Congenital iRHOM2 deficiency causes ADAM17 dysfunction and environmentally directed immunodysregulatory disease.

We report a pleiotropic disease due to loss-of-function mutations in RHBDF2, the gene encoding iRHOM2, in two kindreds with recurrent infections in different organs. One patient had recurrent pneumonia but no colon involvement, another had recurrent infectious hemorrhagic colitis but no lung involvement and the other two experienced recurrent respiratory infections. Loss of iRHOM2, a rhomboid superfamily member that regulates the ADAM17 metalloproteinase, caused defective ADAM17-dependent cleavage and release of cytokines, including tumor-necrosis factor and amphiregulin. To understand the diverse clinical phenotypes, we challenged Rhbdf2-/- mice with Pseudomonas aeruginosa by nasal gavage and observed more severe pneumonia, whereas infection with Citrobacter rodentium caused worse inflammatory colitis than in wild-type mice. The fecal microbiota in the colitis patient had characteristic oral species that can predispose to colitis. Thus, a human immunodeficiency arising from iRHOM2 deficiency causes divergent disease phenotypes that can involve the local microbial environment.

Biallelic TLR4 deficiency in humans.

BACKGROUND: Toll-like receptors (TLRs) mediate functions for host defense and inflammatory responses. TLR4 recognizes LPS, a component of gram-negative bacteria as well as host-derived endogenous ligands such as S100A8 and S100A9 proteins. OBJECTIVE: We sought to report phenotype and cellular function of individuals with complete TLR4 deficiency. METHODS: We performed genome sequencing and investigated exome and genome sequencing databases. Cellular responses were studied on primary monocytes, macrophages, and neutrophils, as well as cell lines using flow cytometry, reporter, and cytokine assays. RESULTS: We identified 2 individuals in a family of Qatari origin carrying a homozygous stop codon variant p.Q188X in TLR4 presenting with a variable phenotype (asymptomatic and inflammatory bowel disease consistent with severe perianal Crohn disease). A third individual with homozygous p.Y794X was identified in a population database. In contrast to hypomorphic polymorphisms p.D299G and p.T399I, the variants p.Q188X and p.Y794X completely abrogated LPS-induced cytokine responses whereas TLR2 response was normal. TLR4 deficiency causes a neutrophil CD62L shedding defect, whereas antimicrobial activity toward intracellular Salmonella was intact. CONCLUSIONS: Biallelic TLR4 deficiency in humans causes an inborn error of immunity in responding to LPS. This complements the spectrum of known primary immunodeficiencies, in particular myeloid differentiation primary response 88 (MYD88) or the IL-1 receptor-associated kinase 4 (IRAK4) deficiency that are downstream of TLR4 and TLR2 signaling.

Pediatric Cervicofacial Actinomycosis: Lessons From a Craniofacial Unit.

Actinomycosis is a rare disease that remains difficult to diagnose and manage. Prompted by 2 recent cases the authors sought evidence-based conclusions about best practice. A systematic review was conducted using standard PRISMA methodology. The study was registered prospectively (PROSPERO: CRD42018115064). Thirty-three children from 23 series are described. The mean age was 8 years (range 3-17). Fifty-five percent were female. Twenty cases involved bone (usually mandible); 13 cases involved cervicofacial soft tissue. Poor dental hygiene and oral trauma were implicated. The median diagnostic delay was 12 weeks (range 1-156 weeks). The median duration of definitive antibiotic therapy was 17 weeks (range 1-130 weeks). Although diagnostic delay did not correlate with number of surgeries, bony involvement was associated with more procedures (P = 0.008, unpaired t test). All (6) cases with residual infection had bony involvement (P = 0.06, Fisher exact test). Neither diagnostic delay nor number of surgeries significantly influenced infection-free outcome which, instead, relies on aggressive surgical debridement and prolonged antibiotic therapy. Mandibular involvement exhibits a higher surgical burden and chronicity in around a third of cases. As dental caries are implicated in mandibular disease, preventative strategies must focus on improving pediatric oral hygiene.

Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy.

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Haemophilus influenzae: a potent perinatal pathogen disproportionately isolated from Indigenous women and their neonates.

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) bacteraemia in pregnant women is strongly associated with pregnancy loss and preterm delivery. However, the clinical significance of isolation of NTHi from nonsterile sites is unknown. AIMS: To examine the hypothesis that isolation of NTHi from any specimen is associated with adverse perinatal outcomes and to investigate the impression that NTHi is disproportionately isolated from indigenous women and their neonates. MATERIALS AND METHODS: Cases where NTHi was isolated from maternal, fetal or neonatal specimens during the period from 1 July 1997 to 1 July 2009 were identified. Demographic and clinical data were extracted from case notes. Histopathological material was re-reviewed by a perinatal pathologist. Demographic and clinical features of the affected group were compared with the hospital obstetric population. RESULTS: NTHi was isolated from maternal, fetal or neonatal specimens in 97 pregnancies. Two women had NTHi isolated during different pregnancies. Two mothers and 10 neonates were bacteraemic. Indigenous women comprised 28% of pregnancies where NTHi was isolated, compared with 6% of the hospital obstetric population (P < 0.001). Pregnancy loss occurred in six cases (6%). Median gestation at delivery was 33 weeks. Of 96 liveborn neonates, 88 (92%) required admission to a neonatal special care unit. Four liveborn neonates died (4%). Chorioamnionitis was confirmed by histology in 31/33 (93.9%) of placentas examined. CONCLUSIONS: Isolation of NTHi occurred more commonly in indigenous women and neonates. Isolation of NTHi from any obstetric or neonatal specimen is associated with chorioamnionitis, preterm birth, pregnancy loss, early-onset neonatal sepsis and neonatal death.

Risk of cancer among children with birth defects: a novel approach.

BACKGROUND: Associations between birth defects (BDs) and childhood cancers have been studied previously and have identified several specific birth defect-cancer associations. No studies have examined the risk after exclusion of known associations. METHODS: We analyzed data from high-quality population-based registers of BDs and cancers for Western Australian births 1982 to 2007. The cohort comprised 641,036 babies still alive at 90 days. Two experts independently reviewed all 120 births with a BD and a cancer to determine whether the cancer was congenital, caused by the BD, known to be associated with the BD or otherwise. These categories were used in sensitivity analyses. Cox regression was used to estimate hazard ratios (HRs) for any cancer and specific cancers associated with any BD and specific BDs. RESULTS: The HR for any cancer among children with any BD was 1.96 (95% confidence interval, 1.59-2.43). The HR for any cancer among children with a BD not known to be related to a cancer (n = 57) was 1.19 (95% confidence interval, 0.91-1.56). The HR for the latter association among children diagnosed with cancer before 5 years of age was 1.74 (95% confidence interval, 1.28-2.37). CONCLUSION: This novel approach aimed to prevent inflated HRs arising from reverse causation, and allow identification of associations beyond those already well documented. Larger studies using this method are needed to explore currently undocumented associations between BDs and cancers.

Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis.

Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis.

Solitary, multifocal and generalized myofibromas: clinicopathological and immunohistochemical features of 114 cases.

AIMS: To report a large series of solitary and multiple myofibromas with systematic clinicopathological correlations. METHODS AND RESULTS: We report on 114 patients with myofibromas, 97 of which were solitary and 17 multifocal. The age at presentation ranged from newborn to 70 years. All multifocal myofibromas and 91% of solitary myofibromas occurred in children. The head and neck region was the most common site (n = 43), followed by the trunk (n = 24), lower limbs (n = 14), upper limbs (n = 11), and viscera (n = 4). Solitary and multifocal myofibromas stained positively for smooth muscle actin (SMA) in 95% and 92% of cases, muscle-specific actin (MSA) in 75% and 50% of cases, and desmin in 10% and 14% of cases, respectively. Regressive features were seen in 34 solitary myofibromas and in nine multifocal myofibromas. Most patients were treated with complete excision (n = 79) or partial excision (n = 12). There were no recurrences after treatment. CONCLUSIONS: Solitary and multiple myofibromas are benign tumours that predominantly occur in infancy and childhood. Myofibromas occur especially in the head and neck region, and are characterized by SMA and, to a lesser extent, MSA expression. The clinical course is self-limiting, and local excision appears to be sufficient.

Fetal akinesia: review of the genetics of the neuromuscular causes.

Fetal akinesia refers to a broad spectrum of disorders in which the unifying feature is a reduction or lack of fetal movement. Fetal akinesias may be caused by defects at any point along the motor system pathway including the central and peripheral nervous system, the neuromuscular junction and the muscle, as well as by restrictive dermopathy or external restriction of the fetus in utero. The fetal akinesias are clinically and genetically heterogeneous, with causative mutations identified to date in a large number of genes encoding disparate parts of the motor system. However, for most patients, the molecular cause remains unidentified. One reason for this is because the tools are only now becoming available to efficiently and affordably identify mutations in a large panel of disease genes. Next-generation sequencing offers the promise, if sufficient cohorts of patients can be assembled, to identify the majority of the remaining genes on a research basis and facilitate efficient clinical molecular diagnosis. The benefits of identifying the causative mutation(s) for each individual patient or family include accurate genetic counselling and the options of prenatal diagnosis or preimplantation genetic diagnosis. In this review, we summarise known single-gene disorders affecting the spinal cord, peripheral nerves, neuromuscular junction or skeletal muscles that result in fetal akinesia. This audit of these known molecular and pathophysiological mechanisms involved in fetal akinesia provides a basis for improved molecular diagnosis and completing disease gene discovery.

Fetal oromandibular limb hypogenesis syndrome following uterine curettage in early pregnancy.

BACKGROUND: Fetal structural abnormalities, particularly limb reduction defects, have been reported after early hypoxic injury, such as chorionic villus sampling (CVS) before 66 day's gestation. CASE: We present a case of uterine curettage in early pregnancy associated with the subsequent prenatal diagnosis of fetal oromandibular limb hypogenesis syndrome. CONCLUSIONS: This case is consistent with previous hypotheses concerning the role of hypoxic trauma in inducing fetal structural defects in early pregnancy.

Human AGR2 Deficiency Causes Mucus Barrier Dysfunction and Infantile Inflammatory Bowel Disease.

BACKGROUND & AIMS: The gastrointestinal epithelium plays a crucial role in maintaining homeostasis with the gut microbiome. Mucins are essential for intestinal barrier function and serve as a scaffold for antimicrobial factors. Mucin 2 (MUC2) is the major intestinal gel-forming mucin produced predominantly by goblet cells. Goblet cells express anterior gradient 2 (AGR2), a protein disulfide isomerase that is crucial for proper processing of gel-forming mucins. Here, we investigated 2 siblings who presented with severe infantile-onset inflammatory bowel disease. METHODS: We performed whole-genome sequencing to identify candidate variants. We quantified goblet cell numbers using H&E histology and investigated the expression of gel-forming mucins, stress markers, and goblet cell markers using immunohistochemistry. AGR2-MUC2 binding was evaluated using co-immunoprecipitation. Endoplasmic reticulum (ER) stress regulatory function of mutant AGR2 was examined by expression studies in Human Embryonic Kidney 293T (HEK293T) using tunicamycin to induce ER stress. RESULTS: Both affected siblings were homozygous for a missense variant in AGR2. Patient biopsy specimens showed reduced goblet cells; depletion of MUC2, MUC5AC, and MUC6; up-regulation of AGR2; and increased ER stress. The mutant AGR2 showed reduced capacity to bind MUC2 and alleviate tunicamycin-induced ER stress. CONCLUSIONS: Phenotype-genotype segregation, functional experiments, and the striking similarity of the human phenotype to AGR2-/- mouse models suggest that the AGR2 missense variant is pathogenic. The Mendelian deficiency of AGR2, termed "Enteropathy caused by AGR2 deficiency, Goblet cell Loss, and ER Stress" (EAGLES), results in a mucus barrier defect, the inability to mitigate ER stress, and causes infantile-onset inflammatory bowel disease.

Caudal dysgenesis and sirenomelia-single centre experience suggests common pathogenic basis.

Abnormally formed lower limbs with varying degrees of fusion are the major feature of sirenomelia whereas maldeveloped lower limbs without fusion are found in association with caudal dysgenesis (CD). The relationship between these two entities has been a topic of debate for many years. The presence of a single umbilical artery originating from the abdominal aorta was considered a major feature distinguishing sirenomelia from CD. Based on this finding, the vascular steal theory was put forward as the causative mechanism of sirenomelia. CD and sirenomelia were considered to be two entirely different entities with distinct pathogenic mechanisms. However, it is now clear that a single umbilical artery can be found in some patients of CD and normal umbilical arteries in some patients of sirenomelia. The hypothesis of primary deficiency of caudal mesoderm caused by early developmental disruption suggests that sirenomelia and CD are two ends of a spectrum of maldevelopment of caudal mesoderm. In this paper we report on the clinical and pathological features of 16 patients of CD and 9 patients of sirenomelia from our institution and review the literature. This series of cases is notable for the significant association with neural tube defects, refining the renal and urogenital pathology associated with these conditions, and supporting the concept of a continuum of the disease spectrum.

Infection and fetal loss in the mid-second trimester of pregnancy.

INTRODUCTION: Chorioamnionitis is a common cause of second trimester pregnancy loss, usually due to ascending infection. This study investigates the prevalence and bacteriology of chorioamnionitis in cases of spontaneous pregnancy loss in previable gestations (16-22 weeks). METHODS: Fetal losses between 16- and 22-week gestation were identified from the institutional database over a three-year period. Cases with an autopsy were identified, pathology reports reviewed, and maternal features noted (clinical symptoms, blood count and vaginal culture results). Second trimester medical termination for fetal abnormality during the same time period served as controls for the confounding influence of labour. RESULTS: A total of 101 cases of spontaneous non-anomalous non-macerated fetal losses and 103 control cases of induced loss for fetal anomaly were identified. Median gestation of cases was 19 weeks (interquartile range (IQR) 17, 21) and of controls was 20 weeks (IQR 19, 21). Maternal white cell count was higher in cases (median 13.6 IQR 10.8, 16.6) than in controls (9.9 IQR 7.6, 11.5) (P < 0.01). Seventy-eight (77.2%) of 101 cases and no controls had histological chorioamnionitis. A fetal reaction was identified in 48.7% of cases with chorioamnionitis, and the frequency of fetal reaction increased as gestation advanced (5.3% at 16-week gestation vs 33.3% at 22-week gestation). In cases with chorioamnionitis 36/76 (47.4%) were culture positive, whereas 4/25 (16%) without chorioamnionitis were culture positive. CONCLUSION: In otherwise normal fetuses, chorioamnionitis is a common finding in mid-trimester pregnancy loss. Routine culture methods have a low sensitivity for isolation of the causative micro-organisms. This inflammatory process seems to predate the onset of labour and appears a primary mechanism in the aetiology of such losses.

Frequency and timing of loss of imprinting at 11p13 and 11p15 in Wilms' tumor development.

Epigenetic changes occur frequently in Wilms' tumor (WT), especially loss of imprinting (LOI) of IGF2/H19 at 11p15. Our previous results have identified imprinted transcripts (WT1-AS and AWT1) from the WT1 locus at 11p13 and showed LOI of these in some WTs. In this article, we set out to test the relationship between LOI at 11p13 and 11p15 and their timing in WT progression relative to other genetic changes. We found a higher level (83%) of 11p13 LOI in WT than of 11p15 LOI (71%). There was no correlation between methylation levels at the 11p13 and 11p15 differentially methylated regions or between allelic expression of WT1-AS/AWT1 and IGF2. Interestingly, retention of normal imprinting at 11p13 was associated with a small group of relatively late-onset, high-stage WTs. An examination of genetic and epigenetic alterations in nephrogenic rests, which are premalignant WT precursors, showed that LOI at both 11p13 and 11p15 occurred before either 16q loss of heterozygosity (LOH) or 7p LOH. This suggests that these LOH events are very unlikely to be a cause of LOI but that LOH may act by potentiating the effects of overexpression of IGF2 and/or WT1-AS/AWT1 that result from LOI.

Function of caspase-14 in trophoblast differentiation.

BACKGROUND: Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored. METHODS: Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways. RESULTS: Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted. CONCLUSION: Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease.

Fetal growth and the risk of childhood CNS tumors and lymphomas in Western Australia.

The etiology of childhood cancers is largely unknown, although the early age at diagnosis has led to particular interest in in utero and perinatal factors. Birth weight is the most frequently studied perinatal factor in relation to risk of childhood cancers, and results have been inconsistent. We investigated whether the risk of CNS tumors and lymphomas in children was associated with three measures of the appropriateness of intra-uterine growth: proportion of optimal birth weight (POBW), birth length (POBL) and weight for length (POWFL). A cohort of 576,633 infants born in Western Australia in 1980-2004 were followed from birth to diagnosis of a CNS tumor (n = 183) or lymphoma (n = 84) before age 15, death, or December 31, 2005, and analyzed with Cox regression. Overall, there was little evidence of any association between fetal growth and risk of CNS tumors, although risk of ependymoma/choroid plexus tumors was positively associated with POBL and negatively associated with POWFL. The risk of Hodgkin and Burkitt lymphoma increased with increasing fetal growth among boys only, whereas the increased risk observed with non-Hodgkin lymphoma was only in girls. These associations between fetal growth and disease risk were also observed among children not classified as high birth weight, suggesting that accelerated growth is more important than birth weight per se. Results were similar when cases were compared with their unaffected siblings, suggesting that the increased growth associated with cancer risk was not general to the family. The associations we observed are consistent with causal pathways involving fetal growth factors.

Placental characteristics and birthweight.

Standard gross placental measures capture dimensions relevant to specific placental functions. Our objective was to determine their accountability independent of placental weight for variance in birthweight, an important proxy for intrauterine 'adequacy' in fetal origins studies. The sample consisted of 24 152 singleton liveborn children of the Collaborative Perinatal Project delivered from 34 to 42 completed weeks gestation, with complete data for six placental measures (placental disc shape, umbilical cord length, distance from cord insertion to nearest margin, large diameter, small diameter, placental thickness) and placental weight. Associations between birthweight and placental measures were examined using multiple linear regression. Placental weight alone accounted for 36.6% of birthweight variation; the six other placental measures accounted for 28.1%. Combined, all placental measures accounted for 39.1% of birthweight variation. Seven maternal characteristics (age, height, weight, parity, socio-economic status, cigarette use, and race) were investigated to determine whether their known associations with birthweight were mediated by placental markers. Analysis suggested that the impact of all maternal characteristics except smoking was consistent with mediation by placental characteristics.

Renal tumours of childhood: an update.

The role of the pathologist has been fundamental in the progress of the treatment of paediatric renal tumours. There are different philosophies in the treatment of these tumours, and there have been many recent advances in the areas of chemotherapy, identification of new entities, prognostic histological criteria following treatment and molecular prognostic and diagnostic features. This review discusses the different approaches of the different treatment protocols from Europe and North America, and reviews staging criteria, prognostic criteria and also the different tumour entities.

The Possible Role of Placental Morphometry in the Detection of Fetal Growth Restriction.

Fetal growth restriction (FGR) is often the result of placental insufficiency and is characterized by insufficient transplacental transport of nutrients and oxygen. The main underlying entities of placental insufficiency, the pathophysiologic mechanism, can broadly be divided into impairments in blood flow and exchange capacity over the syncytiovascular membranes of the fetal placenta villi. Fetal growth restriction is not synonymous with small for gestational age and techniques to distinguish between both are needed. Placental insufficiency has significant associations with adverse pregnancy outcomes (perinatal mortality and morbidity). Even in apparently healthy survivors, altered fetal programming may lead to long-term neurodevelopmental and metabolic effects. Although the concept of fetal growth restriction is well appreciated in contemporary obstetrics, the appropriate detection of FGR remains an issue in clinical practice. Several approaches have aimed to improve detection, e.g., uniform definition of FGR, use of Doppler ultrasound profiles and use of growth trajectories by ultrasound fetal biometry. However, the role of placental morphometry (placental dimensions/shape and weight) deserves further exploration. This review article covers the clinical relevance of placental morphometry during pregnancy and at birth to help recognize fetuses who are growth restricted. The assessment has wide intra- and interindividual variability with various consequences. Previous studies have shown that a small placental surface area and low placental weight are associated with a slower growth of the fetus. Parameters such as placental surface area, placental volume and placental weight in relation to birth weight can help to identify FGR. In the future, a model including sophisticated antenatal placental morphometry may prove to be a clinically useful method for screening or diagnosing growth restricted fetuses, in order to provide optimal monitoring.

Methods to decrease variability in histological scoring in placentas from a cohort of preterm infants.

OBJECTIVE: Reliable semiquantitative assessment of histological placental acute inflammation is problematic, even among experts. Tissue samples in histology slides often show variability in the extent and location of neutrophil infiltrates. We sought to determine whether the variability in pathologists' scoring of neutrophil infiltrates in the placenta could be reduced by the use of 'regions of interest' (ROIs) that break the sample into smaller components. DESIGN: ROIs were identified within stained H&E slides from a cohort of 56 women. ROIs were scored using a semiquantitative scale (0-4) for the average number of neutrophils by at least two independent raters. SETTING: Preterm singleton births at Yale New Haven Hospital. PARTICIPANTS: This study used stained H&E placental slides from a cohort of 56 women with singleton pregnancies who had a clinically indicated amniocentesis within 24 hours of delivery. PRIMARY AND SECONDARY OUTCOME MEASURES: Interrater agreement was assessed with the intraclass correlation coefficient (ICC) and log-linear regression. Predictive validity was assessed using amniotic fluid protein profile scores (neutrophil defensin-2, neutrophil defensin-1, calgranulin C and calgranulin A). RESULTS: Excellent agreement by the ICC was found for the average neutrophil scores within a region of interest. Log-linear analyses suggest that even where there is disagreement, responses are positively associated along the diagonal. There was also strong evidence of predictive validity comparing pathologists' scores with amniotic fluid protein profile scores. CONCLUSIONS: Agreement among observers of semiquantitative neutrophil scoring through the use of digitised ROIs was demonstrated to be feasible with high reliability and validity.