significant difference in overall survival depending on mRNA expression; patients with SCC and high HIF-1α levels survived longer compared to those with low HIF-1α levels, while patients with AC and low GLUT1 levels had a higher average survival time than those with high GLUT1 levels. Further studies are needed to determine the prognostic value of the expression of these factors depending on the histologic type.

Chorioamnionitis has no impact on immunohistochemical expression of IL-6 in placental membranes of the late preterm delivery regardless of the membrane status.

OBJECTIVES: To compare the immunohistochemical expression of IL-6 in placental membranes of late preterm delivery in women with histologically proven chorioamnionitis with and without preterm premature rupture of membranes (PPROM). METHODS: Fetal membranes were collected from 60 women who had late preterm delivery with histologic chorioamnionitis with and without PPROM (30 in each group). Immunohistochemistry for IL-6 was performed on formalin fixed and paraffin-embedded sections. The two groups were matched for age, body mass index and parity. SPSS Version 17.0 was used for statistical analysis. RESULTS: There was no difference in immunohistochemical expression of IL-6 in placental membranes of women with histologic chorioamnionitis regardless of the membrane status. CONCLUSIONS: Chorioamnionitis has no impact on immunohistochemical expression of IL-6 in placental membranes of women with late preterm delivery despite the clinical presentation.

Expression Pattern of iNOS, BCL-2 and MMP-9 in the Hip Synovium Tissue of Patients with Osteoarthritis.

Hip osteoarthritis (HOA) is characterized by degradation of the cartilage and synovitis. However, the pathohistological effects of synovial tissue inflammation on HOA are not clear. The aim of this study was to evaluate the expression of iNOS, BCL-2 and MMP-9 markers in different synovial cell populations. A total of 32 patients were evaluated retrospectively. Age, sex, height, weight, body mass index were recorded and lymphocyte, fibrocytes and macrophages were analysed in tissue sections. Osteoarthritis cartilage histopathology assessment system (OARSI), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Krenn score, Harris Hip Score (HHS) and Kellgren-Lawrence (K-L) grading of the hip joints were performed. Total hip arthroplasty was performed on 32 patients and controls. Patients were divided into two groups according to their disease severity. The tissues were immunohistochemically analysed. K-L grade and Krenn score differ between all three groups, but also between moderate and severe OA. Synovial lining cell layer, resident cells in stroma and especially inflammatory infiltration were increasing with severity of OA. iNOS expression in both intima and subintima was positively correlated with Krenn score in moderate and severe osteoarthritis (OA) groups. Expression of BCL-2 in intima of severe OA patients was positively correlated with Krenn score. In conclusion, iNOS, BCL-2 and MMP-9 are involved in the regulation of HOA. Our study indicates a relationship between the pathohistological features, the synovial inflammation and the cartilage condition at the time of hip replacement due to OA or femoral neck fracture.

Expression and localization of DAB1 and Reelin during normal human kidney development.

AIM: To explore the spatial and temporal expression patterns of DAB1 and Reelin in the developing and postnatal healthy human kidneys as potential determinants of kidney development. METHODS: Paraffin-embedded fetal kidney tissue between the 13/14th and 38th developmental weeks (dw) and postnatal tissue at 1.5 and 7 years were stained with DAB1 and Reelin antibodies by double immunofluorescence. RESULTS: During the fetal kidney development and postnatal period, DAB1 and Reelin showed specific spatial expression pattern and diverse fluorescence intensity. During the fetal period, DAB1 was strongly expressed in the distal convoluted tubules (DCT), with strong reactivity, and diversely in the proximal convoluted tubules (PCT) and glomeruli. In the postnatal period, DAB1 expression decreased. The strongest Reelin expression in early fetal stages was observed in the PCT. In the postnatal period, Reelin expression decreased dramatically in all observed structures. These two markers were colocalized during early developmental stages, mostly in PCT, DCT, and podocytes. CONCLUSION: The appearance of DAB1 and Reelin during fetal kidney development confirms their potential significant role in the formation of kidney structure or function. High DAB1 expression in the DCT implies its regulatory role in tubular formation or function maintenance during development. Reelin was highly expressed in human kidneys at early fetal stages, mostly in the PCT, while at later fetal stages and postnatal period its expression decreased.

Lipocalin-2 and matrix metalloproteinase-9 expression in high-grade endometrial cancer and their prognostic value.

AIMS: To demonstrate lipocalin-2 (LCN-2) immunohistochemical expression together with matrix metalloproteinase-9 (MMP-9) protein in high-grade endometrial cancer and determine their correlations with FIGO (International Federation of Gynecology and Obstetrics) stage, histological subtype, presence of vascular invasion, patient age and overall and disease-free survival. METHODS AND RESULTS: Immunohistochemical staining was performed using LCN-2 and MMP-9 antibodies on high-grade endometrial cancer (n = 85) diagnosed at Split University Hospital Centre during 1998-2010. Immunohistochemical expression was determined on archived paraffin-embedded samples and scored semiquantitatively. Survival time was analysed using the Kaplan-Meier method, and the log-rank test was used to assess between-group differences. The Cox proportional hazard regression model was used on multivariate survival analysis. Patients were followed from the time of primary surgery until death or last follow-up until December 2012. LCN-2 and MMP-9 were highly expressed in high-grade endometrial cancer. Univariate analysis showed positive immunohistochemical staining for LCN-2 and MMP-9 to be associated with shorter survival in patients with high-grade endometrial cancer. Multivariate analysis showed LCN-2 overexpression to be associated with shorter overall and disease-free survival in high-grade endometrial cancer. CONCLUSIONS: Our findings suggest that LCN-2 expression may be an important independent indicator of shorter survival in patients with high-grade endometrial cancer.

Asymmetrical fetal growth is not associated with altered trophoblast apoptotic activity in idiopathic intrauterine growth retardation.

AIM: To investigate whether there is difference in trophoblast apoptosis between infants with asymmetrical idiopathic intrauterine growth retardation (IUGR) and those with symmetrical fetal growth appropriate for gestational age (AGA). METHODS: Data and placentas from 52 singleton term pregnancies with idiopathic IUGR, from which a subgroup of 33 (63.4%) infants with asymmetrical growth and malnutrition was identified using the ponderal index served as a study group. The control group included 60 (86.9%) infants with symmetrical growth, identified by the same criterion among 69 normal singleton pregnancies with AGA. IUGR was defined by birthweight less than the 10th percentile of standard values. Ponderal index value was considered as the measurement of fetal growth proportionality. RESULTS: The proportion of fetal thinness up to ponderal index value was greater in the IUGR group than control (χ(2) = 9.2; P = 0.002). There was no statistically significant difference in the cytotrophoblast proliferation (t = 0.88; P = 0.373), trophoblast expression of the Bcl-2 anti-apoptotic factor (z = 0.66; P = 0.505), total trophoblast apoptotic index (t = 0.45; P = 0.651), as in cytotrophoblast (t = 0.01; P = 0.988) and syncytiotrophoblast apoptotic index (t = 0.34; P = 0.730) between the idiopathic asymmetrical IUGR and control group. CONCLUSION: Asymmetry of fetal growth is a result of rather long-term placental nutritive insufficiency in which trophoblasts have a central role. Although being crucial for its functioning, the proliferative and apoptotic trophoblast activity remains unaltered in the placentas from pregnancies with idiopathic IUGR and asymmetrical fetal growth. The results obtained in this study indicate that placental nutritive insufficiency may develop without any deviation in the physiological trophoblast regeneration via apoptosis.

Bone porosity and longevity in early medieval Southern Croatia.

Porosity of the skull and skeletal remains, especially of the orbital roof, are one of the most frequent pathological findings on ancient human skeletal remains. There are several presumed causes of this condition and anthropologists consider skull porosities as a marker of physical and nutritional stress. A total of 115 graves were discovered at the early-medieval graveyard near Zadar (Croatia) that contained 128 partially preserved skeletons. Average estimated age at death was 37.2 ± 12.6 years for men, 31.9 ± 13.9 for women, and 5.3 ± 3.6 years for subadults. Pathological bone porosity was analysed. Cribra orbitalia was observed on 21 skulls (28.7%), signs of temporal porosity were noticed on six skulls and signs of subperiosteal bleeding on three skulls. Nineteen skulls had bone porosities in other areas. There was a significant difference (p = 0.039) in achieved age of adults with and without cribra orbitalia as those with cribra orbitalia lived on average 8.1 years longer. The bone porosity was probably caused by malnutrition that might have had a beneficial effect on longevity of adults, similar to effects of restricted food intake on extending lifespan through epigenetic signatures influencing gene expression.

The impact of the COVID-19 pandemic on adverse fetal outcomes: A cross-sectional study.

The coronavirus disease 2019 (COVID-19) pandemic has been one of the most damaging pandemics in all of human history. Some of the most vulnerable groups within society such as pregnant women and children have also been affected. This observational research, cross-sectional study was conducted to investigate if there was any difference in the incidence of unfavorable outcomes in pregnancy such as miscarriage, intrauterine fetal demise, and early neonatal death during the year prior to the pandemic and the year of the COVID-19 pandemic. This retrospective study was conducted at the University Hospital of Split at the Department of Pathology, Forensic and Cytology and Department of Obstetrics and Gynecology of the same hospital. All data was collected in the time period from March 1st, 2019, to March 1st, 2021. The study included all pregnant women who had an unfavorable pregnancy outcome such as miscarriage and intrauterine fetal demise, as well as early neonatal death at the University Hospital of Split within the time frame mentioned previously. There was no statistically significant difference in the incidence of adverse pregnancy outcomes in the year prior to the pandemic and during the year of the COVID-19 pandemic. Our study showed that the pandemic did not have a negative effect on pregnant women and their fetuses; there was no increase in miscarriage, intrauterine fetal demise, or perinatal death during the year of the pandemic.

Apoptosis, proliferation and Fas ligand expression in placental trophoblast from pregnancies complicated by HELLP syndrome or pre-eclampsia.

OBJECTIVE: To investigate apoptosis, proliferation and Fas ligand expression of placental trophoblast in the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome and in pre-eclampsia (PE), and to compare this with normal pregnancies. DESIGN: Prospective study. SETTING: University hospital in Croatia. SAMPLE: Placentae from women with HELLP syndrome (n=10), PE (n=10) and normal pregnancies (n=10). METHODS: The HELLP syndrome was diagnosed with platelets <100×10(9) /L, aspartate aminotransferase (AST) and alanine transaminase (ALT) >70 U/L and lactic acid dehydrogenase (LDH) > 600 U/L. Pre-eclampsia was diagnosed at blood pressure >140/90 mmHg, with proteinuria >300 mg/L/24 hours. For detection of apoptosis and proliferation in villous trophoblast, antibodies M30 and Ki-67 were used. Expression of Fas ligand was assessed using immunohistochemistry and the semiquantitative HSCORE method. MAIN OUTCOME MEASURES: Apoptosis, proliferation and Fas ligand expression in villous trophoblast. RESULTS: Apoptosis, proliferation and Fas ligand expression were higher in villous trophoblast in HELLP syndrome than in the PE group (p=0.015, p=0.018 and p=0.002, respectively) and the control group (p=0.000, p=0.012 and p=0.049, respectively). Placentae from the PE group had higher levels of apoptosis (p=0.019), lower Fas ligand expression (p=0.029) and no difference in proliferation (p=0.887) compared with the control group. CONCLUSIONS: There is an increase in apoptosis, proliferation and Fas ligand expression in placentae from women with HELLP syndrome compared with placentae from PE and normal pregnancies. Our findings indicate the possibility of differential mechanisms behind HELLP syndrome and PE.

Results of full postmortem examination in a cohort of clinically unexplained stillbirths: undetected fetal growth restriction and placental insufficiency are prevalent findings.

OBJECTIVE: To analyze a cohort of clinically unexplained stillbirths (CUS) referred for postmortem. STUDY DESIGN: In total, 258 CUS were referred for full postmortem between 2009 and 2015. Relevant Condition at Death (ReCoDe) classification was applied. Statistical analysis included chi-square test and multiple logistic regression. RESULTS: In all, 386 ReCoDe categories identified corresponded to: fetus (99); umbilical cord (48); placenta (165); amniotic fluid (55), and mother (1). No condition was identified in 18 cases. Prevalent conditions were placental insufficiency (101 cases, 39%) and fetal growth restriction (96 cases, 37%), frequently presenting together (41 cases, 15.9%). Significant associations were found between fetal growth restriction and gestational age, asymmetrical fetal growth and placental insufficiency. CONCLUSIONS: In total, 60.5% of CUS were diagnosed at postmortem to have fetal growth restriction and/or placental insufficiency. The mean gestational age of death in which these conditions presented was 32.7 weeks and 35.5 weeks, respectively, suggesting a critical time-frame to monitor to potentially reduce stillbirth occurrence.

Expression of apoptotic and proliferation factors in gastric mucosa of patients with systemic sclerosis correlates with form of the disease.

Despite high prevalence of patients with gastric disease in systemic sclerosis (SSc), its pathogenesis is still poorly understood. We immunohistochemically analysed biopsies of gastric mucosa (GM) in 5 controls and 15 patients with different forms of SSc: limited cutaneous (lc), diffuse cutaneous moderate (sys1) and severe (sys2). The number of positive cells was analysed by a Kruskall-Wallis test, P < 0.05 was considered statistically significant. Percentage of proliferating (Ki-67 positive) cells was highest in sys1 (3% in superficial and 4,6% in deeper parts of GM), which dropped to 1% in sys2. Percentage of α-smooth muscle actin (α-SMA) positive cells was 5% in controls, 9% in superficial GM, while in deeper GM rose from 7% to 19% in sys1 and sys2, thus indicating increased myofibroblast population. Caspase-3 positive apoptotic cells characterized 1,5-2% of controls, 8% of superficial and 6% of deeper GM cells in sys1. In sys2, apoptosis affected 50% of surface epithelial and gland cells and 30% of deeper glands, and correlated with increased fibrosis and decreased syndecan-1 expression. Our data demonstrate that sys1 is the most "active" proliferating form of SSc. Sys2 characterize collagen deposition, surface epithelium defects, extensive apoptosis and low proliferation, GM atrophy and loss of function.

Involvement of T lymphocytes in the placentae with villitis of unknown etiology from pregnancies complicated with preeclampsia.

OBJECTIVE: The aim of the study was to compare immunohistochemical expression of different T type lymphocytes in foci of villitis of placentae with villitis of unknown etiology (VUE) without and with preeclampsia (PE). METHODS: Fifty-four placentae were collected from women who had VUE with (N = 27) and without (N = 27) PE. Immunohistochemistry for types of T lymphocytes was performed on formalin fixed and paraffin-embedded sections by use of the CD3, CD4, FOXP3, CD25, CD8 and CD68 antibodies. All data analyses were done by R Development Core Team. RESULTS: There was higher immunohistochemical CD4 positive T lymphocyte count and CD4 positive/CD8 positive ratio in placentae with VUE complicated with PE compared to control group. CONCLUSION: The higher immunohistochemical CD4 positive T lymphocyte count and CD4 positive/CD8 positive ratio in placentae with VUE complicated with PE could point to their role in ethiopathogenesis of PE.

Fas and FasL expression in placentas complicated with intrauterine growth retardation with and without preeclampsia.

OBJECTIVE: To compare the level of Fas and FasL immunohistochemical expression in villous trophoblast (VT), extravillous trophoblast (EVT) cells, decidual cells (DC), endothelial cells (EC) of villous blood vessels and spiral arteries between the study groups of intrauterine growth retardation (IUGR) placentas with and without preeclampsia (PE). METHODS: The study included 17 placentas from pregnancies complicated by IUGR + PE and 17 placentas from pregnancies complicated by idiopathic IUGR (I-IUGR). Seventeen placentas from normal pregnancies served as a control group. CD31 was used to detect endothelial cells (EC). Immunohistochemical expression of Fas and FasL was assessed in all examined parts of placenta using the semi-quantitative HSCORE method. RESULTS: FasL expression was significantly higher in all examined parts of placenta in I-IUGR as compared to IUGR + PE and control group. Placentas with IUGR + PE had the significantly lowest expression of FasL in VT and EC of villi vessels. Expression of Fas did not differ significantly between the study groups. CONCLUSION: Different expression of FasL in placentas from I-IUGR and IUGR + PE suggests that FasL probably has a different role in the etiology of these two syndromes.

Flow cytometric DNA hypertetraploidy tends to be more frequent in male than in female breast cancers.

The aim of the study was to explore possible differences in DNA flow cytometric characteristics, particularly differences in distribution of DNA indices of aneuploid clones, between male and female breast cancers. We retrospectively analyzed 31 male breast cancers. Clinicopathological and DNA flow cytometric characteristics of male breast cancers (patient age, tumor size, histological type, histological grade, axillary lymph node status, hormone receptor expression, ploidy, and S-phase fraction) were compared with that of the control group of matched female breast cancers. Hormone receptors and HER-2/neu were investigated immunohistochemically with additional chromogenic in situ hybridization (CISH) analysis of HER-2/neu 2+ cases. Ploidy and S-phase fraction were determined by DNA flow cytometry. Comparison with clinicopathological features was made using χ (2) and t test. Aneuploidy was found in 78% of the cases, with the predomination of hypotetraploid clones (39%), followed by tetraploid (23%) and hypertetraploid clones (16%). We found higher frequency of hypertetraploidy in male breast cancers (16 and 6%, respectively) than in the control group of matched female breast cancers. Clinicopathological features of hypertetraploid male breast cancers did not differ from that of non-hypertetraploid cancers. Higher frequency of hypertetraploidy among male breast cancers might indicate different cytogenetical evolutionary pathway between male and female breast cancer.