INTRODUCTION:Preeclampsia, one of the Great Obstetrical Syndromes, remains a major contributor to maternal and fetal morbidity worldwide. Its clinical heterogeneity, classically reflected in "early" versus "late" presentations, underscores the need for biologically grounded sub-classifications to guide diagnosis and management. OBJECTIVES:To characterize the cellular and molecular mechanisms underlying preeclampsia subtypes using advanced single-cell and single-nuclei transcriptomic approaches, and to evaluate their alignment with a proposed integrated model distinguishing Type I (placental dysfunction) from Type II (maternal cardiovascular maladaptation) preeclampsia. METHODS:We performed single-cell and single-nuclei RNA sequencing on approximately 90,000 placental cells, encompassing 46 distinct cell populations, including trophoblast, lymphoid, myeloid, stromal, and endothelial lineages. Comparative transcriptomic analyses were conducted between early-onset and late-onset preeclampsia and control pregnancies. RESULTS:Profound cellular dysregulation was identified in early-onset preeclampsia across multiple placental cell types, consistent with widespread placental dysfunction, angiogenic imbalance, inflammatory response and apoptosis. In contrast, late-onset preeclampsia exhibited comparatively subtle transcriptional changes, suggestive of a distinct pathophysiological process driven by maternal cardiovascular maladaptation, placental aging, cellular senescence and oxidative stress. These findings support the classification of preeclampsia into Type I (placental-dominant, early-onset) and Type II (maternal-dominant, late-onset) forms, each characterized by specific risk factors, biomarkers, and therapeutic targets. CONCLUSIONS:This placental molecular atlas integrates single-cell and single-nuclei transcriptomic data with clinical phenotypes, providing mechanistic support for the Type I and Type II preeclampsia framework. These insights inform the development of targeted interventions, including RNA-based therapies to modulate placental angiogenic pathways in early-onset disease, and maternal cardiovascular optimization strategies in late-onset disease.

INTRODUCTION:Early onset preeclampsia (EOPE), a complex complication of pregnancy, is a major contributor to maternal and fetal morbidity and mortality. The only symptom consistently observed in EOPE is new maternal hypertension. Other symptoms vary among patients, posing challenges for early detection. There are currently no clinically accepted molecular markers other than FLT1, which is highly upregulated and displays a small change in its splicing ratio in EOPE placentas. However, an exhaustive search for changes in transcript use in EOPE has not been performed. METHODS:Differential transcript usage (DTU) analysis was conducted between placental samples of women with and without EOPE for four public RNA sequencing datasets. To identify changes due to placental age, DTU events were compared to preterm placentas. Selected DTUs were experimentally validated in an independent cohort. RESULTS:We identified 43 DTU events between term-birth and EOPE placentas in more than one dataset, and attributed 3 of the 43 to placental age. The genes that carry those DTUs were enriched for cell junction and cell membrane-associated pathways, which were previously suggested to be altered in preeclampsia at the gene expression level, although the alteration was mediated by other genes. DISCUSSION:Alterations in transcript usage are part of the EOPE molecular mechanism. The validated DTUs in FLT4, ADGRG6, MXI1, and LCP1 may contribute to the development of EOPE. Surprisingly, the splicing change in FLT4 takes place in the intracellular domain, whereas the preeclampsia-associated changes in its paralog FLT1 are in the extracellular domain.

OBJECTIVE:Maternal diabetes increases offspring risk for neurodevelopmental disorders, but its association with delayed developmental milestones is unknown. We examined milestone attainment in offspring of mothers with gestational diabetes mellitus (GDM) and pregestational type 1 and type 2 diabetes compared with unexposed control individuals. RESEARCH DESIGN AND METHODS:We examined a nationwide retrospective cohort study of 466,462 term singleton infants born January 2018 to December 2021 using linked national maternal-child health clinic and hospital discharge data. Developmental data were obtained from nurse-administered structured assessments at routine well-child visits through 24 months of age. Adjusted hazard ratios (aHRs) and 95% CIs for failure to attain developmental milestones were estimated by diabetes type and infant sex. RESULTS:Pregestational diabetes was associated with increased risk for delayed milestone attainment in language, personal-social, and gross motor domains compared with control groups. Offspring of mothers with type 1 diabetes showed the highest risk, followed by those with type 2 diabetes. The association between type 1 diabetes and developmental delay was primarily driven by female offspring, with elevated risks in personal-social (aHR, 1.64; 95% CI, 1.14-2.36), language (aHR, 1.44; 95% CI, 1.09-1.89), and gross motor (aHR, 1.41; 95% CI, 1.05-1.89) domains. GDM was associated with modestly increased risk limited to the fine motor domain (aHR, 1.11; 95% CI, 1.06-1.16). CONCLUSIONS:Pregestational diabetes is associated with delayed milestone attainment, with risk varying by diabetes type, developmental domain, and infant sex. Further research should explore long-term trajectories to inform early intervention for children exposed to maternal diabetes.

Ferroptosis, a regulated form of iron-dependent cell death driven by lipid peroxidation, has recently gained attention as a potential contributor to the pathophysiology of preeclampsia, a major cause of maternal and perinatal morbidity and mortality. This review synthesizes current evidence linking ferroptosis to placental dysfunction and explores its relevance to the pathogenesis of preeclampsia. We examine how disrupted iron metabolism, oxidative stress, and impaired antioxidant defenses create a permissive environment for ferroptotic damage in the syncytiotrophoblast. Key biochemical pathways, including redox phospholipid metabolism, glutathione-GPX4 activity, and mitochondrial function, are reviewed in detail. We also discuss emerging data connecting placental ferroptosis to systemic maternal manifestations via sFlt-1 release and extracellular vesicle-mediated endothelial injury. Finally, we highlight therapeutic implications, including pharmacological inhibition of ferroptosis and drug repurposing strategies. By integrating diverse mechanistic insights, ferroptosis emerges as a unifying paradigm that may reshape our understanding and treatment of preeclampsia.

Objective The COVID-19 pandemic raised concerns regarding the effects of maternal SARS-CoV-2 infection during pregnancy on infant growth and neurodevelopment. Prior evidence has been inconsistent, limited by small sample sizes, short follow-up, and confounding by prematurity. We evaluated growth and developmental outcomes through 24 months among term-born children exposed in utero to maternal SARS-CoV-2 infection compared with unexposed controls. Methods We conducted a nationwide retrospective matched cohort study including 66,285 term infants born in Israel between March 2020 and March 2022. Maternal SARS-CoV-2 infection during pregnancy defined exposure, with exposed infants (n=22,096) matched to unexposed controls by delivery date. National registries provided standardized growth and developmental data. Outcomes included infant growth and attainment of 31 developmental milestones up to 24 months, analyzed using adjusted stratified Cox regression models. Results Infant growth trajectories, developmental milestone attainment, and referral rates were similar between exposed and unexposed children. Findings were consistent across sub-analyses by sex, trimester of infection, maternal disease severity, and during the pre-vaccine period. Conclusion This large nationwide study did not identify a significant association between maternal SARS-CoV-2 infection during pregnancy and early childhood growth or development among term neonates. Based on nationwide data with two-year follow-up, these findings offer reassuring evidence regarding outcomes.

Preeclampsia, one of the great obstetrical syndromes, manifests through diverse maternal and fetal complications and remains a leading contributor to adverse perinatal outcomes. In this review, we describe our work on single-cell and single-nuclei RNA sequencing to elucidate the molecular mechanisms that underlie early- and late-onset preeclampsia. Analysis of 46 cell types, encompassing approximately 90,000 cells from placental tissues collected after delivery, demonstrated cellular dysregulation in early-onset preeclampsia, whereas late-onset preeclampsia showed comparatively subtle changes. These findings were observed in all cell lines, including all types of trophoblast, lymphoid, myeloid, stromal, and endothelial cells. Key findings in early-onset preeclampsia included disrupted syncytiotrophoblast and extravillous trophoblast angiogenic signaling, characterized by an up-regulation of FLT1 and down-regulation of PGF, consistent with an angiogenic imbalance. The stromal and vascular compartments exhibited stress-induced transcriptomic shifts. Both endothelial cells and pericytes showed evidence of stress, including up-regulation of heat shock proteins and markers of apoptosis. In addition, the inflammation- and stress-responsive states were more abundant in early-onset preeclampsia than in matched controls. Inflammatory pathways were markedly up-regulated in both the maternal and fetal immune cells; for example, we observed a marked increase in pro-inflammatory cytokines, including secreted phosphoprotein 1 and C-X-C motif chemokine ligand 2 and 3. Conversely, late-onset preeclampsia retained adaptive placental features with localized dysregulation of extracellular matrix remodeling and angiogenic markers, underscoring its possible maternal cardiovascular etiology. Single-cell and single-nuclei RNA sequencing investigations of placental tissues support the proposed classification of preeclampsia into a placental dysfunction type, primarily presenting early in pregnancy, and a maternal cardiovascular maladaptation type, primarily presenting later in pregnancy, each with distinct biomarkers, risk factors, and therapeutic targets. The early-onset preeclampsia findings advocate for interventions that target angiogenic pathways, such as RNA-based therapies that target specific cells of the placenta, to modulate soluble fms-like tyrosine kinase-1 levels. In contrast, late-onset preeclampsia management may benefit from maternal cardiovascular optimization, including individualized antihypertensive and metabolic treatments. These results underscore the heterogeneity of preeclampsia, emphasizing the need for individualized diagnostic and therapeutic strategies. This molecular atlas of preeclampsia advances our understanding of the complex interplay among elements of the maternal-placental-fetal array, thereby bridging clinical phenotypes and cellular mechanisms. Future research should focus on integrating these insights into longitudinal studies to develop precision medicine approaches for preeclampsia to enhance outcomes for mothers and neonates.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation, has a key role in organ injury, degenerative disease, and vulnerability of therapy-resistant cancers. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we demonstrate that macrophages co-cultured with ferroptotic cancer cells from various types effectively mitigate cell death induced by GPX4 inhibitors (RSL3 and ML162), GPX4 silencing via shRNA, or the Xc- system inhibitor IKE. Furthermore, macrophages effectively reduced lipid peroxidation in ferroptotic cells. Importantly, macrophage function relies on direct cell-to-cell contact and is affected by their differentiation. Specifically, polarization into M1 macrophages, but not M2, greatly hinders their protective capabilities. Interestingly, unlike apoptotic cells, ferroptotic cells retain elevated levels of the 'don't eat me' signal, CD47, and conversely, fail to present the "eat me" signal phosphatidylserine (PS) on the outer layer of the plasma membrane, providing an opportunity for their rescue. Furthermore, in placental villi explants, macrophages protect trophoblasts from ferroptotic death. These results underscore the intricate interplay between ferroptotic cells and their microenvironment and provide compelling evidence of a yet-unrecognized anti-ferroptotic activity of macrophages as a cell-extrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

To investigate the impact of virtual reality (VR) exposure on anxiety levels in patients undergoing planned oocyte cryopreservation. Participants were randomized into a routine management group or a VR group. The VR group underwent a 20-min VR session featuring scenic movies before entering the operating room. All procedures were conducted under anesthesia. Demographic data collected included age, BMI, cycle number, expected and retrieved eggs, and whether patients were accompanied. The primary outcome was anxiety change, measured using the State-Trait Anxiety Inventory (STAI). A sample size of 12 patients per group was calculated to detect a 5-point reduction in STAI scores. Secondary measures included anxiety-related vital signs and visual analogue scale (VAS, 1–10). Data were collected at admission, pre-procedure, and post-recovery. Of the 30 patients recruited, 16 were assigned to routine management and 14 to the VR group. Baseline anxiety scores (S-STAI) of both groups were low and almost identical (VR group 24.8 ± 10.2, Routine management group 24.75 ± 11.1, p = 0.897). The VR group showed a significant reduction in STAI scores from admission to pre-procedure, while the routine group experienced an increase (p = 0.02). Systolic blood pressure decreased more in the VR group post-recovery (− 9.7 ± 12.3 mmHg vs. − 1.3 ± 13.1 mmHg, p = 0.08), although not reaching a statistical significance. VAS scores also showed a greater reduction in the VR group from admission to pre-procedure (p = 0.04). VR exposure before oocyte retrieval significantly reduced anxiety, as indicated by STAI scores, systolic blood pressure, and VAS scores. Clinical-Trials NCT06280222, Registered online 07.12.2023.

How can cfChIP-seq analysis be used to characterize functional gene expression signatures in follicular fluid and be used to assess ovarian function and oocyte competence? CfChIP-seq analysis revealed intact modified nucleosomes with distinct active promoters, including hallmarks of granulosa cells and signs of immune modulation in unexplained infertility samples. While follicular fluid analysis offers potential insights into oocyte quality, traditional approaches have been limited to proteomics, metabolomics, and hormone levels as well as quantitative cell-free DNA measurements. Cell-free chromatin immunoprecipitation followed by sequencing (cfChIP-seq) moves beyond standard DNA detection to reveal functional signatures of gene expression and active biological processes. An ongoing prospective clinical study in a single university-affiliated IVF unit. So far, over the past two years, 47 patients have been recruited. Patients included were either diagnosed with infertility or were patients performing oocyte cryopreservation from choice. Patients were approached before oocyte retrieval procedure and were asked to provide a 5-ml serum sample along with a uni-follicular sample of follicular fluid, collected after oocyte separation. The samples underwent cfChIP-seq assay, including immunoprecipitation to enrich nucleosomes with active chromatin marks, followed by DNA sequencing of these nucleosomes. Collected patient data included patient clinical characteristics and treatment details. We analyzed follicular fluid (FF) samples from 47 patients: 20 were presumably fertile (controls), 8 had polycystic ovarian syndrome (PCOS), 9 had unexplained infertility (UE), and 6 had diminished ovarian reserve (DOR). CfChIP-seq analysis revealed intact modified nucleosomes with distinct gene expression signatures. When comparing serum samples to FF, there was a unique signature pattern to FF. These active promoter patterns showed hallmarks of granulosa cells, including FSHR and HSPG2 (Perlecan) and even active promoters not previously reported in ovarian samples, such as GLP1R. Analysis of gene activation showed no remarkable alterations in PCOS and DOR, but UE demonstrated significant changes: SLC17A8 and GAB4 upregulated, ARHGEF35 and LIPF downregulated. Non-negative matrix factorization successfully distinguished between clinical conditions, revealing unique molecular signatures in patients with unexplained infertility. Furthermore, enrichment analysis revealed distinct patterns of immune modulation in UE samples. To the best of our knowledge, qualitative analysis of cell-free DNA was not previously done on follicular fluid, and we have yet to determine the clinical implications of our results, thus caution is mandatory. Since the sample size is limited, our findings require further validation. This study introduces a novel qualitative tool for analyzing follicular fluid. Future applications of this methodology could enable more personalized approaches to medical care tailored to specific patient populations. Yes

BACKGROUND:Preeclampsia is characterized by hypertension, proteinuria, and elevated antiangiogenic sFlt-1 (soluble fms-like tyrosine kinase-1) levels. Despite extensive research, mechanisms underlying sFlt-1 dysregulation remain unclear. This hypothesis-testing study investigated whether ferroptosis, a lipid peroxidation-driven cell death mechanism, contributes to preeclamptic placental pathogenesis and sFlt-1 release, and whether drug repurposing could identify novel therapeutic options. METHODS:We analyzed oxidized phosphatidylethanolamines in human preeclamptic and healthy placental tissues using redox phospholipidomics. In placental explants, we evaluated ferroptosis effects on sFlt-1 release using Ferrostatin-1 and deferoxamine as inhibitors. We screened 6520 drugs and compounds to identify effective ferroptosis inhibitors in primary trophoblasts. Statistical analyses used Student t test and 1-way ANOVA with multiple comparison corrections. RESULTS:Preeclamptic placentas showed significant accumulation of oxidized phosphatidylethanolamines compared with controls. Inducing ferroptosis in placental explants increased sFlt-1 release, while inhibition using Ferrostatin-1 and deferoxamine reduced sFlt-1 levels (P<0.01). Our screen identified dipyridamole and promethazine as potent ferroptosis inhibitors, reducing lipid peroxidation, preserving glutathione levels, and decreasing sFlt-1 release in preeclamptic explants. CONCLUSIONS:This study establishes placental ferroptosis as a key mechanism in early preeclampsia and demonstrates its direct link to sFlt-1 dysregulation. Our systematic drug screening approach identified approved drugs with antiferroptotic activity, suggesting a novel therapeutic strategy for preeclampsia management through drug repurposing. Further research is needed to establish optimal dosing and confirm efficacy in vivo.

Hybrid immunity, acquired through vaccination followed or preceded by a COVID-19 infection, elicits robust antibody augmentation. We hypothesize that maternal hybrid immunity will provide greater infant protection than other forms of COVID-19 immunity in the first 6 months of life. We conducted a case-control study in Israel, enrolling 661 infants up to 6 months of age, hospitalized with COVID-19 (cases) and 59,460 age-matched non-hospitalized infants (controls) between August 24, 2021, and March 15, 2022. Infants were grouped by maternal immunity status at delivery: Naïve (never vaccinated or tested positive, reference group), Hybrid-immunity (vaccinated and tested positive), Natural-immunity (tested positive before or during the study period), Full-vaccination (two-shot regimen plus 1 booster), and Partial-vaccination (less than full three shot regimen). Applying Cox proportional hazards models to estimate the hazard ratios, which was then converted to percent vaccine effectiveness, and using the Naïve group as the reference, maternal hybrid-immunity provided the highest protection (84% [95% CI 75-90]), followed by full-vaccination (66% [95% CI 56-74]), natural-immunity (56% [95% CI 39-68]), and partial-vaccination (29% [95% CI 15-41]). Maternal hybrid-immunity was associated with a reduced risk of infant hospitalization for Covid-19, as compared to natural-immunity, regardless of exposure timing or sequence. These findings emphasize the benefits of vaccinating previously infected individuals during pregnancy to reduce COVID-19 hospitalizations in early infancy.

(Abstracted from Nat Commun 2024;15:2846) Infants with COVID-19, especially the Omicron variant, are at increased risk of acute respiratory failure and hospitalization. Pfizer and Moderna mRNA vaccines significantly reduce this risk but are not approved for infants under 6 months.

The vast majority of membrane phospholipids (PLs) include two asymmetrically positioned fatty acyls: oxidizable polyunsaturated fatty acids (PUFA) attached predominantly at the sn2 position, and non-oxidizable saturated/monounsaturated acids (SFA/MUFA) localized at the sn1 position. The peroxidation of PUFA-PLs, particularly sn2-arachidonoyl(AA)- and sn2-adrenoyl(AdA)-containing phosphatidylethanolamines (PE), has been associated with the execution of ferroptosis, a program of regulated cell death. There is a minor subpopulation (≈1–2 mol %) of doubly PUFA-acylated phospholipids (di-PUFA-PLs) whose role in ferroptosis remains enigmatic. Here we report that 15-lipoxygenase (15LOX) exhibits unexpectedly high pro-ferroptotic peroxidation activity towards di-PUFA-PEs. We revealed that peroxidation of several molecular species of di-PUFA-PEs occurred early in ferroptosis. Ferrostatin-1, a typical ferroptosis inhibitor, effectively prevented peroxidation of di-PUFA-PEs. Furthermore, co-incubation of cells with di-AA-PE and 15LOX produced PUFA-PE peroxidation and induced ferroptotic death. The decreased contents of di-PUFA-PEs in ACSL4 KO A375 cells was associated with lower levels of di-PUFA-PE peroxidation and enhanced resistance to ferroptosis. Thus, di-PUFA-PE species are newly identified phospholipid peroxidation substrates and regulators of ferroptosis, representing a promising therapeutic target for many diseases related to ferroptotic death.