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Supplementary Table 1 from Sorting Nexin 1 Down-Regulation Promotes Colon Tumorigenesis

Purpose: Colon cancer is one of the most common human malignancies, yet studies have only begun to identify the multiple mechanisms that underlie the development of this tumor. In this study, we have identified a novel mechanism, dysregulation of endocytic sorting, which promotes colon cancer development.Experimental Design: Immunohistochemical and microarray analyses were done on human colon cancer tissue specimens to determine the levels of one endocytic protein, sorting nexin 1 (SNX1). SW480 cells, a human colon cancer cell line that retains a relatively high level of SNX1 expression, were used to assess the effects of down-regulating this protein by small hairpin RNA. Activation of signal transduction cascades was evaluated in these cells using Western blotting, and multiple functional assays were done.Results: We determined by immunohistochemistry that the level of SNX1 was significantly down-regulated in 75% of human colon cancers. In corroborative studies using microarray analysis, SNX1 message was significantly decreased (log2 ratio less than −1) for 8 of 19 colon carcinomas. Cell lines with reduced SNX1 levels showed increased proliferation, decreased apoptosis, and decreased susceptibility to anoikis. They also showed increased activation of epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 in response to epidermal growth factor. This increased activation was abolished by inhibition of endocytosis.Conclusions: These data suggest that loss of SNX1 may play a significant role in the development and aggressiveness of human colon cancer, at least partially through the mechanism of increased signaling from endosomes. Further, these findings suggest that dysregulation of endocytic proteins may represent a new paradigm in the process of carcinogenesis.

The transforming growth factor-β (TGF-β) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-β is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-β has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-β reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-β receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-β involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-β. These data suggest new roles for the TGF-β pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation. [Cancer Res 2007;67(18):8643–52]

Supplementary Figures 1-5 from Transforming Growth Factor-β Can Suppress Tumorigenesis through Effects on the Putative Cancer Stem or Early Progenitor Cell and Committed Progeny in a Breast Cancer Xenograft Model

Supplementary Table 1 from Transforming Growth Factor-β Can Suppress Tumorigenesis through Effects on the Putative Cancer Stem or Early Progenitor Cell and Committed Progeny in a Breast Cancer Xenograft Model

Background: Adverse pregnancy outcomes (APOs) identify pregnant people at increased risk of later cardiovascular disease. Poor antenatal cardiovascular health (CVH) quantified using components of the Life’s Simple 7 ideal CVH framework has been associated with higher APO incidence, indicating modifiable aspects of CVH during pregnancy may be related to pregnancy health. Placental pathology is also common in APOs, but it is unclear if antenatal CVH is directly related to placental health. Further, clinically-used dichotomous measures of placental pathology limit investigation of associations. Thus, the aim of this study was to examine the relationship between antenatal CVH and a novel, continuous marker of placental vascularization. Hypothesis: We hypothesized higher (healthier) antenatal CVH scores would be associated with higher (healthier) vascularization in the placenta. Methods: Participants enrolled in the Magee Obstetric Maternal & Infant Biobank and one of two prospective observational cohort studies examining activity patterns in pregnancy (MoM Health or Pregnancy 24/7). Antenatal CVH was quantified with a pregnancy-adapted Life’s Essential 8 framework assessed during each trimester and averaged across gestation. Components included sleep, diet, smoking, objective physical activity, pre-pregnancy BMI, blood pressure, 50g glucose challenge test results, and gestational weight gain. Component scores were averaged for a composite score (possible range, 0-100; higher indicated better CVH). Immunohistochemistry of placenta tissue was performed. Sections were stained with CD34 antibody to highlight vascular endothelial cells and counterstained with hematoxylin. Whole-slide images were digitized. Software computed the number of pixels positive for CD34 (numerator) and the total pixels (denominator); the ratio was the outcome of fetal vascular percentage (FV%), or the proportion of villous tissue occupied by fetal vessels. Linear regression associated CVH scores with FV%. Results: Placenta tissue was obtained from 64 participants (mean±SD age = 32±4.9 years). CVH score averaged across gestation was 72.6±10.7 points and decreased significantly from the first to third trimester (72.8±12.7 vs. 65.1±11.9, p Conclusion: Antenatal CVH was not significantly associated with placental vasculature, though a small sample size limits conclusions. Replication in a fully-powered sample is warranted.

Women with adverse pregnancy outcomes later experience excess hypertension and cardiovascular disease, but how the events are linked is unknown. Examination of the placenta may provide clues to vascular impairments after delivery. Maternal vascular malperfusion lesions (MVMs) were abstracted from clinical reports, validated and characterized using clinical guidelines and severity score. A total of 492 women (170 with MVMs and 322 without MVMs) participated in a study visit 8 to 10 years after delivery to assess blood pressure, cardiometabolic factors, and sublingual microvascular features using sidestream dark field imaging. Covariates included age, race, adverse pregnancy outcomes (preeclampsia, small for gestational age, and preterm birth), and health behaviors. Women with versus without MVM had a distinct sublingual microvascular profile comprised of (1) lower microvascular density (−410 μm/mm 2 , P =0.015), (2) higher red blood cell filling as a marker of perfusion (2%, P =0.004), and (3) smaller perfused boundary region (−0.07 µm, P =0.025) as a measure of glycocalyx integrity, adjusted for covariates including adverse pregnancy outcomes. Women with MVM also had higher adjusted diastolic blood pressure (+2.6 mm Hg, P =0.021), total and LDL (low-density lipoprotein)-cholesterol (+11.2 mg/dL, P =0.016; +8.7 mg/dL, P =0.031). MVM associations with subsequent cardiovascular measures did not vary by type of adverse pregnancy outcome, except among women with preterm births where blood pressure was higher only among those with MVM. Results were similar when evaluated as MVM severity. A decade after delivery, women with placental vascular lesions had an adverse cardiovascular profile comprised of microvascular rarefaction, higher blood pressure and more atherogenic lipids. Placental histopathology may reveal a woman’s early trajectory toward subsequent vascular disease.

Accurate placenta pathology assessment is essential for managing maternal and newborn health, but the placenta’s heterogeneity and temporal variability pose challenges for histology analysis. To address this issue, we developed the ‘Histology Analysis Pipeline.PY’ (HAPPY), a deep learning hierarchical method for quantifying the variability of cells and micro-anatomical tissue structures across placenta histology whole slide images. HAPPY differs from patch-based features or segmentation approaches by following an interpretable biological hierarchy, representing cells and cellular communities within tissues at a single-cell resolution across whole slide images. We present a set of quantitative metrics from healthy term placentas as a baseline for future assessments of placenta health and we show how these metrics deviate in placentas with clinically significant placental infarction. HAPPY’s cell and tissue predictions closely replicate those from independent clinical experts and placental biology literature.

Background Hypertensive disorders of pregnancy (HDP) are associated with short-term cardiac structure and function abnormalities, but later life changes are not well studied. Objectives This study aimed to determine if HDP history is associated with echocardiographic differences 8 to 10 years after delivery, and if subgroups with placental maternal vascular malperfusion (MVM) lesions or current hypertension may be particularly affected. Methods Women with pregnancies delivered from 2008 to 2009 were selected from a clinical cohort with abstracted pregnancy and placental pathology data to undergo transthoracic echocardiography (2017 to 2020). Medical history, blood pressure, and weight were measured at the study visit. Results The authors enrolled 132 women (10 ± 1 years post-delivery, age 38 ± 6 years): 102 with normotensive pregnancies and 30 with HDP: pre-eclampsia (n = 21) or gestational hypertension (n = 9). Compared with women with normotensive pregnancies, those with HDP history were more likely to have current hypertension (63% vs. 26%; p Conclusions Women with both HDP history and current hypertension have pronounced differences in left ventricular structure and function a decade after pregnancy, warranting continued surveillance and targeted therapies for cardiovascular disease prevention.

POINTS CLES Une femme de 40 ans (gravida 2, para 1) a ete admise dans un hopital de soins tertiaires de Toronto, en Ontario. On a note chez elle : neutropenie familiale, diabete de grossesse et probleme d’infections bacteriennes a repetition, dont 3 episodes (sinusite, dermatite et

Introduction Wharton's jelly (WJ) is the mucoid connective tissue that surrounds the vessels in the human umbilical cord and provides protection from compression and torsion in response to fetal movement. WJ is known to be altered in the presence of pregnancy complications such as gestational diabetes mellitus and preeclampsia. The present study examined associations between the cross-sectional area of WJ measured by ultrasound and postpartum placental pathology and morphometry. Methods The area of WJ was measured by ultrasound in 156 eligible participants between 23 and 37 weeks’ gestation. Morphometric assessment of fixed cord cross sections was conducted, together with assessment of the cord and placenta for specific pathologies using standard criteria. Results From 156 participants, 123 ultrasound images met the data quality requirements and pathology reporting was completed for 99 placentas. 17 of the participants (14%) delivered a small for gestational age neonate and 32 of the 99 placentas examined (32%) had significant placental pathology findings. Area of WJ was associated with low birth weight (p = 0.002) and was associated with specific placental pathology (p = 0.01). WJ area was positively associated with placental dimensions such as width, length and surface area. Discussion Decreased WJ area is associated with clinically-significant placental pathology and WJ area scales proportionally with placental size. These findings suggest that WJ area correlates with functional capacity of the placenta and thus merits further evaluation alongside currently-available tests of placental function in clinical practice.

Cardiovascular disease remains the leading killer of women, with sex-specific manifestation, mechanisms, and morbidity. Preeclampsia, fetal growth restriction, and a subset of preterm births demonstrate aberrancies in the maternal vessels supplying the placenta and damage to the placental parenchyma consistent with hypoxic/ischemic or oxidative injury. This constellation of findings, maternal vascular malperfusion (MVM) lesions, may hold the key to understanding and identifying the elevated risk for early cardiovascular disease in women who experience adverse pregnancy outcomes. This intriguing possibility has only begun to be examined, but accumulating evidence is compelling and is reviewed here.

Introduction: Women with adverse pregnancy outcomes (APO, preeclampsia, preterm birth, small for gestational age neonates), especially those with placental maternal vascular malperfusion (MVM), are at increased cardiovascular and cognitive risk, perhaps due to poor cardiovascular health (CVH) and other vascular pathology. Hypothesis: Including history of APO and MVM in CVH scores identifies women with lower cognitive function and circulating angiogenic biomarker profiles indicating ongoing vascular pathophysiology. Methods: CVH was evaluated in 39 women 8-10 years after pregnancy (mean age 39.8 ±6 years, 36% Black) using standard metrics (smoking, BMI, blood pressure, total cholesterol, fasting glucose, and physical activity; higher score is healthier). APO history was abstracted from the medical record and added to CVH (no APO [n=14], APO-MVM [n=13], APO+MVM [n=13] to create a CVH-APO score. We evaluated cognitive function (memory, processing speed and executive function) as domain z scores using standard tests. Angiogenic biomarkers (VEGF-A, VEGF-C, VEGF-D, Tie-2, sFlt1, PlGF, and bFGF) were quantified using Angiogenesis Panel 1 (Meso Scale Diagnostics). Associations of CVH with cognition and biomarkers were evaluated using Pearson correlations. CVH groups (poor [CVH ≤7] vs. healthy [CVH >8) were compared with and without APO history using t-test. Results: Women with APO+MVM had lower CVH scores compared to those with no APO, adjusted for age and race (-1.5, p=0.03). Poor CVH was associated with slower processing speed (r=0.350, p=0.031) and reduced executive function (r=0.353, p=0.03) but not memory. When APO history was added to CVH, associations became stronger (r=0.402, p=0.012 for processing speed; 0.422, p=0.008 for executive function). Poorer CVH scores also correlated with higher VEGF A (-0.501 p=0.001), VEGF C (-0.653, p Discussion: Poor CVH is associated with lower cognition and higher circulating angiogenic biomarker profiles in women, and APO history may further contribute to the cumulative vascular pathophysiological burden a decade after pregnancy.

Background Evidence of placental maternal vascular malperfusion is associated with significant perinatal outcomes such as preeclampsia, intrauterine growth restriction and preterm birth. Elevations in pre-pregnancy blood pressure increase the risk for poor perinatal outcomes; however, the evidence linking pre-pregnancy blood pressure and placental malperfusion is sparse. Materials and methods We conducted a retrospective case-control study of women with singleton gestations with placental evaluations who delivered at Magee-Womens Hospital in 2012. Charts from 100 deliveries with placental malperfusion lesions (vasculopathy, advanced villous maturation, infarct, or fibrin deposition) and 102 deliveries without placental malperfusion were randomly selected for screening. Blood pressure, demographic, and clinical data were abstracted from pre-pregnancy electronic medical records and compared between women with and without subsequent placental malperfusion lesions. Results Overall, 48% of women had pre-pregnancy records, and these were similarly available for women with and without placental malperfusion. Women with placental malperfusion demonstrated a reduction in their pre- to early pregnancy decrease in diastolic blood pressure (DBP). Adjusted for race, pre-pregnancy BMI, age, pre-conception interval, and gestational age at the first prenatal visit, the difference in pre- to early pregnancy DBP was significantly less in women with placental malperfusion compared to those without this pathologic finding (− 1.35 mmHg drop vs − 5.6mmg, p Conclusion A blunted early gestation drop in DBP may be a risk factor for placental malperfusion, perhaps related to early pregnancy vascular maladaptation. The ability of the electronic medical record to provide pre-pregnancy data serves as an underutilized approach to study pre-pregnancy health.

Placental pathology assessment following delivery in pregnancies complicated by preeclampsia, fetal growth restriction, abruption, and stillbirth reveals a range of underlying diseases. The most common pathology is maternal vascular malperfusion, characterized by high-resistance uterine artery Doppler waveforms and abnormal expression of circulating maternal angiogenic growth factors. Rare placental diseases (massive perivillous fibrinoid deposition and chronic histiocytic intervillositis) are reported to have high recurrence risks, but their associations with uterine artery Doppler waveforms and angiogenic growth factors are presently ill-defined.To characterize the patterns of serial placental growth factor measurements and uterine artery Doppler waveform assessments in pregnancies that develop specific types of placental pathology to gain insight into their relationships with the timing of disease onset and pregnancy outcomes.A retrospective cohort study conducted between January 2017 and November 2021 included all singleton pregnancies with at least 1 measurement of maternal circulating placental growth factor between 16 and 36 weeks' gestation, delivery at our institution, and placental pathology analysis demonstrating diagnostic features of maternal vascular malperfusion, fetal vascular malperfusion, villitis of unknown etiology, chronic histiocytic intervillositis, or massive perivillous fibrinoid deposition. Profiles of circulating placental growth factor as gestational age advanced were compared between these placental pathologies. Maternal and perinatal outcomes were recorded.A total of 337 pregnancies from 329 individuals met our inclusion criteria. These comprised placental pathology diagnoses of maternal vascular malperfusion (n=109), fetal vascular malperfusion (n=87), villitis of unknown etiology (n=96), chronic histiocytic intervillositis (n=16), and massive perivillous fibrinoid deposition (n=29). Among patients who developed maternal vascular malperfusion, placental growth factor levels gradually declined as pregnancy progressed (placental growth factor10th percentile at 16-20 weeks' gestation in 42.9%; 20-24 weeks in 61.9%; 24-28 weeks in 77%; and 28-32 weeks in 81.4%) accompanied by mean uterine artery Doppler pulsatility index95th percentile in 71.6% cases. Patients who developed either fetal vascular malperfusion or villitis of unknown etiology mostly exhibited normal circulating placental growth factor values in association with normal uterine artery Doppler waveforms (mean [standard deviation] pulsatility index values: fetal vascular malperfusion, 1.14 [0.49]; villitis of unknown etiology, 1.13 [0.45]). Patients who developed either chronic histiocytic intervillositis or massive perivillous fibrinoid deposition exhibited persistently low placental growth factor levels from the early second trimester (placental growth factor10th centile at 16-20 weeks' gestation in 80% and 77.8%, respectively; 20-24 weeks in 88.9% and 63.6%; 24-28 weeks in 85.7% and 75%), all in combination with normal uterine artery Doppler waveforms (mean pulsatility index95th centile: chronic histiocytic intervillositis, 25%; massive perivillous fibrinoid deposition, 37.9%). Preeclampsia developed in 83 of 337 (24.6%) patients and was most common in those developing maternal vascular malperfusion (54/109, 49.5%) followed by chronic histiocytic intervillositis (7/16, 43.8%). There were 29 stillbirths in the cohort (maternal vascular malperfusion, n=10 [9.2%]; fetal vascular malperfusion, n=5 [5.7%]; villitis of unknown etiology, n=1 [1.0%]; chronic histiocytic intervillositis, n=7 [43.8%]; massive perivillous fibrinoid deposition, n=6 [20.7%]). Most patients experiencing stillbirth exhibited normal uterine artery Doppler waveforms (21/29, 72.4%) and had nonmaternal vascular malperfusion pathologies (19/29, 65.5%). By contrast, 28 of 29 (96.5%) patients experiencing stillbirth had ≥1 low placental growth factor values before fetal death.Serial circulating maternal placental growth factor tests, in combination with uterine artery Doppler waveform assessments in the second trimester, may indicate the likely underlying type of placental pathology mediating severe adverse perinatal events. This approach has the potential to test disease-specific therapeutic strategies to improve clinical outcomes. Serial placental growth factor testing, compared with uterine artery Doppler studies, identifies a greater proportion of patients destined to have a poor perinatal outcome because diseases other than maternal vascular malperfusion are characterized by normal uteroplacental circulation.

Neonatal morbidity attributable to prematurity predominantly occurs among early preterm births (32 weeks) rather than late preterm births (32 to37 weeks). Methods to distinguish early and late preterm births are lacking given the heterogeneity in pathophysiology and risk factors, including maternal obesity. Although preterm births are often characterized by clinical presentation (spontaneous or clinically indicated), classifying deliveries by placental features detected on histopathology reports may help identify subgroups of preterm births with similar etiology and risk factors. Latent class analysis is an empirical approach to characterize preterm births on the basis of observed combinations of placental features.To identify histopathologic markers that can distinguish early (32 weeks) and late preterm births (32 to37 weeks) that are also associated with maternal obesity and neonatal outcomes.Women with a singleton preterm birth at University of Pittsburgh Medical Center Magee-Womens Hospital (Pittsburgh, PA) from 2008 to 2012 and a placental evaluation (89% of preterm births) were stratified into early (n=900, 61% spontaneous) and late preterm births (n=3362, 57% spontaneous). Prepregnancy body mass index was self-reported at first prenatal visit and 16 abstracted placental features were analyzed. Placental subgroups (ie, latent classes) of early and late preterm births were determined separately by latent class analysis of placental features. The optimal number of latent classes was selected by comparing fit statistics. The probability of latent class membership across prepregnancy body mass indexes was estimated in early preterm births and in late preterm births by an extension of multinomial regression called pseudo-class regression, adjusting for race, smoking, education, and parity. The frequencies of severe neonatal morbidity (composite outcome: respiratory distress, bronchopulmonary dysplasia, intraventricular hemorrhage, necrotizing enterocolitis, periventricular leukomalacia, patent ductus arteriosus, and retinopathy of prematurity), small-for-gestational-age, and length of neonatal intensive care unit stay were compared across latent classes by chi-square and Kruskal-Wallis tests.Early preterm births were grouped into 4 latent classes based on placental histopathologic features: acute inflammation (38% of cases), maternal vascular malperfusion with inflammation (29%), maternal vascular malperfusion (25%), and fetal vascular thrombosis with hemorrhage (8%). As body mass index increased from 20 to 50kg/mObesity may predispose women to early but not late preterm birth through placental vascular impairment. Latent class analysis of placental histopathologic data provides an evidence-based approach to group preterm births with shared underlying etiology and risk factors.

OBJECTIVES—: To determine the relationship between blood flow in the fetal descending aorta and discordant umbilical arteries (UAs). METHODS—: Pulsed wave Doppler of both UAs and the descending aorta was performed at 4-weekly intervals between 14 and 40 weeks of gestation in 209 pregnant women. In datasets with discordant UAs, a linear mixed effects model was used to determine the categorical relationship between the UA pulsatility index (PI) (high, low and average) and the descending aorta PI. RESULTS—: Of the 209 cases, 81 had a discordance of greater than 25% in UA PI during one of their visits. There were no differences in birth outcomes between the groups with concordant and discordant UA PIs. In the cases with discordant UA PIs, the descending aorta PI was most strongly associated with both the average UA PI (P = .008), and with the UA with the lower PI (P = .008). CONCLUSIONS—: The relationship between blood flow in the descending aorta and UAs is consistent with the law for combining resistances in parallel. Measurements of the descending aorta PI, particularly in a scenario with discordant UAs, may inform the stability of the feto-placental circulation where discordant UA PIs are found.

Patients at high risk of severe preeclampsia and fetal growth restriction have low circulating levels of placental growth factor and features of maternal vascular malperfusion placental pathology at delivery. Multimodal screening and commencement of aspirin prophylaxis at 11 to 13 weeks' gestation markedly reduces the risk of preterm delivery with preeclampsia. However, the additional role of low-molecular-weight heparin and mechanisms of action remain uncertain. Because low-molecular-weight heparin augments the production and release of placental growth factor in vitro by both placental villi and vascular endothelium, it may be effective to suppress the risk of severe preeclampsia in a niche group of high-risk patients with low circulating placental growth factor in the early second trimester.This study aimed to define a gestational age-specific reference range for placental growth factor and to test the hypothesis that prophylactic low-molecular-weight heparin administered in the early second trimester may restore deficient circulating placental growth factor levels and thereby prolong pregnancy.Centile curves for circulating placental growth factor levels from 12 to 36 weeks' gestation were derived using quantile regression of combined data from a published cohort of 4207 unselected nulliparous patients in Cambridge, United Kingdom, at 4 sampling time points (12, 20, 28, and 36 weeks' gestation) and the White majority (n=531) of a healthy nulliparous cohort in Toronto, Canada, at 16 weeks' gestation using the same test platform. Within a specialty high-risk clinic in Toronto, a niche group of 7 patients with a circulating placental growth factor at the10th centile in the early second trimester received daily prophylactic low-molecular-weight heparin (enoxaparin; 40 mg subcutaneously) and were followed up until delivery (group 1). Their baseline characteristics, delivery details, and placental pathologies were compared with 5 similar patients who did not receive low-molecular-weight heparin during the observation period (group 2) and further with 21 patients who delivered with severe preeclampsia (group 3) in the same institution.A gestational age-specific reference range for placental growth factor levels at weekly intervals between 12 and 36 weeks was established for White women with singleton pregnancies. Within group 1, 5 of 7 patients demonstrated a sustained increase in circulating placental growth factor levels, whereas placental growth factor levels did not increase in group 2 or group 3 patients who did not receive low-molecular-weight heparin. Group 1 patients receiving low-molecular-weight heparin therapy exhibited a later gestation at delivery, relative to groups 2 and 3 (36 weeks [33-37] vs 23 weeks [22-26] and 28 weeks [27-31], respectively), and consequently had higher birthweights (1.93 kg [1.1-2.7] vs 0.32 kg [0.19-0.39] and 0.73 kg [0.52-1.03], respectively). The incidence of stillbirth was lowest in group 1 (14% [1 of 7]), relative to groups 2 and 3 (80% [4 of 5] and 29% [6 of 21], respectively). Maternal vascular malperfusion was the most common placental pathology found in association with abnormal uterine artery Doppler.In patients at high risk of a serious adverse pregnancy outcome owing to placental disease, the addition of low-molecular-weight heparin to aspirin prophylaxis in the early second trimester may restore deficient circulating placental growth factor to mediate an improved perinatal outcome. These data support the implementation of a multicenter pilot randomized control trial where patients are recruited primarily based on the assessment of placental function in the early second trimester.

Background: The umbilical artery (UA) Doppler pulsatility index is used clinically to detect elevated feto-placental vascular resistance. However, this metric is confounded by variation in fetal cardiac function and is only moderately predictive of placental pathology. Our group developed a novel ultrasound methodology that measures wave reflections in the UA, thereby isolating a component of the Doppler signal that is specific to the placenta. The present study examined whether wave reflections in the UA are predictive of placental vascular pathology. Methods: Standard clinical Doppler ultrasound of the UAs was performed in 241 pregnant women. Of these, 40 women met narrowly defined preset criteria for the control group, 36 had maternal vascular malperfusion (MVM) and 16 had fetal vascular malperfusion (FVM). Using a computational procedure, the Doppler waveforms were decomposed into a pair of forward and backward propagating waves. Findings: Compared to controls, wave reflections were significantly elevated in women with either MVM (p

Context.—The number of neonates with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is increasing, and in a few there are reports of intrauterine infection.Objective.—To characterize the placental pathology findings in a preselected cohort of neonates infected by transplacental transmission arising from maternal infection with SARS-CoV-2, and to identify pathology risk factors for placental and fetal infection.Design.—Case-based retrospective analysis by a multinational group of 19 perinatal specialists of the placental pathology findings from 2 cohorts of infants delivered to mothers testing positive for SARS-CoV-2: live-born neonates infected via transplacental transmission who tested positive for SARS-CoV-2 after delivery and had SARS-CoV-2 identified in cells of the placental fetal compartment by molecular pathology, and stillborn infants with syncytiotrophoblast positive for SARS-CoV-2.Results.—In placentas from all 6 live-born neonates acquiring SARS-CoV-2 via transplacental transmission, the syncytiotrophoblast was positive for coronavirus using immunohistochemistry, RNA in situ hybridization, or both. All 6 placentas had chronic histiocytic intervillositis and necrosis of the syncytiotrophoblast. The 5 stillborn/terminated infants had placental pathology findings that were similar, including SARS-CoV-2 infection of the syncytiotrophoblast, chronic histiocytic intervillositis, and syncytiotrophoblast necrosis.Conclusions.—Chronic histiocytic intervillositis together with syncytiotrophoblast necrosis accompanies SARS-CoV-2 infection of syncytiotrophoblast in live-born and stillborn infants. The coexistence of these 2 findings in all placentas from live-born infants acquiring their infection prior to delivery indicates that they constitute a pathology risk factor for transplacental fetal infection. Potential mechanisms of infection of the placenta and fetus with SARS-CoV-2, and potential future studies, are discussed.

Objective To describe the evolution and evaluation of protocol-based multidisciplinary quality improvement (QI) in women undergoing cesarean hysterectomy for radiologically suspected and pathologically confirmed placenta accreta spectrum (PAS) disorders. Methods A single-center, retrospective cohort study was conducted of all patients undergoing cesarean hysterectomy for PAS disorders between March 2009 and June 2018. Two distinct periods were defined to compare outcomes: 2009-2011 (initial period) and 2017-2018 (current period). Primary outcomes included blood loss and administration of blood products. Secondary outcomes included perioperative levels of hemoglobin, adverse events and complications, time to mobilization, and length of hospitalization. Results Among the 105 consecutive patients identified, there were 26 in the initial period and 32 in the current period. With the implementation of all QI care bundles, median estimated surgical blood loss halved from 2000 mL in the initial period to 1000 mL in the current period, and fewer patients required allogenic blood transfusion (61.5% vs 25%). Patients in the current period demonstrated improved postoperative levels of hemoglobin compared to those in the initial period (101 g/L vs 89 g/L) and had a shorter median postoperative hospital stay (3 days vs 5 days). Conclusion These results support the implementation of a multifaceted QI and patient care initiative for women with PAS disorders.

Background Fetal sex is known to affect pregnancy outcomes. In current clinical practice, monitoring of fetal well-being is based on Doppler ultrasound measurements of major placental and fetal vessels. The objective of this study was to investigate the effect of fetal sex on Doppler parameters throughout gestation in healthy pregnancy. Methods A prospective study was conducted in 240 pregnant women with ultrasound examinations at a 4-weekly interval between 12 and 38 weeks of gestation. Pulsed Doppler spectra were collected for the umbilical arteries (UAs), middle cerebral artery (MCA), descending abdominal aorta (DAo), and ductus venosus (DV). Linear mixed effects models were used to determine if the pulsatility indices (PIs) of these vessels depended on gestational age and fetal sex. Results While there were no differences in the MCA PI and DV PIV over gestation between female and male fetuses, the trajectory of the UA and DAo PIs differed by fetal sex (p = 0.02 and p = 0.01, respectively). Conclusions Doppler ultrasound parameters were found to be dependent on fetal sex for some vessels and not for others in healthy pregnancies. Further investigations are needed to understand the physiological mechanisms for these sex differences and the relevance for disease processes in pregnancy.

The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.

Placental dysfunction is a major contributing factor to fetal growth restriction. Placenta-mediated fetal growth restriction occurs through chronic fetal hypoxia owing to poor placental perfusion through a variety of mechanisms. Maternal vascular malperfusion is the most common placental disease contributing to fetal growth restriction; however, the role of rare placental diseases should not be overlooked. Although the features of maternal vascular malperfusion are identifiable on placental pathology, antepartum diagnostic methods are evolving. Placental imaging and uterine artery Doppler, used in conjunction with angiogenic growth factors (specifically placenta growth factor and soluble fms-like tyrosine kinase-1), play an increasingly important role.

Introduction Women with adverse pregnancy outcomes (APOs) have excess risk of later life cardiovascular disease (CVD) perhaps related to an underlying high-risk vascular phenotype. We sought to determine if placental evidence of maternal malperfusion in uncomplicated pregnancies is associated with an increased risk of APOs in subsequent pregnancies. Methods 536 women with more than one delivery and an initial uncomplicated pregnancy with placental pathology examination between 2008 and 2012 were included. APOs (small for gestational age, preterm delivery, or preeclampsia) were identified for each delivery. Multivariable log-binomial regression was used to estimate the risk of an APO in a subsequent pregnancy associated with MVM lesions in index pregnancy with adjustment for covariates. Results Placental pathology from the initial pregnancy was compared between women with no APO in any pregnancy (-APO/-APO; n = 403) and women with an initial uncomplicated pregnancy and a subsequent adverse outcome (-APO/+APO; n = 133). Women with MVM lesions had an increased risk of an APO in a subsequent pregnancy relative to women with no MVM lesions after adjusting for covariates (aOR = 1.61; 95%CI = 1.06–2.46). Decidual vasculopathy was found in 13/133 (9.8%) of -APO/+APO women compared with 16/403 (4.0%) of -APO/-APO women, with an adjusted odds ratio of 2.51 (95% CI = 1.31–4.80). Discussion MVM lesions found in placentas in uncomplicated pregnancies are associated with an increased risk of an adverse outcome in a subsequent pregnancy. Placental evidence of vascular malperfusion could offer a novel approach to risk stratification for subsequent pregnancy complications and perhaps future CVD.

Triploidy is the presence of an extra haploid set of chromosomes and can exist in complete or mosaic form. The extra haploid set of chromosomes in triploid cells can be of maternal or paternal origin. Diploid/triploid mixoploidy is a unique form of triploid mosaicism that requires the aberrant segregation of entire parental genomes into distinct blastomere lineages (heterogoneic cell division) at the earliest zygotic divisions. Here we report on eight cases of diploid/triploid mixoploidy from our institution and conduct a comprehensive review of the literature. The parental origin of the extra set of chromosomes was determined in two cases; and, based on phenotypic evidence we propose the parental origin in the other cases. One case with complex mixoploidy appears to have a digynic origin in addition to the involvement of two different sperm. Of our eight cases, only one resulted in the birth of a live healthy child. The other pregnancies ended in miscarriage, elective termination of pregnancy, intrauterine fetal demise or neonatal death. A review of the literature and the results of our cases show that a preponderance of recognized cases of diploid/triploid mixoploidy has a digynic origin.

Introduction The biological mechanisms that underlie racial disparities in placenta-mediated pregnancy complications remain unknown. Placental evidence of maternal vascular malperfusion (MVM), a common pathologic feature of these outcomes, represents hypoxic-ischemic damage to the placenta. We sought to separately estimate the risk of MVM and individual lesions associated with maternal race. Methods This was a retrospective cohort study of black and white women with singleton live births and placental pathology data at Magee-Womens Hospital during 2008–2012 (n = 15,581). MVM consisted of ≥1 individual lesions: low placental weight, decidual vasculopathy, accelerated villous maturation, infarcts, and fibrinoid deposition. We separately compared the incidence of MVM and individual lesions in black and white women using logistic regression with generalized estimating equations. Results After adjusting for covariates, black women had increased risks of MVM (aOR 1.14, 95% CI 1.05–1.23), low placental weight (aOR 1.41, 95% CI 1.28–1.55), and decidual vasculopathy (aOR 1.58, 95% CI 1.36–1.83), also observed in uncomplicated, preterm, and term births. Conversely, black women had decreased risk of infarcts (aOR 0.84, 95% CI 0.75–0.95) compared with white women, also observed in uncomplicated and full-term births. Race was not associated with accelerated villous maturation or fibrinoid deposition. Inverse probability weighting to account for potential selection bias generated similar results. Discussion Our findings suggest that excess risks of MVM, specifically low placental weight and decidual vasculopathy in black women may be due to a pathological susceptibility to an underlying high-risk vascular phenotype. The clinical significance of race differences in the occurrence of infarcts warrants further investigation.

The placenta is one of the most exciting organs. It is dynamic; its morphology and function continuously develop and adjust over its brief life span. It mediates the physiology of two distinct yet highly interconnected individuals. The pathology that develops in the placenta, and the adaptations the placenta undergoes to mitigate this pathology, may influence the later life health of the mother and baby (Circ Res, 116, 2015, 715; Hum Reprod Update, 17, 2011, 397; Nutr Rev 71, 2013, S88; Placenta, 36, 2015, S20). Pathological placenta examination may reveal macroscopic and microscopic patterns that provide valuable information to the obstetricians, neonatologists, and pediatricians caring for the family. The placenta often plays a key role in understanding adverse fetal outcomes such as hypoxic brain injury, cerebral palsy, fetal growth restriction, stillbirth, and neonatal death (Placenta, 35, 2014, 552; Placenta, 52, 2017, 58; Placenta, 30, 2009, 700; Obstet Gynecol, 114, 2009, 809; Clin Perinatol, 33, 2006, 503; Pediatr Dev Pathol, 11, 2008, 456; Arch Pathol Lab Med, 124, 2000, 1785). Moreover, it may help to understand the pathophysiology of pregnancy, improve management of subsequent pregnancies, and assist in medicolegal assessment. Placental pathologic examination may even provide evidence of susceptibility to adult-onset diseases such as diabetes (Pediatr Dev Pathol, 6, 2003, 54; Diabetes Metab, 36, 2010, 682; BJOG, 113, 2006, 1126; Int J Gynaecol Obstet, 104, 2009, S25; Zentralbl Gynakol, 97, 1975, 875). Pathologic examination of the placenta may thus be of tremendous value, particularly for those women experiencing an adverse pregnancy outcome. However, this potential utility may be entirely wasted, if the findings are not communicated in an effective manner to the appropriate clinicians. An optimized, readily understandable report of pathological findings is essential for clinical utility.

The placental pathologies that develop in the context of hypoxic insults to the fetus and placenta are termed maternal vascular malperfusion (MVM). On molecular analysis, these lesions primarily show evidence of oxidative damage, suggesting that they arise in the context of hypoxia-reperfusion injury. The earliest abnormalities are likely incomplete or absent remodeling of maternal spiral arteries (decidual arteriopathy). These vascular remodeling defects then lead to subsequent damage patterns, including accelerated villous maturation, distal villous hypoplasia, increased syncytial knots, villous infarction, retroplacental hemorrhage, and placental hypoplasia. MVM may occur in a surprisingly wide array of clinical disorders, but the relation between these clinical disorders and placental MVM is complex. It seems likely that these clinical disorders represent final common pathways for multiple etiologies, only some of which result in MVM. Birth Defects Research 109:1345-1357, 2017.© 2017 Wiley Periodicals, Inc.

Introduction The genetics and pathology of diploid complete and triploid partial hydatidiform moles have been well established. Enlarged cystic placenta often indicates an underlying etiology and is frequently associated with adverse pregnancy outcome. Several imprinted genes are strongly expressed in placental tissues and essential for normal placental growth and development. Disruption of these imprinted genes can lead to abnormal placental pathology and placental stunting or overgrowth. We present the genetic etiologies of five unusual mosaic cases of enlarged cystic placentas and report a novel etiology, mosaicism for deletion of the maternal GRB10 gene. Methods Five mosaic placental mesenchymal dysplasia cases with discrete populations of “cystic” and “normal” villi and/or atypical p57 KIP2 immunostaining were evaluated by genetic analysis; including G-banded karyotyping, fluorescence in situ hybridization (FISH), whole genome CGH + SNP microarray, conventional Sanger sequencing, and STR microsatellite analysis. Results Genetic etiologies ranged from genome-wide changes, including mosaic androgenetic isodisomy and mosaic diandric triploidy, to a novel microdeletion of the maternally-expressed GRB10 gene. An abnormal mosaic population of cells was also detected in the fetus in two cases. Discussion Four cases were mosaic for either diandric triploidy or an androgenetic cell population, and the enlarged cystic placentas were likely due to an excess of paternally-expressed growth promoting genes and also the absence of maternally-expressed growth restricting genes. Also we identified mosaicism for a novel microdeletion of the maternal GRB10 allele, a potent growth inhibitor, which resulted in placental overgrowth in the cystic area of one placenta. We advocate the use of ancillary techniques to investigate complex mosaic cases of enlarged cystic placentas to discover atypical genetic etiologies and to increase our understanding of the placental genome.

During pregnancy, placental trophoblasts at the feto-maternal interface produce a broad repertoire of microRNA (miRNA) species. These species include miRNA from the primate-specific chromosome 19 miRNA cluster (C19MC), which is expressed nearly exclusively in the placenta. Trafficking of these miRNAs among the maternal, placental, and fetal compartments is unknown. To determine miRNA expression and trafficking patterns during pregnancy, we sequenced miRNAs in triads of human placenta and of maternal and fetal blood and found large subject-to-subject variability, with C19MC exhibiting compartment-specific expression. We therefore created humanized mice that transgenically express the entire 160-kb human C19MC locus or lentivirally express C19MC miRNA members selectively in the placenta. C19MC transgenic mice expressed a low level of C19MC miRNAs in diverse organs. When pregnant, female C19MC mice exhibited a strikingly elevated (>40-fold) expression of C19MC miRNA in the placenta, compared with other organs, that resembled C19MC miRNAs patterns in humans. Our mouse models showed that placental miRNA traffic primarily to the maternal circulation and that maternal miRNA can traffic to the placenta and even into the fetal compartment. These findings define an extraordinary means of nonhormonal, miRNA-based communication between the placenta and feto-maternal compartments.—Chang, G., Mouillet, J.-F., Mishima, T., Chu, T., Sadovsky, E., Coyne, C. B., Parks, W. T., Surti, U., Sadovsky, Y. Expression and trafficking of placental microRNAs at the feto-maternal interface.

Although preeclampsia is a common and serious complication of pregnancy, insight into its pathobiology and diagnosis is lacking. Circulating plasma exosomes, which contain RNA and other molecules and have recently become accessible for diagnostics, may be informative in this regard. We tested the hypothesis that preeclampsia may affect the miRNA cargo within circulating maternal blood exosomes. We collected plasma from 60 pregnant women at term, including 20 women with pregnancy complicated by preeclampsia, and 20 women with fetal growth restriction and 20 with healthy pregnancy, serving as controls. We isolated exosomes from the maternal plasma by continuous density gradient ultracentrifugation. Our main outcome variable was exosomal miRNA cargo, analyzed by quantitative polymerase chain reaction-based TaqMan advanced miRNA assay in a card format and the expression of differentially expressed exosomal miRNA in whole plasma from the same participants. We found that 7 miRNA species were differentially expressed in exosomes from women with preeclampsia and those from controls. In contrast, there was no significant difference in exosomal miRNA expression between women with fetal growth restriction and controls. The results were not affected by fetal sex. Only one of the preeclampsia-related, differentially expressed exosomal miRNAs was significantly different in whole plasma miRNA analysis. We concluded that unlike whole plasma miRNA, exosomes extracted from the plasma of women with preeclampsia exhibit a unique miRNA profile, suggesting that plasma exosomal miRNA could provide insight into the pathophysiology of preeclampsia, and may play a role in disease diagnostics.

Introduction Preterm birth is a heterogeneous phenotype, with placental abnormalities underlying many cases. The etiology of preterm births that occur in the absence of placental abnormalities, however, remain enigmatic and we considered that early pregnancy biomarkers may provide clues. Methods Women from a hospital-based cohort (2008–2012, n = 397) were randomly selected within 6 strata of term and preterm birth with and without placental decidual vasculopathy (arteriopathy), intrauterine inflammation/infection (acute chorioamnionitis), or no lesions. Lipids and inflammatory markers were analyzed in first trimester samples (12.5 ± 0.6 weeks) and related to outcome groups (referent, term births with no lesions). Factor analysis then clustered analytes and related these to preterm birth groups, adjusted for covariates and stratified by pre-pregnancy obesity. Results Three biomarker patterns were identified. Immune activation cytokines (33% of the variance) were associated with preterm birth with no lesions (aOR 1.5, 95%CI 1.1–2.1), particularly among obese women. In contrast, inflammatory chemokines (9% of variance) were associated with term and preterm vasculopathy among non-obese women (aOR 2.6 [1.3, 4.7] and 2.0 [1.1, 3.0], respectively). Discussion The early pregnancy maternal immune profile is related to preterm births classified according to placental lesions, and these associations vary according to obesity status.

The quality of the intrauterine environment, in which the placenta plays a critical role, affects birth outcomes and lifelong health. The effect of metal contaminants on the growth and functioning of the placenta have not been widely reported but may provide insights into how metal exposures lead to these outcomes. We examined relationships between placental concentrations of cadmium (Cd), arsenic (As), mercury (Hg) and lead (Pb) and measures of placental growth and functioning (placental weight, placental efficiency (the log ratio of placental weight and birth weight), chorionic disc area and disc eccentricity) as part of the New Hampshire Birth Cohort Study (N = 1159). We additionally examined whether these associations were modified by placental concentrations of essential elements zinc (Zn) and selenium (Se). Associations were evaluated using generalized linear models. Multivariable-adjusted differences in placental weight were − 7.81 g (95% CI: −15.42, −2.48) with every ng/g increase in the Cd concentration of placenta (p-Value = 0.0009). Greater decrements in placental weight and efficiency associated with placental Cd were observed for females. For placentae with below median Zn and Se concentrations, decrements in placental weight were − 8.81 g (95% CI: −16.85, −0.76) and − 13.20 g (95% CI: −20.70, −5.70) respectively. The Cd concentration of placenta was also associated with reductions in placental efficiency both overall, and in Zn- and Se-stratified models. No appreciable differences were observed with other elements (As, Hg or Pb) and with other placental measures (chorionic disc area and disc eccentricity). In structural equation models, placental weight was a mediator in the relation between placental Cd concentration and reduced birth weight. Our findings suggest a role of interacting essential and contaminant elements on birth weight that may be mediated by changes in the growth and function of the placenta. Keywords: Placenta, Weight, Efficiency, Arsenic, Cadmium, Zinc, Selenium

Introduction: Women with placentally mediated pregnancy complications such as preeclampsia have excess hypertension and cardiovascular disease later in life, but the underlying pathophysiology linking these conditions is unknown. Hypothesis: We considered that histological examination of the placenta may reveal maternal vascular impairments that identify a group susceptible to blood pressure (BP) elevations a decade after delivery, including elevations that may only be detectable using out of office BP measurement. Method: Women with a singleton live birth at Magee-Womens Hospital (Pittsburgh, PA) during 2008-2009 were enrolled 8-10 years after delivery (n=248). Pregnancy features and placental pathology were abstracted from medical records. Women with placental vasculopathy (several related lesions arising from impaired remodeling of maternal myometrial and decidual arteries, n=24) or infarcts (n=32) were compared to women with neither lesion (n=192). Clinic BP was measured three times by trained research staff using a standard protocol and a validated home BP device. Women were trained to measure their BP twice daily for 7 days using the same device mentioned above. Subsequently, time and date stamped BP measures were downloaded (mean 12.2 measures ± 4.5). BP status (elevated, stage 1, stage 2) was identified using the 2018 ACC/AHA guidelines for clinic and home BP measures. Sustained normotensive, white coat, masked, and sustained hypertension status were classified. Results: Women were, on average, 37 years of age at measurement. In analysis controlled for age and race (p-value < 0.05) those with placental vasculopathy had higher mean diastolic BP in clinic and at home (80.5 ± 13.3 mmHg, 79.9 ± 10.4, respectively) than women without vascular lesions in the placenta (75.4 ± 9.8 mmHg and 74.3 ±8.6, respectively). In addition, women with placental vasculopathy had a higher prevalence of masked hypertension (higher elevated BP status measured at home vs. in clinic; 37.5% vs. 14.1%) that persisted after accounting for maternal age and race (adjusted OR 4.7, 95% confidence interval 1.6, 13.8). Vasculopathy associations were similar after excluding women with preeclampsia (n=42), the pregnancy complication most strongly associated with placental vascular pathology. There were modest, non-significant associations between placental infarcts and all measures of maternal blood pressure. Conclusions: Irrespective of prior preecclampsia, women with placental evidence of vasculopathy have excess hypertension and particularly masked hypertension in the decade after pregnancy. Our results emphasize the value of out of office blood pressure measurement, and indicate that placental vasculopathy may be pathophysiologically related to development of hypertension before age 50.

Maternal vascular malperfusion (MVM) lesions represent hypoxic-ischemic damage to the placenta, and they are associated with adverse pregnancy outcomes. Women with gestational diabetes (GDM) are at increased risk for pregnancy complications, so we set out to characterize the prevalence and clinical correlates of MVM lesions in this cohort.This was a retrospective cohort study of 1187/1374 (86.4%) women with GDM delivered between 2009 and 2012 who had placental pathology available. Placental lesions of all types were tabulated and grouped into constructs of related entities. MVM lesions specifically included villous infarcts, decidual vasculopathy, increased syncytial knots, perivillous fibrin, and fibrin deposition. We compared maternal characteristics between women with and without MVM lesions, and we also assessed the impact of these lesions on birth weight, preterm birth, and pre-eclampsia using multivariable logistic regression analysis.MVM lesions were the most common placental lesion type in women with GDM (n = 362, 30.5%). Excess gestational weight gain was independently associated with MVM lesions (aOR 1.42, 95% CI 1.06-1.91, p = 0.02) after adjusting for maternal characteristics. MVM lesions were associated with lower birth weight (-90.3 g, 95% CI -148.0 to -32.7, p = 0.002), as well as a 2-fold increased risk for delivery of a small for gestational age infant (10.8 vs 5.9%, p = 0.01) in overweight and obese women. MVM lesions were also associated with increased risk for preterm birth34 weeks (adjusted OR 2.36, 95% CI 1.31-4.23, p = 0.004) and hypertensive disorders of pregnancy (HDP; adjusted OR 1.58, 95% CI 1.13-2.22, p = 0.02).Placental maternal vascular malperfusion lesions may be one pathway linking excess gestational weight gain to adverse pregnancy outcomes in women with GDM, and future studies are needed to identify metabolic factors that may explain this association.

Prominent syncytial knots (SK) in placentas signal advanced gestation or placental malperfusion, reflecting exposures that adversely affect placental development and pregnancy outcomes. Molecular-level interrogations of syncytiotrophoblast have altered perceptions of and raised questions about the function and disposition of SK. Quantifying SK and achieving acceptable levels of interrater reliability have been challenging. Our objective was to develop a simple, reproducible protocol for counting SK and demonstrate interrater reliability overall and within 3 parameters, ie, preterm vs term delivery, presence vs absence of diffuse prominent SK (DPSK), and SK relationship with a lesion, all of which could influence measurement reproducibility and interpretation. Criteria for defining SK and a grid system drawn on glass slides were developed for counting percentage of villi with SK. One disc section each from 151 placentas, sampled from 8 groups defined by the 3 parameters, was assessed by 2 pretrained pathologists. The resulting weighted kappa statistic for overall interrater agreement was 0.60 (very good) and Spearman correlation coefficient for ranking quartiles was >0.70. Agreement was best for preterm placentas, kappa = 0.61, and those only showing DPSK associated with a lesion, kappa = 0.67. Agreement was low in the absence of DPSK, kappa = 0.22, or when DPSK was present in a placenta not associated with a lesion, kappa = 0.32. The proposed method offers a potentially reliable approach for categorizing SK counts as normal vs abnormal or providing continuous measure counts. More extensive pretraining, focused on placentas with few SK and those without an associated lesion, is recommended to improve agreement.