Placental pathology and neonatal morbidity: exploring the impact of gestational age at birth.

AIM: To evaluate placental pathology in term and post-term births, investigate differences in clinical characteristics, and assess the risk of adverse neonatal outcome. METHODS: This prospective observational study included 315 singleton births with gestational age (GA) > 36 weeks + 6 days meeting the local criteria for referral to placental histopathologic examination. We applied the Amsterdam criteria to classify the placentas. Births were categorized according to GA; early-term (37 weeks + 0 days to 38 weeks + 6 days), term (39 weeks + 0 days to 40 weeks + 6 days), late-term (41 weeks + 0 days to 41 weeks + 6 days), and post-term births (≥ 42 weeks + 0 days). The groups were compared regarding placental pathology findings and clinical characteristics. Adverse neonatal outcomes were defined as 5-minute Apgar score < 7, umbilical cord artery pH < 7.0, admission to the neonatal intensive care unit or intrauterine death. A composite adverse outcome included one or more adverse outcomes. The associations between placental pathology, adverse neonatal outcomes, maternal and pregnancy characteristics were evaluated by logistic regression analysis. RESULTS: Late-term and post-term births exhibited significantly higher rates of histologic chorioamnionitis (HCA), fetal inflammatory response, clinical chorioamnionitis (CCA) and transfer to neonatal intensive care unit (NICU) compared to early-term and term births. HCA and maternal smoking in pregnancy were associated with adverse outcomes in an adjusted analysis. Nulliparity, CCA, emergency section and increasing GA were all significantly associated with HCA. CONCLUSIONS: HCA was more prevalent in late and post-term births and was the only factor, along with maternal smoking, that was associated with adverse neonatal outcomes. Since nulliparity, CCA and GA beyond term are associated with HCA, this should alert the clinician and elicit continuous intrapartum monitoring for timely intervention.

Maternal physical activity affects yolk sac size and growth in early pregnancy, but girls and boys use different strategies.

This longitudinal study investigated the impact of actigraphy-measured maternal physical activity on yolk sac size during early development. The yolk sac, a transient extraembryonic organ, plays a crucial role in embryonic development and is involved in metabolism, nutrition, growth, and hematopoiesis. Prospectively collected data from 190 healthy women indicated that their total daily physical activity, including both light and moderate-vigorous activity, was associated with yolk sac growth dynamics depending on embryonic sex and gestational age. Higher preconception maternal physical activity was linked to a larger yolk sac at 7 weeks (95% CI [0.02-0.13 mm]) and a smaller yolk sac at 10 weeks' gestation (95% CI [- 0.18 to - 0.00]) in male embryos; in female embryos, the yolk sac size was increased at 10 weeks' gestation (95% CI [0.06-0.26]) and was, on average, 24% larger than that in male embryos (95% CI [0.12-0.38]). Considering the pattern of other maternal effects on yolk sac size-e.g., body composition and sleep duration-we suggest that physiological yolk sac adaptations occur in short, sex-specific time windows and can be influenced by various maternal factors.

Histologic chorioamnionitis in extremely preterm births, microbiological findings and infant outcome.

BACKGROUND: Histologic chorioamnionitis (HCA) is most often caused by ascending bacterial infection originating from the cervicovaginal tract. OBJECTIVES: To investigate whether HCA with a fetal inflammatory response (FIR) has a worse clinical outcome than HCA alone. Further, if FIR or a positive maternal microbiologic culture obtained prior to birth were related to adverse neonatal outcomes in a cohort of extremely preterm (EP) neonates. METHODS: Prospective observational cohort study recruiting EP singleton pregnancies (gestational age at birth ≤28 weeks) with confirmed HCA. FIR was defined by fetal neutrophils in the chorionic vessels and/or umbilical vessels. Positive culture was defined as growth of potentially pathogenic bacteria in a sample from the cervicovaginal tract prior to birth, or if a cervicovaginal culture was lacking, a culture result from the placenta was used. Logistic regression was used to estimate odds ratios and 95% confidence intervals for the associations between FIR, a positive culture result and adverse outcomes, defined as bronchopulmonary dysplasia (BPD), brain pathology assessed by magnetic resonance imaging, retinopathy of prematurity, necrotizing enterocolitis, early-onset neonatal sepsis, and perinatal death. A composite outcome variable included one or more adverse outcomes. RESULTS: We included 71 cases with HCA, of which 51 (72%) had FIR. Maternal age, rate of clinical chorioamnionitis (CCA), preterm pre-labor rupture of membranes (PPROM), the number of women receiving antenatal steroids and antibiotics, and the rate of positive maternal cultures of potentially pathogenic bacteria were all significantly higher in the HCA with FIR. Neonates in the FIR group had significantly higher levels of blood leukocytes compared to those without. FIR was associated with a longer interval from PPROM to delivery (log-rank test: p = .022). Microbiological sampling had been performed in 63 (89%) cases, of which 60 (95%) were cervicovaginal samples. No associations were found between a positive culture and adverse neonatal outcomes, in contrast to FIR, that was significantly associated to BPD and brain pathology. CONCLUSIONS: In a cohort of EP pregnancies with confirmed HCA, the presence of FIR was associated with advanced maternal age, CCA, PPROM, antenatal steroids and antibiotics, and a positive maternal culture of potentially pathogenic bacteria. However, the presence of FIR, and not a positive culture, was associated with adverse neonatal outcomes.

Recurrence of postpartum hemorrhage, maternal and paternal contribution, and the effect of offspring birthweight and sex: a population-based cohort study.

PURPOSE: This study examines individual aggregation of postpartum hemorrhage (PPH), paternal contribution and how offspring birthweight and sex influence recurrence of PPH. Further, we wanted to estimate the proportion of PPH cases attributable to a history of PPH or current birthweight. METHODS: We studied all singleton births in Norway from 1967 to 2017 using data from Norwegian medical and administrational registries. Subsequent births in the parents were linked. Multilevel logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (CI) for PPH defined as blood loss > 500 ml, blood loss > 1500 ml, or the need for blood transfusion in parous women. Main exposures were previous PPH, high birthweight, and fetal sex. We calculated adjusted population attributable fractions for previous PPH and current high birthweight. RESULTS: Mothers with a history of PPH had three- and sixfold higher risks of PPH in their second and third deliveries, respectively (adjusted OR 2.9; 95% CI 2.9-3.0 and 6.0; 5.5-6.6). Severe PPH (> 1500 ml) had the highest risk of recurrence. The paternal contribution to recurrence of PPH in deliveries with two different mothers was weak, but significant. If the neonate was male, the risk of PPH was reduced. A history of PPH or birthweight ≥ 4000 g each accounted for 15% of the total number of PPH cases. CONCLUSION: A history of PPH and current birthweight exerted strong effects at both the individual and population levels. Recurrence risk was highest for severe PPH. Occurrence and recurrence were lower in male fetuses, and the paternal influence was weak.

Metformin exposure, maternal PCOS status and fetal venous liver circulation: A randomized, placebo-controlled study.

BACKGROUND: Metformin is prescribed to women with polycystic ovary syndrome (PCOS) to prevent pregnancy complications. Children exposed to metformin vs. placebo in utero, have increased head circumference at birth and are more overweight and obese at 8 years of age. Also, maternal PCOS-status seems to alter the long-term cardio-metabolic health of offspring. We hypothesized that the long-term effects of metformin-exposure and/or maternal PCOS may be mediated by circulatory adaptations during fetal life. MATERIAL AND METHODS: This is a sub-study of a larger double-blinded, placebo-controlled trial, where women with PCOS were randomized to metformin (2g/day) or placebo in pregnancy, a total of 487 women. A sub-group of participants (N = 58) took part in this sub-study and had an extended ultrasound examination at gestational week 32, including blood flow velocity and diameter measurements of the umbilical vein (UV), the ductus venosus (DV) and the portal vein (PV). Blood flow volume was calculated and adjusted for estimated fetal weight (EFW) (normalized flow). Metformin exposed fetuses were compared to placebo exposed fetuses. Fetuses of mothers with PCOS (metformin [n = 30] and placebo [n = 28]) were compared to a low-risk reference population (N = 160) by z-score statistics. RESULTS: There was no difference in fetal liver flow between metformin vs. placebo-exposed fetuses. Fetuses of mothers with PCOS had higher EFW (0.63 [95% CI 0.44-0.83] p<0.001), lower normalized UV, DV, PV, and lower total venous liver blood flows than the reference population. CONCLUSION: Metformin during pregnancy did not affect fetal liver blood-flow. In our population, maternal PCOS-status was associated with reduced total venous liver blood-flow, which may explain altered growth and metabolism later in life.

Placental histology predicted adverse outcomes in extremely premature neonates in Norway-population-based study.

AIM: We evaluated the role of placental pathology in predicting adverse outcomes for neonates born extremely preterm (EPT) before 28 weeks of gestation. METHODS: This was a prospective observational study of 123 extremely preterm singletons born in a hospital in western Norway, and the placentas were classified according to the Amsterdam criteria. The associations between histologic chorioamnionitis (HCA), by the presence or the absence of a foetal inflammatory response (FIR+ or FIR-), maternal vascular malperfusion (MVM) as a whole and adverse neonatal outcomes were evaluated by logistic regression analyses. Adverse outcomes were defined as perinatal death, necrotising enterocolitis (NEC), bronchopulmonary dysplasia (BPD), brain pathology by magnetic resonance imaging at term-equivalent age, retinopathy of prematurity and early-onset neonatal sepsis. The results are reported as odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: HCA was associated with NEC (OR 12.2, 95% CI 1.1 to 137.1). HCA/FIR+ was associated with BPD (OR 14.9, 95% CI 1.8-122.3) and brain pathology (OR 9.8, 95% CI 1.4-71.6), but HCA/FIR- was not. The only neonatal outcome that MVM was associated with was low birthweight. CONCLUSION: Placental histology provided important information when assessing the risk of adverse neonatal outcomes following EPT birth.

Recurrence of postpartum hemorrhage in relatives: A population-based cohort study.

INTRODUCTION: Studies on the family aggregation of postpartum hemorrhage (PPH) are scarce and with inconsistent results, and to what extent current birthweight influences recurrence between relatives remains to be studied. Further, family aggregation of PPH has been studied from an individual, but not from a public heath perspective. We aimed to investigate family aggregation of PPH in Norway, how birthweight influences these effects, and to estimate the proportion of PPH cases attributable to a family history of PPH and current birthweight. MATERIAL AND METHODS: Using data from the Medical Birth Registry of Norway, Statistics Norway, and Central Population Registry of Norway we identified individuals as newborns, parents, grandparents, and full and half-siblings, and studied 1 002 687 mother-offspring, 841 164 father-offspring, and 761 011 both-parents-offspring pairs. We used multilevel logistic regression to calculate odds ratios (OR) with 95% CI. RESULTS: If the birth of the mother but not of the father involved PPH, then the OR of PPH (>500 mL) in the next generation was 1.44 (95% CI 1.39-1.49). If the birth of the father but not of the mother involved PPH, then OR was 1.12 (95% CI 1.08-1.16). These effects were stronger in severe PPH. Recurrence between siblings was highest between full sisters (OR 1.47, 95% CI 1.41-1.52), followed by maternal half-sisters, paternal half-sisters, and partners of full brothers. A family history of PPH or birthweight of 4000 g or more accounted for ≤5% and 15% of the total number of PPH cases, respectively. CONCLUSIONS: A history of PPH in relatives influenced the recurrence risk of PPH in a dose-response pattern consistent with the anticipated proportion of shared genes. The recurrence was highest through the maternal line.

Automated Clinical Grade Expansion of Regulatory T Cells in a Fully Closed System.

Adoptive transfer of T regulatory cells (Treg) has been successfully exploited in the context of graft-versus-host disease, transplantation, and autoimmune disease. For the majority of applications, clinical administration of Treg requires laborious ex vivo expansion and typically involves open handling for culture feeds and repetitive sampling. Here we show results from our approach to translate manual Treg manufacturing to the fully closed automated CliniMACS Prodigy® system reducing contamination risk, hands-on time, and quality variation from human intervention. Polyclonal Treg were isolated from total nucleated cells obtained through leukapheresis of healthy donors by CD8+ cell depletion and subsequent CD25high enrichment. Treg were expanded with the CliniMACS Prodigy® device using clinical-grade cell culture medium, rapamycin, IL-2, and αCD3/αCD28 beads for 13-14 days. We successfully integrated expansion bead removal and final formulation into the automated procedure, finalizing the process with a ready to use product for bedside transfusion. Automated Treg expansion was conducted in parallel to an established manual manufacturing process using G-Rex cell culture flasks. We could prove similar expansion kinetics leading to a cell yield of up to 2.12 × 109 cells with the CliniMACS Prodigy® and comparable product phenotype of >90% CD4+CD25highCD127lowFOXP3+ cells that had similar in vitro immunosuppressive function. Efficiency of expansion bead depletion was comparable to the CliniMACS® Plus system and the final ready-to-infuse product had phenotype stability and high vitality after overnight storage. We anticipate this newly developed closed system expansion approach to be a starting point for the development of enhanced throughput clinical scale Treg manufacture, and for safe automated generation of antigen-specific Treg grafted with a chimeric antigen receptor (CAR Treg).

The bispecific immunoligand ULBP2-aCEA redirects natural killer cells to tumor cells and reveals potent anti-tumor activity against colon carcinoma.

NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody-derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re-directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell-mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti-tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD45+ immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti-tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.

Rescue of impaired NK cell activity in hodgkin lymphoma with bispecific antibodies in vitro and in patients.

Natural killer (NK) cells represent a key component of the innate immune system against cancer. Nevertheless, malignant diseases arise in immunocompetent individuals despite tumor immunosurveillance. Hodgkin lymphoma (HL) is characterized by CD30(+) tumor cells and a massive infiltration of immune effector cells in affected lymph nodes. The latter obviously fail to eliminate the malignant cell population. Here, we tested for functional NK cell defects in HL and suggest an improvement of NK function by therapeutic means. We demonstrate that peripheral NK cells (pNK) from patients with HL fail to eliminate HL cell lines in ex vivo killing assays. Impaired NK cell function correlated with elevated serum levels of soluble ligands for NK cell receptors NKp30 (BAG6/BAT3) and NKG2D (MICA), factors known to constrict NK cell function. In vitro, NK cell cytotoxicity could be restored by an NKG2D/NKp30-independent bispecific antibody construct (CD30xCD16A). It artificially links the tumor receptor CD30 with the cytotoxicity NK cell receptor CD16A. Moreover, we observed that NK cells from patients treated with this construct were generally activated and displayed a restored cytotoxicity against HL target cells. These data suggest that reversible suppression of NK cell activity contributes to immune evasion in HL and can be antagonized therapeutically.

Soluble ligands for NK cell receptors promote evasion of chronic lymphocytic leukemia cells from NK cell anti-tumor activity.

Natural killer (NK) cells are a major component of the anti-tumor immune response. NK cell dysfunctions have been reported in various hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here we investigated the role of tumor cell-released soluble and exosomal ligands for NK cell receptors that modulate NK cell activity. Soluble CLL plasma factors suppressed NK cell cytotoxicity and down-regulated the surface receptors CD16 and CD56 on NK cells of healthy donors. The inhibition of NK cell cytotoxicity was attributed to the soluble ligand BAG6/BAT3 that engages the activating receptor NKp30 expressed on NK cells. Soluble BAG6 was detectable in the plasma of CLL patients, with the highest levels at the advanced disease stages. In contrast, NK cells were activated when BAG6 was presented on the surface of exosomes. The latter form was induced in non-CLL cells by cellular stress via an nSmase2-dependent pathway. Such cells were eliminated by lymphocytes in a xenograft tumor model in vivo. Here, exosomal BAG6 was essential for tumor cell killing because BAG6-deficient cells evaded immune detection. Taken together, the findings show that the dysregulated balance of exosomal vs soluble BAG6 expression may cause immune evasion of CLL cells.

A novel BAT3 sequence generated by alternative RNA splicing of exon 11B displays cell type-specific expression and impacts on subcellular localization.

BACKGROUND: The human lymphocyte antigen (HLA) encoded BAT3/BAG6 recently attracted interest as a regulator of protein targeting and degradation, a function that could be exerted in the cytosol and in the nucleus. The BAT3 gene was described to consist of 25 exons. Diversity of transcripts can be generated by alternative RNA splicing, which may control subcellular distribution of BAT3. METHODOLOGY/PRINCIPAL FINDINGS: By cDNA sequencing we identified a novel alternatively spliced sequence of the BAT3 gene located between exons 11 and 12, which was designated as exon 11B. Using PCR and colony hybridization we identified six cDNA variants, which were produced by RNA splicing of BAT3 exons 5, 11B and 24. In four examined cell types the content of BAT3 splice variants was examined. Most of the cDNA clones from monocyte-derived dendritic cells contain exon 11B, whereas this sequence was almost absent in the B lymphoma Raji. Exon 5 was detected in most and exon 24 in approximately half of the cDNA clones. The subcellular distribution of endogenous BAT3 largely correlates with a cell type specific splicing pattern. In cells transfected with BAT3 variants, full-length and Δ24 BAT3 displayed nearly exclusive nuclear staining, whereas variants deleted of exon 11B showed substantial cytosolic expression. We show here that BAT3 is mainly expressed in the cytosol of Raji cells, while other cell types displayed both cytosolic and nuclear staining. Export of BAT3 from the nucleus to the cytosol is inhibited by treatment with leptomycin B, indicating that the Crm1 pathway is involved. Nuclear expression of BAT3 containing exon 11B suggests that this sequence plays a role for nuclear retention of the protein. CONCLUSIONS/SIGNIFICANCE: Cell type-specific subcellular expression of BAT3 suggests distinct functions in the cytosol and in the nucleus. Differential expression of BAT3 variants may reconcile the multiple roles described for BAT3.