Radiotherapy is a cornerstone of cancer treatment; however, its effects on healthy ovarian somatic cells remain largely unexplored. This study addresses this gap by investigating how human cortical and medullary primary ovarian cells (cPOCs and mPOCs, respectively) respond to acute, high-dose X-ray exposure in vitro. Ovarian tissue was obtained from eight patients (aged 23–36 years) undergoing gender-affirming surgery at Karolinska University Hospital in Huddinge, Sweden. The tissue was separated into cortex and medulla and dissociated into cPOCs and mPOCs. Monolayer cultures of cPOCs and mPOCs were exposed to 10 Gy X-rays upon reaching confluency, or left unexposed as paired controls. Following irradiation, cells were assessed for ATP content and mitochondrial dehydrogenase activity, followed by immunofluorescence staining, bulk RNA sequencing (Illumina Stranded mRNA Prep Ligation protocol; sequencing on the Illumina NovaSeq 6000 platform), bulk proteomic analysis (liquid chromatography–tandem mass spectrometry), and a functional assay for assessing their ability to form 3D Silk-Ovarioids. While irradiation did not significantly affect cell viability, immunofluorescence analyses revealed alterations in DNA damage response, apoptosis, and cell cycle regulation. Transcriptomic analysis showed minimal changes at 1 h post-irradiation in both cPOCs and mPOCs. However, marked shifts in transcriptomic profiles were observed at 4 h (2,810 and 2,540 DEGs in cPOCs and mPOCs, respectively) and at 24 h (2,462 and 2,802 DEGs, respectively), including upregulation of the p53 pathway and downregulation of MYC targets, E2F targets, the G2/M checkpoint, and the mTORC1 pathway. At the proteomic level, differentially expressed proteins associated with cell adhesion, focal adhesion, and cadherin binding were detected at 24 h post-irradiation. Functionally, irradiated cells demonstrated an impaired capacity to self-organize into 3D Silk-Ovarioids, indicating compromised cell–cell adhesion. These findings reveal a novel mechanism by which radiotherapy may damage ovarian tissue independently of follicular loss, underscoring the need for targeted strategies to preserve somatic cell function in fertility preservation protocols.

Cohesin is a DNA tethering complex essential for chromosome structure and function. In fission yeast, defects in the cohesin loader Mis4 result in chromosome segregation defects and dysregulated expression of genes near chromosome ends. A genetic screen for suppressors of the thermosensitive growth defect of mis4-G1487D identified several hypomorphic mutants of the Target of Rapamycin Complex 1 (TORC1), a conserved kinase that integrates cellular signals to regulate growth and metabolism through substrate-specific phosphorylation. Here, we demonstrate that the TORC1 pathway modulates cohesin functions in chromosome segregation and gene expression. In the context of compromised cohesin loading, the incidence of chromosome segregation defects was modulated by the growth medium in a TORC1-dependent manner. Pharmacological or genetic down-regulation of TORC1 activity restored cohesin binding to its chromosomal sites and improved mitotic chromosome segregation. Notably, reduced TORC1 activity also increased cohesin binding and chromosome transmission fidelity in wild-type cells. These results suggest that environmental cues influence chromosome stability via TORC1. Biochemically, TORC1 co-purified with cohesin and reduced TORC1 activity correlated with decreased phosphorylation of specific residues on Mis4 and cohesin. Mutations in cohesin that mimic the non-phosphorylated state mirrored the effects of TORC1 downregulation, showing that TORC1 is part of the network that controls cohesin phosphorylation to modulate its functions. Finally, we show that the functional interaction between TORC1 and Mis4 extends to the regulation of stress-responsive genes. Our findings reveal a TORC1-cohesin link that may facilitate cellular adaptation to environmental changes. Given that TORC1 inhibitors and calorie restriction extend lifespan in diverse species, this connection raises the intriguing possibility that cohesin-mediated changes in chromosome structure contribute to these effects.

ABSTRACT Ovarian tissue cryopreservation enables fertility preservation in females undergoing gonadotoxic therapies, restoring fertility in adults. Although offered even before puberty, the childhood ovary and its vulnerability to therapy remain poorly characterized. Here, ovarian tissue from 16 patients undergoing fertility preservation (aged 1-16 years) and 11 adult controls (aged 22-32 years) was analyzed using single-cell RNA sequencing, spatial transcriptomics, and multiplex immunostaining. In chemotherapy-naïve samples, 13 somatic cell populations underwent extracellular matrix remodeling, vascular, neural, and stromal maturation during puberty, whereas changes in germline related to chromatin remodeling. Spatial transcriptomics resolved 23 clusters across, revealing distinct tissue organization and follicular niche composition between children and adults. Chemotherapy exposure depleted perifollicular and vascular cells, suppressed intercellular signaling, and dysregulated over half of puberty-associated genes, converging on stress responses and extracellular matrix remodeling, with SEPTIN7 as a potential biomarker. These findings uncover critical developmental vulnerabilities of the pediatric ovary relevant to fertility preservation.

STUDY QUESTION: How do human cortical (cPOCs) and medullary (mPOCs) primary ovarian cells respond to acute X-ray exposure? SUMMARY ANSWER: Acute high-dose X-ray exposure causes a shift in cPOCs and mPOCs transcriptomic profiles and impairs significantly their cell-cell adhesion ability. WHAT IS KNOWN ALREADY: Radiotherapy is a leading cancer treatment, due to its effectiveness in targeting malignant cells. However, it can also affect healthy cells, potentially causing organ dysfunction, among which ovaries. When targeted radiotherapy is not feasible, fertility preservation is recommended to avoid premature ovarian insufficiency. In addition, the effects of irradiation on ovarian somatic cells remain poorly understood. STUDY DESIGN, SIZE, DURATION: Ovarian tissue was obtained from patients undergoing gender-affirming surgery at Karolinska University Hospital Huddinge, Sweden. The ovarian tissue was separated into cortex and medulla, then individually dissociated into single-cell suspensions using mechanical and enzymatic methods. Monolayer cultures from cPOCs and mPOCs were exposed to a single dose of 10 Gy X-ray irradiation or left unexposed as paired controls. Following irradiation, the cells were cultured at various time-points for further molecular and morphological evaluation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue from 8 patients (age 23-36 years) was used. Dissociated cPOCs and mPOCs were cultured to 80% confluence and irradiated with 10 Gy (1.33 Gy/min), with non-irradiated controls. Cellular ATP and mitochondrial activity were assessed, followed by immunofluorescence staining for canonical irradiation-induced effects in cells: DNA damage, apoptosis and cell cycle progression. Bulk RNA-sequencing was performed on controls and irradiated samples. Libraries were prepared using the Illumina Stranded mRNA Prep Ligation protocol and sequenced on Illumina NovaSeq6000 platform. Genes were considered to be differentially expressed under the cut-off of false discovery rate (FDR) < 0.05. Subsequently, affected biological pathway was predicted using all expressed genes ranked by log2 fold change again hallmark gene sets. To further investigate the potential upstream regulators, transcription factor enrichment analysis were performed based on DEGs. To assess changes at protein level, we mapped the proteomic profile using liquid chromatography-tandem mass spectrometry. Peptides were considered to be differentially expressed (DEPs) under the cut-off of p-value < 0.01. Finally, we measured the ability of cPOCs and mPOCs to form 3D aggregates after seeding irradiated and non-irradiated cells on Biosilk scaffolds. MAIN RESULTS AND THE ROLE OF CHANCE: Following irradiation, ATP levels and mitochondrial activity in cPOCs and mPOCs were comparable to controls, indicating minimal irradiation impact on cell viability and proliferation. Immunofluorescence analysis confirmed the modulation of canonical pathways, such as DNA damage, apoptosis and cell cycle in both cPOCs and mPOCs. Transcriptomic analysis showed that cPOCs and mPOCs at 1 h post-irradiation clustered together with the related 1 h control. However, a shift in transcriptomic profile was observed after 4 h and even more after 24 h post-irradiation in both cPOCs and mPOCs. Gene set enrichment analysis (GSEA) indicated upregulation of the p53 pathway at 4 h and 24 h post-irradiation, alongside downregulation of MYC targets, E2F targets, the G2/M checkpoint and mTORC1 pathway. Gene pattern analysis showed irradiation-dependent trends related to extracellular matrix (ECM) organisation, p53-mediated apoptotic mechanisms and chromosome segregation during the 24 h period following irradiation. Additionally, transcription factor enrichment analysis based on DEGs suggested p53 and MYC as potential upstream regulators. On a proteomic level, DEPs associated with ECM organisation and cytoskeleton formation were detected at 4 and 24 h post-irradiation. Finally, X-ray exposure hindered the cell-cell adhesion ability of both cPOCs and mPOCs, leading to impaired formation of Silk-Ovarioids. LARGE SCALE DATA: The RNA sequencing count matrix is deposited in Gene Expression Omnibus (GEO) with accession number GSE291604. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD061796. The code used for the analysis can be found at https://github.com/tialiv/X-Ovary. LIMITATIONS, REASONS FOR CAUTION: The ovarian tissue was obtained from gender-affirming surgery patients who received androgen treatment before removal. Even though unlikely, this hormonal treatment might influence ovarian environment and alter cellular response to irradiation. Additionally, in this study the impact of X-ray exposure was assessed on a monolayer cell model, thus limiting the extrapolation power of our results to ovary in vivo. Lastly, the impact of irradiation was focused on the somatic cell populations that are essential for ovarian function. Further studies are needed to investigate the effects of X-ray exposure on ovarian follicles and their function. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the roles of MYC, p53 and cell adhesion factors in response to irradiation could guide the development of future ovarian protective strategies. These findings lay the foundation for further studies on ovarian tissue protection and fertility preservation in cancer patients. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the European Union's HORIZON 2020 research and innovation programme (MATER) under the Marie Sklodowska-Curie Actions (grant agreement No: 813707), the Estonian Research Council (grants PRG1076 and PSG608), the Orion Research Foundation sr personal grant, the Research grant from the Center for Innovative Medicine (CIMED) and the Karolinska Institutet Consolidator Grant. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACTProblemRecurrent vulvovaginal candidiasis (RVVC) affects 5%−10% of all women, negatively impacting their reproductive health and quality of life. Herein, we investigated the molecular effects of RVVC on the vaginal mucosa of otherwise healthy women.Method of StudyGene expression analysis was performed on vaginal tissue biopsies from women with RVVC, including those with a current episode of vulvovaginal candidiasis (VVC, n = 19) and women between infections (culture negative RVVC [CNR], n = 8); women asymptomatically colonized with Candida albicans (asymptomatic [AS], n = 7); and healthy controls (n = 18). Gene expression profiles were compared between groups and correlated with clinical data retrieved from questionnaires and gynecologic examinations.ResultsOf 20 171 genes identified in vaginal biopsies, 6506 were differentially expressed in the RVVC group, compared to healthy controls. Gene expression pathway analysis revealed an association between RVVC and pathways of inflammatory responses, especially genes involved in neutrophil recruitment and activation. Expression of genes involved in inflammation and neutrophil recruitment increased with increasing clinical severity of VVC, whereas expression of some genes involved in epithelial integrity decreased with increasing clinical severity of infection. Gene expression profiles of both the CNR and AS groups were comparable to those of healthy controls.ConclusionsThe clinical severity of RVVC during active infection correlates with increased expression of genes involved in molecular inflammation and neutrophil activation in the vaginal mucosa. The lack of differences between healthy controls and women with RVVC who were between acute infections indicates that the molecular effects observed in the RVVC group are only present during active infection.

Schizophrenia (SCZ) displays a heterogeneous etiology. In about half of the patients, first-episode psychosis (FEP) associates with increased levels of striatal dopamine (DA), while cognitive and negative symptoms associate with reduced DA function in the prefrontal cortex (PFC). However, the mechanism underlying these changes in DA is unclear. Abnormal increase in glial cell-line derived neurotrophic factor (GDNF) leads to similar SCZ-like symptoms in mice, but its relevance in patients has remained uncertain. Here, we use omics to examine three patient cohorts and identify a subgroup of patients with elevated GDNF. High GDNF associates with increased DA in cerebrospinal fluid, reduced DA-related PFC gene expression, aggravated negative SCZ symptoms, and a specific serum proteomics pattern. Post-mortem analysis revealed a GDNF-high specific striatal gene expression with significant overlap between mice and patients. Collectively, our data suggest a mechanism for DA imbalance in a subgroup of patients with SCZ. ### Competing Interest Statement EW was invited to advisory boards from Recordati, Teva and Boehringer Ingelheim. PF received speaker fees by Boehringer-Ingelheim, Janssen, Otsuka, Lundbeck, Recordati, and Richter and was a member of advisory boards of these companies and Rovi. The other authors declare no competing interests. ### Funding Statement During this study, JOA, IR and AD have been supported by Center of Innovative Medicine (CIMED), Hjärnfonden, Team Rynkeby-God Morgon Skolloppet, Åhlén-stiftelsen, Swedish Research Council (grants no. 2019- 01578 and 2022-01093). In addition, JOA and AM have been funded by the Academy of Finland (grants no. 297727, JOA and 350678, JOA), Sigrid Juselius Foundation, ERA-NET NEURON grant nr 352077, JOA and AM, Helsinki Institute of Life Science Research Fellow, JAES Foundation grant no. 240034, JOA, and by HORIZON-RIA grant nr 101188432 DTRIP4H, to JOA. DRG has been funded by University of Helsinki Doctoral Programme Brain and Mind. Furthermore, this research was supported by the Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung (BMBF)) with the EraNet project GDNF UpReg (01EW2206) to PF, AS, and VY. The study was endorsed by the BMBF within the initial phase of the German Center for Mental Health (DZPG) (grant: 01EE2303A, 01EE2303F to PF, AS). The study was funded by the Supplement to BMBF funding for the German Centre for Mental Health (DZPG) by the Bavarian State Ministry for Science and the Arts with the Grant for the research project "Improving Infrastructures for DZPG and NAKO Cohorts" to PF. The study was funded by the EU HORIZON-INFRA-2024-TECH-01-04 project DTRIP4H 101188432 to PF, AS and FR. VY was supported by the Residency/PhD track of the International Max Planck Research School for Translational Psychiatry (IMPRS-TP). VY was supported by the Faculty of Medicine at LMU Munich (FöFoLe Reg.-Nr. 1226/2024). JM was supported by the Faculty of Medicine at LMU Munich (FöFoLe Reg.-Nr. 1167). FJR received funding from the Pesl-Alzheimer-Stiftung (2024-2025), from the Lisa Oehler-Stiftung (2022-2024) and from the Verum-Stiftung (2024-2025). C.M.S (KaSP cohort) was supported by grants from Erling Persson Family Foundation, Swedish Research Council (2023-02827), Swedish Society for Medical Research (CG-23-0298-B), and Region Stockholm (FoUI-986509). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Stockholm Regional Ethics Committee of Swedish Ethical Review Authority gave ethical approval for this work. (Dnr 2010/879-31/1) The ethical committee of LMU Munich gave ethical approval of this work. (reference numbers 21-1139 and 21-0183) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The code used for the analysis is available upon request. CSF from LMU is available upon reasonable request and will require MTA/DTA agreements.

STUDY QUESTION:What is the best protocol to establish a long-term stable three-dimensional (3D) model for human primary ovarian cells? SUMMARY ANSWER:We developed and characterized long-term cultured 3D models of primary ovarian somatic cells isolated from adult tissues, using Biosilk as a scaffold. WHAT IS KNOWN ALREADY:In vitro models that mimic ovaries are crucial for elucidating the biological mechanisms underlying follicle activation and growth, hormonal activity, ovarian angiogenesis, damage in response to toxic exposures, and other biological mechanisms that enable the functionality of this complex organ. Three-dimensional systems are particularly relevant because they replicate heterogeneity and cell-cell communication among different ovarian cell types. However, complex models using human ovarian primary cells are yet to be developed. STUDY DESIGN SIZE DURATION:Ovarian tissue samples were collected from five patients (age 26 ± 5 years) who underwent gender-affirming surgery. The cortex and medulla were separated and dissociated into single-cell suspensions using mechanical and enzymatic methods. Three approaches were tested to establish a 3D model culture system: matrix-free ovarian spheroids (MFOS), a Matrigel-based three-layer gradient system (3LGS), and Biosilk scaffolds (Silk-Ovarioid). In parallel, paired controls from each patient and ovarian area were cultured in a standard 2D system for the same duration. PARTICIPANTS/MATERIALS SETTING METHODS:The 3D culture systems were monitored every second day to detect signs of aggregation and growth. Freshly fixed tissue, as well as 2D- and 3D-cultured samples were further processed for transcriptomic profiling after 42 days of culture using RNA sequencing. The culture of the 3D system was further characterized, regarding its protein profile and steroid and cytokine production, through proteomics and liquid chromatography-tandem mass spectrometry and the Luminex platform, respectively. The key findings from the high-throughput assays were finally validated through RNA fluorescent in situ hybridization (RNA-FISH) and immunofluorescence staining. MAIN RESULTS AND THE ROLE OF CHANCE:The 3D model systems MFOS (n = 120) and 3LGS (n = 18) failed to form aggregates capable of long-term maintenance in culture (MFOS: maximum of 15 days for both cortex and medulla; 3LGS: maximum of 11 days for medulla only). In contrast, we successfully established ovarian cortex- and medulla-derived 3D systems using Biosilk, termed Silk-Ovarioids (n = 120). Silk-Ovarioids were maintained for up to 42 days as free-floating culture without any signs of cell death, as confirmed by the absence of TUNEL, γ-H2A.X, and cleaved caspase 3 fluorescent signals. The presence of key ovarian somatic cell types, including granulosa, stromal, endothelial, and perivascular cells, was confirmed by transcriptomics and proteomics in the majority of Silk-Ovarioids. Validation through RNA-FISH and immunostaining was performed using the following markers: AMHR2 for granulosa cells, PDGFRα for stromal cells, CLDN5 and GPIHBP1 for endothelial cells, GJA4/Cx37 and MCAM for perivascular cells. Notably, Silk-Ovarioids exhibited the formation of a pro-angiogenic hypoxic core, as evidenced by the transcriptomic and proteomic data and visualized by the expression of hypoxia markers MMP2 and PDGFRβ. This hypoxic environment led to development of vessel-like structures after 4-6 weeks of culture, which were positive for the angiogenic markers TGFBR2, BMP2, and PDGFα. The functionality of Silk-Ovarioids was further confirmed by the identification of de novo extracellular matrix secretion (Col1α1 and Lamα1), and by the detection of pro-angiogenic cytokines (e.g. IL-6, IL-8, and GM-CSF) and steroids (e.g. pregnenolone and epitestosterone) in the culture media. LARGE SCALE DATA:The RNA-sequencing count matrix is deposited in Gene Expression Omnibus with accession number GSE253571. Raw data are deposited in Swedish National Data Service with the DOI https://doi.org/10.48723/h8cm-bs19. Single-cell RNA-seq data have been downloaded from the ArrayExpress database at EMBL-EBI with the accession codes 'E-MTAb - 8381'. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048710. The code used for the analysis can be found in https://github.com/tialiv/Silk-Ovarioid_project. LIMITATIONS REASONS FOR CAUTION:The ovarian samples were collected from patients undergoing androgen treatment, raising the concern that androgen exposure may alter the behavior of cells in Silk-Ovarioids compared to those derived from androgen-unstimulated patients. Furthermore, the cell culture media used in this study were supplemented with fetal bovine serum and did not contain any supplements or growth factors that could be essential for the resemblance of Silk-Ovarioids to the tissue of origin. WIDER IMPLICATIONS OF THE FINDINGS:The Silk-Ovarioids exhibited low intra-batch variability and long-term culture stability, underscoring their potential as a robust step toward developing a bioengineered, patient-specific artificial ovary. In addition, Silk-Ovarioids could be utilized as the first ovarian angiogenesis in vitro model, function as biological scaffold for in vitro folliculogenesis, and be used for toxicological and pharmacological studies targeting the ovaries. STUDY FUNDING/COMPETING INTERESTS:This study was funded by: a research grant from the Center for Innovative Medicine (CIMED) at Karolinska Insitutet; European Union's Horizon 2020 Research and Innovation Programme (project ERIN no. 952516); a Horizon Europe grant (NESTOR, grant no. 101120075) of the European Commission; the Swedish Research Council for Sustainable Development FORMAS (2018-02280, 2020-01621); StratRegen Funding from Karolinska Institute, Swedish Research Council VR (grant no. 2020-02132); Swedish Childhood Cancer Fund (Reference PR2017-0044, PR2020-0096); Estonian Research Council (grant no. PRG1076); Swedish Research Council (grant no. 2024-02530); Novo Nordisk Foundation (grant no. NNF24OC0092384); European Union's H2020 project Sinfonia (no. 857253) (INL research); and SbDToolBox, with reference NORTE-01-0145-FEDER-000047, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (INL research). The authors have no conflicts of interest to declare.

Spaceflight negatively impacts skeletal muscle mass and function, and current countermeasures fail to completely offset those alterations. Innovative approaches are then needed to understand the mechanisms governing space-induced muscle changes. This study explores short-term microgravity (suborbital flight) effects on transcriptional profile of skeletal muscle mesenchymal cells and intramuscular adipose tissue. We identified a set of molecular factors and biological processes impacted by microgravity in these cell types, warranting further investigation.

Precision medicine, which tailors strategies to individual tumor characteristics, is transforming cancer treatment. Immunotherapy, targeting immune suppression in the tumor microenvironment (TME) to activate cytotoxic immune cells, shows great promise. However, sex-specific immune responses in the TME are poorly understood, partly due to the underrepresentation of women in clinical trials, leading to generalized treatments that overlook key differences. To explore sex-specific differences driving immunosuppression and tumor progression, we analyzed data from 172 pancreatic ductal adenocarcinoma (PDAC) patients using bulk and single-cell transcriptomics, proteomics, and functional assays. We identified a macrophage subpopulation linked to immune-exclusive tumor phenotypes, immune cell dysfunction, and metastasis, driven by the G-protein coupled receptor FPR2. FPR2 expression correlated with poor outcomes in females but not males, underscoring sex-specific TME influences. Single-cell analyses revealed distinct immune landscapes: females had reduced NK cells and increased Th17 cells, while males exhibited reduced Th1 cells. Chromosomal region differences extended beyond sex chromosomes. These findings reveal critical sex-based discrepancies shaping the TME, highlighting the need for tailored therapies. We are developing immunotherapy antibodies for women with PDAC, advancing “genderized medicine” to improve outcomes through personalized, sex-specific strategies. Swedish Cancer Society Karolinska Institutets funds Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

The Target of Rapamycin Complex 1 (TORC1) integrates cellular cues and adapts cell growth and metabolism through substrate-specific phosphorylation. A genetic screen for suppressors of a conditional mutant of the cohesin loader Mis4 identified hypomorphic mutants of TORC1. Downregulation of TORC1 enhanced the binding of cohesin and its loader to their regular sites on chromosomes. In the context of impaired cohesin loading, TORC1 downregulation rescued chromosome segregation whereas upregulation had the opposite effect, suggesting environmental cues impinge on the robustness of chromosome segregation. TORC1 co-purified with cohesin from cellular extracts and the phosphorylation level of specific residues on Mis4 and cohesin were reduced in TORC1 mutants. Cohesin mutations mimicking the non-phosphorylated state mirrored the effects of TORC1 downregulation. Challenging cells with various conditions revealed that Mis4 and TORC1 regulate a common set of genes involved in the response to environmental changes. These genes are preferentially located far from centromeres and close to telomeres. We propose that cohesin is an effector of TORC1, orchestrating alterations in chromosome structure that facilitate cellular adaptation to environmental changes. ### Competing Interest Statement The authors have declared no competing interest.

Abstract: Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, responsible for approximately 466,000 deaths globally in 2020. Its incidence increases by about 1% annually, with a higher occurrence in males than females. While differences in immune responses and tumor biology between sexes have been explored, the role of the microbiome in gender-specific PDAC progression is still unclear. Investigating these differences could offer crucial insights for personalized treatment strategies for males and females. Methods: This study reanalyzed oral and gut microbiome data from BioProject: PRJNA832909, comprising 191 samples from PDAC patients and healthy controls. Using shotgun metagenomic sequencing, we examined gender-specific bacterial signatures. Alpha diversity (richness) and beta diversity (community composition) were analyzed. Differentially abundant bacterial taxa were identified via LEfSe, and gender-specific bacterial panels were validated using CombiROC. Results: Alpha diversity analysis revealed significant differences in microbial richness, particularly between male and female PDAC patients and their healthy controls. Beta diversity demonstrated distinct microbial shifts between the PDAC and control groups across genders. LEfSe identified several pathogenic bacteria contributing to gender-specific dysbiosis, including Streptococcus, Fusobacterium, and Prevotella. Shared and sex-specific bacterial species in PDAC were highlighted through Venn diagram analysis. CombiROC validated the predictive ability of these bacterial markers, with AUC values exceeding 0.90 for both sexes. Conclusion: This study uncovered gender-specific microbial patterns in PDAC patients, potentially influenced by sex-specific immune responses. These findings provide important insights into the progression of PDAC and support sex-targeted diagnostic and therapeutic interventions. ### Competing Interest Statement The authors have declared no competing interest.

Cognitive functions, neuropsychiatric disorders and behaviors from feeding to mood relate to the serotonin (5-hydroxytryptamine; 5-HT) system. We report that expression of glial cell line-derived neurotrophic factor (GDNF), a known potent stimulator of the brain dopamine system, correlates with serotonergic markers in humans, and increased GDNF defines a subset of psychiatric patients with a characteristic 5-HT-related gene expression pattern. A similar ~1.5- to 2-fold upregulation of endogenous GDNF expression in mice increases brain 5-HT levels and function, both developmentally and during adulthood, and modulates response to fluoxetine. Notably, increasing GDNF more than approximately 2-fold does not increase 5-HT further and instead produces an inverted U- shaped curve of 5-HT levels, suggesting why the GDNF/5-HT correlation has remained controversial. Collectively, our data indicate that GDNF levels fine-tune 5-HT system development and adult function while excess GDNF exclusively associates with neuropsychiatric illness, making it an important target for future research. ### Competing Interest Statement The authors have declared no competing interest.

In vitro models that mimic ovaries are crucial for elucidating the biological mechanisms underlying follicle activation and growth. Three-dimensional (3D) systems are particularly relevant because they can replicate the heterogeneity and cell-cell communication between different ovarian cell types. However, complex models using human ovarian primary cells have not yet been established. In this study, we developed and characterized long-term cultured 3D models of primary ovarian somatic cells isolated from adult tissues, using Biosilk as a scaffold. We successfully established both ovarian cortex- and medulla-derived 3D systems, termed Silk-Ovarioids. The presence of key ovarian somatic cell types - including granulosa, stromal, endothelial, and perivascular cells - was confirmed by transcriptomics, proteomics, and immunostaining. Notably, Silk-Ovarioids exhibited the formation of a pro-angiogenic hypoxic core, as evidenced by the development of vessel-like structures after six weeks of culture. The Silk-Ovarioids demonstrated low intra-batch variability and long-term culture stability, underscoring their potential as a robust step towards creating a bioengineered, patient-specific artificial ovary.

Phthalates are found in everyday items like plastics and personal care products. There is an increasing concern that continuous exposure can adversely affect female fertility. However, experimental data are lacking to establish causal links between exposure and disease in humans. To address this gap, we tested the effects of a common phthalate metabolite, mono-(2-ethylhexyl) phthalate (MEHP), on adult human ovaries in vitro using an epidemiologically determined human-relevant concentration range (2.05nM – 20.51mM). Histomorphological assessments, steroid and cytokine measurements were performed on human ovarian tissue exposed to MEHP for 7 days in vitro. Cell viability and gene expression profile were investigated following 7 days of MEHP exposure using the human granulosa cancer cell lines (KGN, COV434), the germline tumor cell line (PA-1), and human ovarian primary cells. Selected differentially expressed genes (DEGs) were validated by RT-qPCR and immunofluorescence in human ovarian tissue. MEHP exposure reduced follicular growth (20.51nM) and increased follicular degeneration (20.51mM) in ovarian tissue, while not affecting steroid and cytokine production. Out of the 691 unique DEGs identified across all the cell types and concentrations, CSRP2 involved in cytoskeleton organization and YWHAE as well as CTNNB1 involved in the Hippo pathway, were chosen for further validation. CSRP2 was upregulated and CTNNB1 downregulated in both ovarian tissue and cells, whereas YWHAE was downregulated in cells only. In summary, one-week MEHP exposure of human ovarian tissue can perturb the development and survival of human follicles through mechanisms likely involving dysregulation of cytoskeleton organization and Hippo pathway.

Phthalates are endocrine disrupting chemicals (EDCs) found in common consumer products such as soft plastics and cosmetics. Although the knowledge regarding the adverse effects of phthalates on female fertility are accumulating, information on the hormone sensitive endometrium is still scarce. Here, we studied the effects of phthalates on endometrial cell proliferation and gene expression. Human endometrial primary epithelial and stromal cells were isolated from healthy fertile-aged women (n=3), and were compared to endometrial cell lines T-HESC and Ishikawa. Three different epidemiologically relevant phthalate mixtures were used, defined by urine samples in the Midlife Women Health Study (MWHS) cohort. Mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) was used as a single phthalate control. Cells were harvested for proliferation testing and transcriptomic analyses after 24 h exposure. Even though all cell models responded differently to the phthalate exposures, many overlapping differentially expressed genes (DEGs, FDR<0.1), related to cell adhesion, cytoskeleton and mitochondria were found in all cell types. The qPCR analysis confirmed that MEHHP significantly affected cell adhesion gene vinculin (VCL) and NADH:ubiquinone oxidoreductase subunit B7 (NDUFB7), important for oxidative phosphorylation. Benchmark dose modelling showed that MEHHP had significant concentration-dependent effects on cytoskeleton gene actin-beta (ACTB). In conclusion, short 24 h phthalate exposures significantly altered gene expression cell-specifically in human endometrial cells, with six shared DEGs. The mixture effects were similar to those of MEHHP, suggesting MEHHP could be the main driver in the mixture. Impact of phthalate exposures on endometrial functions including receptivity should be addressed.

Exposure to persistent organic pollutants (POPs), such as dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCBs), has historically been linked to population collapses in wildlife. Despite international regulations, these legacy chemicals are still currently detected in women of reproductive age, and their levels correlate with reduced ovarian reserve, longer time -to -pregnancy, and higher risk of infertility. However, the specific modes of action underlying these associations remain unclear. Here, we examined the effects of five commonly occurring POPs - hexachlorobenzene (HCB), p,p '-dichlorodiphenyldichloroethylene (DDE), 2,3,3 ' ,4,4 ' ,5-hexachlorobiphenyl (PCB156), 2,2 ' ,3,4,4 ' ,5,5 ' -heptachlorobiphenyl (PCB180), perfluorooctane sulfonate (PFOS) - and their mixture on human ovaries in vitro . We exposed human ovarian cancer cell lines COV434, KGN, and PA1 as well as primary ovarian cells for 24 h, and ovarian tissue containing unilaminar follicles for 6 days. RNA -sequencing of samples exposed to concentrations covering epidemiologically relevant levels revealed significant gene expression changes related to central energy metabolism in the exposed cells, indicating glycolysis, oxidative phosphorylation, fatty acid metabolism, and reactive oxygen species as potential shared targets of POP exposures in ovarian cells. Alpha-enolase ( ENO1 ), lactate dehydrogenase A ( LDHA ), cytochrome C oxidase subunit 4I1 ( COX4I1 ), ATP synthase F1 subunit alpha ( ATP5A ), and glutathione peroxidase 4 ( GPX4 ) were validated as targets through qPCR in additional cell culture experiments in KGN. In ovarian tissue cultures, we observed significant effects of exposure on follicle growth and atresia as well as protein expression. All POP exposures, except PCB180, decreased unilaminar follicle proportion and increased follicle atresia. Immunostaining confirmed altered expression of LDHA, ATP5A, and GPX4 in the exposed tissues. Moreover, POP exposures modified ATP production in KGN and tissue culture. In conclusion, our results demonstrate the disruption of cellular energy metabolism as a novel mode of action underlying POP -mediated interference of follicle growth in human ovaries.

Defects in adipocyte lipolysis drive multiple aspects of cardiometabolic disease, but the transcriptional framework controlling this process has not been established. To address this, we performed a targeted perturbation screen in primary human adipocytes. Our analyses identified 37 transcriptional regulators of lipid mobilization, which we classified as (i) transcription factors, (ii) histone chaperones, and (iii) mRNA processing proteins. On the basis of its strong relationship with multiple readouts of lipolysis in patient samples, we performed mechanistic studies on one hit, ZNF189, which encodes the zinc finger protein 189. Using mass spectrometry and chromatin profiling techniques, we show that ZNF189 interacts with the tripartite motif family member TRIM28 and represses the transcription of an adipocyte-specific isoform of phosphodiesterase 1B (PDE1B2). The regulation of lipid mobilization by ZNF189 requires PDE1B2, and the overexpression of PDE1B2 is sufficient to attenuate hormone-stimulated lipolysis. Thus, our work identifies the ZNF189-PDE1B2 axis as a determinant of human adipocyte lipolysis and highlights a link between chromatin architecture and lipid mobilization.

STUDY QUESTION:What is the effect of the chemical in vitro activation (cIVA) protocol compared with fragmentation only (Frag, also known as mechanical IVA) on gene expression, follicle activation and growth in human ovarian tissue in vitro? SUMMARY ANSWER:Although histological assessment shows that cIVA significantly increases follicle survival and growth compared to Frag, both protocols stimulate extensive and nearly identical transcriptomic changes in cultured tissue compared to freshly collected ovarian tissue, including marked changes in energy metabolism and inflammatory responses. WHAT IS KNOWN ALREADY:Treatments based on cIVA of the phosphatase and tensin homolog (PTEN)-phosphatidylinositol 3-kinase (PI3K) pathway in ovarian tissue followed by auto-transplantation have been administered to patients with refractory premature ovarian insufficiency (POI) and resulted in live births. However, comparable effects with mere tissue fragmentation have been shown, questioning the added value of chemical stimulation that could potentially activate oncogenic responses. STUDY DESIGN SIZE DURATION:Fifty-nine ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section (C-section). The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic)+740Y-P (Frag+cIVA group) during the first 24 h of culture, while the other half were cultured with medium only (Frag group). Subsequently, both groups were cultured with medium only for an additional 6 days. Tissue and media samples were collected for histological, transcriptomic, steroid hormone, and cytokine/chemokine analyses at various time points. PARTICIPANTS/MATERIALS SETTING METHODS:Effects on follicles were evaluated by counting and scoring serial sections stained with hematoxylin and eosin before and after the 7-day culture. Follicle function was assessed by quantification of steroids by ultra-performance liquid chromatography tandem-mass spectrometry at different time points. Cytokines and chemokines were measured by multiplex assay. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the initial 24-h culture. Selected differentially expressed genes (DEGs) were validated by quantitative PCR and immunofluorescence in cultured ovarian tissue as well as in KGN cell (human ovarian granulosa-like tumor cell line) culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE:Compared to the Frag group, the Frag+cIVA group exhibited a significantly higher follicle survival rate, increased numbers of secondary follicles, and larger follicle sizes. Additionally, the tissue in the Frag+cIVA group produced less dehydroepiandrosterone compared to Frag. Cytokine measurement showed a strong inflammatory response at the start of the culture in both groups. The RNA-seq data revealed modest differences between the Frag+cIVA and Frag groups, with only 164 DEGs identified using a relaxed cut-off of false discovery rate (FDR) <0.1. Apart from the expected PI3K-protein kinase B (Akt) pathway, cIVA also regulated pathways related to hypoxia, cytokines, and inflammation. In comparison to freshly collected ovarian tissue, gene expression in general was markedly affected in both the Frag+cIVA and Frag groups, with a total of 3119 and 2900 DEGs identified (FDR < 0.001), respectively. The top enriched gene sets in both groups included several pathways known to modulate follicle growth such as mammalian target of rapamycin (mTOR)C1 signaling. Significant changes compared to fresh tissue were also observed in the expression of genes encoding for steroidogenesis enzymes and classical granulosa cell markers in both groups. Intriguingly, we discovered a profound upregulation of genes related to glycolysis and its upstream regulator in both Frag and Frag+cIVA groups, and these changes were further boosted by the cIVA treatment. Cell culture experiments confirmed glycolysis-related genes as direct targets of the cIVA drugs. In conclusion, cIVA enhances follicle growth, as expected, but the mechanisms may be more complex than PI3K-Akt-mTOR alone, and the impact on function and quality of the follicles after the culture period remains an open question. LARGE SCALE DATA:Data were deposited in the GEO data base, accession number GSE234765. The code for sequencing analysis can be found in https://github.com/tialiv/IVA_project. LIMITATIONS REASONS FOR CAUTION:Similar to the published IVA protocols, the first steps in our study were performed in an in vitro culture model where the ovarian tissue was isolated from the regulation of hypothalamic-pituitary-ovarian axis. Further in vivo experiments will be needed, for example in xeno-transplantation models, to explore the long-term impacts of the discovered effects. The tissue collected from patients undergoing C-section may not be comparable to tissue of patients with POI. WIDER IMPLICATIONS OF THE FINDINGS:The general impact of fragmentation and short (24 h) in vitro culture on gene expression in ovarian tissue far exceeded the effects of cIVA. Yet, follicle growth was stimulated by cIVA, which may suggest effects on specific cell populations that may be diluted in bulk RNA-seq. Nevertheless, we confirmed the impact of cIVA on glycolysis using a cell culture model, suggesting impacts on cellular signaling beyond the PI3K pathway. The profound changes in inflammation and glycolysis following fragmentation and culture could contribute to follicle activation and loss in ovarian tissue culture, as well as in clinical applications, such as fertility preservation by ovarian tissue auto-transplantation. STUDY FUNDING/COMPETING INTERESTS:This study was funded by research grants from European Union's Horizon 2020 Research and Innovation Programme (Project ERIN No. 952516, FREIA No. 825100), Swedish Research Council VR (2020-02132), StratRegen funding from Karolinska Institutet, KI-China Scholarship Council (CSC) Programme and the Natural Science Foundation of Hunan (2022JJ40782). International Iberian Nanotechnology Laboratory Research was funded by the European Union's H2020 Project Sinfonia (857253) and SbDToolBox (NORTE-01-0145-FEDER-000047), supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. No competing interests are declared.

Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA + CD142 − /low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA + CD142 − /low fibroblasts foster monocyte transition to CCR2 + CD206 + macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2 + CD206 + cells from co-cultures have a phenotype similar to intestinal CCR2 + CD206 + macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.