Human blastoids model blastocyst development and implantation.

One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid1. Here we show that naive human pluripotent stem cells cultured in PXGL medium2 and triply inhibited for the Hippo, TGF-ß and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development3,4.

Coordination of germ layer lineage choice by TET1 during primed pluripotency.

Gastrulation in the early postimplantation stage mammalian embryo begins when epiblast cells ingress to form the primitive streak or develop as the embryonic ectoderm. The DNA dioxygenase Tet1 is highly expressed in the epiblast and yet continues to regulate lineage specification during gastrulation when its expression is diminished. Here, we show how Tet1 plays a pivotal role upstream of germ layer lineage bifurcation. During the transition from naive pluripotency to lineage priming, a global reconfiguration redistributes Tet1 from Oct4-cobound promoters to distal regulatory elements at lineage differentiation genes, which are distinct from high-affinity sites engaged by Oct4. An altered chromatin landscape in Tet1-deficient primed epiblast-like cells is associated with enhanced Oct4 expression and binding to Nodal and Wnt target genes, resulting in collaborative signals that enhance mesendodermal and inhibit neuroectodermal gene expression during lineage segregation. A permissive role for Tet1 in neural fate induction involves Zic2-dependent engagement at neural target genes at lineage priming, is dependent on the signaling environment during gastrulation, and impacts neural tube closure after gastrulation. Our findings provide mechanistic information for epigenetic integration of pluripotency and signal-induced differentiation cues.

Local glucose elevation activates pyroptosis via NLRP3 inflammasome in ovarian granulosa cells of overweight patients.

Overweight, with an increasing prevalence worldwide, significantly impairs the clinical outcomes following in vitro fertilization (IVF). Hyperglycemia, hyperlipidemia, and metabolic disorders are always accompanied by the majority of overweight patients. The association between granulosa cell function and metabolic alterations in follicular fluid including lipids, proteins, and growth factors has been extensively documented. However, the effects of higher glucose level on ovarian granulosa cells (GCs), remain largely unknown. In this study, we identified that overweight women had elevated follicular glucose level which profoundly activated NLRP3 inflammasome and pyroptosis. An in vitro correlation between follicular high glucose, NLRP3 inflammasome and pyroptosis was also established. More importantly, in granulosa cells of overweight patients, the activation of the NLRP3 inflammasome and pyroptosis induced by high glucose was involved in the dysregulation of estradiol synthesis. Our study may provide new options to interpretate and improve IVF outcomes in overweight women.

Interleukin-6 is an activator of pituitary stem cells upon local damage, a competence quenched in the aging gland.

Stem cells in the adult pituitary are quiescent yet show acute activation upon tissue injury. The molecular mechanisms underlying this reaction are completely unknown. We applied single-cell transcriptomics to start unraveling the acute pituitary stem cell activation process as occurring upon targeted endocrine cell-ablation damage. This stem cell reaction was contrasted with the aging (middle-aged) pituitary, known to have lost damage-repair capacity. Stem cells in the aging pituitary show regressed proliferative activation upon injury and diminished in vitro organoid formation. Single-cell RNA sequencing uncovered interleukin-6 (IL-6) as being up-regulated upon damage, however only in young but not aging pituitary. Administering IL-6 to young mice promptly triggered pituitary stem cell proliferation, while blocking IL-6 or associated signaling pathways inhibited such reaction to damage. By contrast, IL-6 did not generate a pituitary stem cell activation response in aging mice, coinciding with elevated basal IL-6 levels and raised inflammatory state in the aging gland (inflammaging). Intriguingly, in vitro stem cell activation by IL-6 was discerned in organoid culture not only from young but also from aging pituitary, indicating that the aging gland's stem cells retain intrinsic activatability in vivo, likely impeded by the prevailing inflammatory tissue milieu. Importantly, IL-6 supplementation strongly enhanced the growth capability of pituitary stem cell organoids, thereby expanding their potential as an experimental model. Our study identifies IL-6 as a pituitary stem cell activator upon local damage, a competence quenched at aging, concomitant with raised IL-6/inflammatory levels in the older gland. These insights may open the way to interfering with pituitary aging.

Protease secretions by the invading blastocyst induce calcium oscillations in endometrial epithelial cells via the protease-activated receptor 2.

BACKGROUND: Early embryo implantation is a complex phenomenon characterized by the presence of an implantation-competent blastocyst and a receptive endometrium. Embryo development and endometrial receptivity must be synchronized and an adequate two-way dialogue between them is necessary for maternal recognition and implantation. Proteases have been described as blastocyst-secreted proteins involved in the hatching process and early implantation events. These enzymes stimulate intracellular calcium signaling pathways in endometrial epithelial cells (EEC). However, the exact molecular players underlying protease-induced calcium signaling, the subsequent downstream signaling pathways and the biological impact of its activation remain elusive. METHODS: To identify gene expression of the receptors and ion channels of interest in human and mouse endometrial epithelial cells, RNA sequencing, RT-qPCR and in situ hybridization experiments were conducted. Calcium microfluorimetric experiments were performed to study their functional expression. RESULTS: We showed that trypsin evoked intracellular calcium oscillations in EEC of mouse and human, and identified the protease-activated receptor 2 (PAR2) as the molecular entity initiating protease-induced calcium responses in EEC. In addition, this study unraveled the molecular players involved in the downstream signaling of PAR2 by showing that depletion and re-filling of intracellular calcium stores occurs via PLC, IP3R and the STIM1/Orai1 complex. Finally, in vitro experiments in the presence of a specific PAR2 agonist evoked an upregulation of the 'Window of implantation' markers in human endometrial epithelial cells. CONCLUSIONS: These findings provide new insights into the blastocyst-derived protease signaling and allocate a key role for PAR2 as maternal sensor for signals released by the developing blastocyst.

Organoids from human tooth showing epithelial stemness phenotype and differentiation potential.

Insight into human tooth epithelial stem cells and their biology is sparse. Tissue-derived organoid models typically replicate the tissue's epithelial stem cell compartment. Here, we developed a first-in-time epithelial organoid model starting from human tooth. Dental follicle (DF) tissue, isolated from unerupted wisdom teeth, efficiently generated epithelial organoids that were long-term expandable. The organoids displayed a tooth epithelial stemness phenotype similar to the DF's epithelial cell rests of Malassez (ERM), a compartment containing dental epithelial stem cells. Single-cell transcriptomics reinforced this organoid-ERM congruence, and uncovered novel, mouse-mirroring stem cell features. Exposure of the organoids to epidermal growth factor induced transient proliferation and eventual epithelial-mesenchymal transition, highly mimicking events taking place in the ERM in vivo. Moreover, the ERM stemness organoids were able to unfold an ameloblast differentiation process, further enhanced by transforming growth factor-ß (TGFß) and abrogated by TGFß receptor inhibition, thereby reproducing TGFß's known key position in amelogenesis. Interestingly, by creating a mesenchymal-epithelial composite organoid (assembloid) model, we demonstrated that the presence of dental mesenchymal cells (i.e. pulp stem cells) triggered ameloblast differentiation in the epithelial stem cells, thus replicating the known importance of mesenchyme-epithelium interaction in tooth development and amelogenesis. Also here, differentiation was abrogated by TGFß receptor inhibition. Together, we developed novel organoid models empowering the exploration of human tooth epithelial stem cell biology and function as well as their interplay with dental mesenchyme, all at present only poorly defined in humans. Moreover, the new models may pave the way to future tooth-regenerative perspectives.

Prednisone vs Placebo and Live Birth in Patients With Recurrent Implantation Failure Undergoing In Vitro Fertilization: A Randomized Clinical Trial.

Importance: Implantation failure remains a critical barrier to in vitro fertilization. Prednisone, as an immune-regulatory agent, is widely used to improve the probability of implantation and pregnancy, although the evidence for efficacy is inadequate. Objective: To determine the efficacy of 10 mg of prednisone compared with placebo on live birth among women with recurrent implantation failure. Design, Setting, and Participants: A double-blind, placebo-controlled, randomized clinical trial conducted at 8 fertility centers in China. Eligible women who had a history of 2 or more unsuccessful embryo transfer cycles, were younger than 38 years when oocytes were retrieved, and were planning to undergo frozen-thawed embryo transfer with the availability of good-quality embryos were enrolled from November 2018 to August 2020 (final follow-up August 2021). Interventions: Participants were randomized (1:1) to receive oral pills containing either 10 mg of prednisone (n = 357) or matching placebo (n = 358) once daily, from the day at which they started endometrial preparation for frozen-thawed embryo transfer through early pregnancy. Main Outcomes and Measures: The primary outcome was live birth, defined as the delivery of any number of neonates born at 28 or more weeks' gestation with signs of life. Results: Among 715 women randomized (mean age, 32 years), 714 (99.9%) had data available on live birth outcomes and were included in the primary analysis. Live birth occurred among 37.8% of women (135 of 357) in the prednisone group vs 38.8% of women (139 of 358) in the placebo group (absolute difference, -1.0% [95% CI, -8.1% to 6.1%]; relative ratio [RR], 0.97 [95% CI, 0.81 to 1.17]; P = .78). The rates of biochemical pregnancy loss were 17.3% in the prednisone group and 9.9% in the placebo group (absolute difference, 7.5% [95% CI, 0.6% to 14.3%]; RR, 1.75 [95% CI, 1.03 to 2.99]; P = .04). Of those in the prednisone group, preterm delivery occurred among 11.8% and of those in the placebo group, 5.5% of pregnancies (absolute difference, 6.3% [95% CI, 0.2% to 12.4%]; RR, 2.14 [95% CI, 1.00 to 4.58]; P = .04). There were no statistically significant between-group differences in the rates of biochemical pregnancy, clinical pregnancy, implantation, neonatal complications, congenital anomalies, other adverse events, or mean birthweights. Conclusions and Relevance: Among patients with recurrent implantation failure, treatment with prednisone did not improve live birth rate compared with placebo. Data suggested that the use of prednisone may increase the risk of preterm delivery and biochemical pregnancy loss. Our results challenge the value of prednisone use in clinical practice for the treatment of recurrent implantation failure. Trial Registration: Chinese Clinical Trial Registry Identifier: ChiCTR1800018783.

Transnasal transsphenoidal pituitary surgery in a large tertiary hospital, a retrospective study.

OBJECTIVES: Pituitary adenomas (PAs), although being small tumours, can have quite an impact on patients' lives causing hormonal and visual disturbances, for which surgery must be performed. As a large peripheral hospital with specialists in pituitary surgery, an assessment of the efficacy and safety of transnasal transsphenoidal pituitary surgery was made. METHODS: A retrospective analysis of neurosurgical reports as well as pre and postoperative imaging was made to evaluate the presenting symptoms, tumoural variables, peri-operative morbidity, and long-term outcome. RESULTS: This cohort included 105 patients who were operated for PAs over a 9-year period, with a slight male predominance. Adenomas had a mean maximum diameter of almost 25 mm, with one-third of tumours presenting with a Knosp-grade 3 or 4. As expected, most patients presented with either visual (32.4%) or hormonal (40.0%) disturbances. After surgery, 85.3% had complete resolution of visual deficits, and 97.1% had normalisation of hormonal hypersecretion. Postoperative hormonal insufficiency requiring substitution was observed in 43.1% and was significantly more frequent in males and in non-functioning pituitary adenomas (NFAs). Postoperative cerebrospinal fluid (CSF) leakage was observed in 2.9%, and merely one patient developed meningitis. Tumour recurrence was significantly more frequent in patients with partial resection as compared to complete resection (25.6 vs. 7.9%). CONCLUSIONS: This study demonstrates that transnasal transsphenoidal pituitary surgery can be performed safely and effectively in a large non-university hospital, improving visual and/or hormonal disturbances as well as providing long-term tumour control. Patients with larger adenomas are at an increased risk to develop postoperative hypopituitarism.

Endometriosis organoids: prospects and challenges.

Endometriosis is a sex hormone-dependent, painful disease that affects 10-15% of women worldwide with no definitive cure, and current treatments are not always effective. This limitation is mainly due to gaps in our knowledge about the mechanisms involved in the pathogenesis of endometriosis at the cellular and molecular levels. Hormonal dysregulation appears to be responsible for inflammation, angiogenesis, endometrial non-receptivity, embryo implantation failure and infertility in women with endometriosis. Although correlative evidence about possible causes of hormonal dysregulations exists, the functional mechanisms remain unknown. Reliable research models of endometriosis are needed to investigate the exact mechanisms that underlie hormone disruptions. This Commentary discusses the available in-vivo and in-vitro systems for studying endometriosis. The authors emphasize the recently developed human endometriosis organoids as cutting-edge and innovative research models for endometriosis investigations, discuss their advantages and describe challenges that must be addressed to yield a reliable in-vitro model of human endometriosis. Moreover, it discusses microfluidic technology to address the present challenges for producing advanced endometriosis organoids and how to benefit from CRISPR technology to improve our knowledge about disturbed hormonal function in patients with endometriosis.

Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability.

The endometrium, which is of crucial importance for reproduction, undergoes dynamic cyclic tissue remodeling. Knowledge of its molecular and cellular regulation is poor, primarily owing to a lack of study models. Here, we have established a novel and promising organoid model from both mouse and human endometrium. Dissociated endometrial tissue, embedded in Matrigel under WNT-activating conditions, swiftly formed organoid structures that showed long-term expansion capacity, and reproduced the molecular and histological phenotype of the tissue's epithelium. The supplemented WNT level determined the type of mouse endometrial organoids obtained: high WNT yielded cystic organoids displaying a more differentiated phenotype than the dense organoids obtained in low WNT. The organoids phenocopied physiological responses of endometrial epithelium to hormones, including increased cell proliferation under estrogen and maturation upon progesterone. Moreover, the human endometrial organoids replicated the menstrual cycle under hormonal treatment at both the morpho-histological and molecular levels. Together, we established an organoid culture system for endometrium, reproducing tissue epithelium physiology and allowing long-term expansion. This novel model provides a powerful tool for studying mechanisms underlying the biology as well as the pathology of this key reproductive organ.

Organoids can be established reliably from cryopreserved biopsy catheter-derived endometrial tissue of infertile women.

RESEARCH QUESTION: Can organoids be established from endometrial tissue of infertile women and does tissue cryopreservation allow for establishment of organoids comparable to organoids derived from freshly biopsied endometrial tissue? DESIGN: Endometrial tissue was obtained from six infertile women through minimally invasive biopsy using a Pipelle catheter and subjected to organoid development, immediately after biopsy as well as after tissue cryopreservation. Organoid formation efficiency, morphology, expandability potential, endometrial marker expression (immunostaining and reverse transcription quantitative real-time polymerase chain reaction) and hormonal responsiveness (after oestradiol and progesterone treatment) were assessed. RESULTS: Organoids established from both fresh and frozen tissue at comparable efficiency could be passaged long-term and showed similar morphology, i.e. cystic with a central lumen lined by a single epithelial cell layer. They also exhibited comparable expression of endometrial markers and proliferative activity (Ki67 expression). Finally, organoids from freshly biopsied and cryopreserved endometrial tissue showed similar responses to oestradiol and progesterone treatment. CONCLUSIONS: Organoids can be established from cryopreserved endometrial tissue of infertile women and cryopreservation of the biopsy does not affect organoid formation and overall organoid characteristics. Cryopreservation of biopsies for later organoid development facilitates sample collection from any fertility clinic, not just the ones near an organoid laboratory.

Traumatic brain injury and resultant pituitary dysfunction: insights from experimental animal models.

PURPOSE: Traumatic brain injury (TBI) is a major worldwide cause of disability, often burdening young people with serious lifelong health problems. A frequent clinical complication is post-traumatic hypopituitarism (PTHP) manifesting in several hypothalamus-pituitary axes. The head trauma-induced mechanisms underlying PTHP remain largely unknown. Several hypotheses have been proposed including direct damage to the pituitary gland and hypothalamus, vascular events and autoimmunity. This review aims to provide a summary of the currently limited number of studies exploring hypothalamus-pituitary dysfunction in experimental animal TBI models. RESULTS: Although the impact of different forms of TBI on a number of hypothalamus-pituitary axes has been investigated, consequences for pituitary tissue and function have only scarcely been described. Moreover, mechanisms underlying the endocrine dysfunctions remain under explored. CONCLUSIONS: Studies on TBI-induced pituitary dysfunction are still scarce. More research is needed to acquire mechanistic insights into the pathophysiology of PTHP which may eventually open up the horizon toward better treatments, including pituitary-regenerative approaches.

Laminin-332 sustains chemoresistance and quiescence as part of the human hepatic cancer stem cell niche.

BACKGROUND & AIMS: Cancer stem cells (CSCs) are thought to be persistent in tumours due to their chemoresistance and to cause relapse and metastasis. Hepatic carcinomas displaying hepatic progenitor cell (HPC) features have been associated with a poor prognosis, though it remains unclear how CSCs relate to these different histological subtypes. METHODS: Candidate CSCs were isolated using the side population (SP) technique from primary tissue samples diagnosed as keratin(K)19-negative or -positive hepatocellular carcinoma (HCC) or as combined hepatocellular/cholangiocarcinoma and analysed for gene and protein expression. The effect of laminin-332 was analysed in vitro by using HCC cell lines and in vivo using a xenograft mouse model. RESULTS: The size of the SP correlated with the degree of HPC features found in human hepatic cancer, and also showed an elevated mRNA expression of biliary/HPC markers and the extracellular matrix marker LAMC2, the gene encoding the laminin γ2-chain. Immunopositivity for the γ2-chain of laminin-332 was seen in the extracellular matrix surrounding small HPC-like tumour cells with a low proliferation rate. In vitro, laminin-332 increased K19 expression, phosphorylated mTOR and decreased phospho-histone H3 expression, indicating reduced cell mitosis. The effect of laminin-332 was enhanced upon mTORC1 inhibition and diminished when inhibiting mTORC1+C2. Resistance to doxorubicin and sorafenib treatment, and the SP fraction increased in the coated condition. In vivo, laminin-332 reduced tumour growth and sustained K19 expression. CONCLUSIONS: In this study we identified a prominent role for laminin-332 as part of the specialised CSC niche in maintaining and supporting cell 'stemness', which leads to chemoresistance and quiescence.

Keratin 19: a key role player in the invasion of human hepatocellular carcinomas.

OBJECTIVE: Keratin (K)19, a biliary/hepatic progenitor cell (HPC) marker, is expressed in a subset of hepatocellular carcinomas (HCC) with poor prognosis. The underlying mechanisms driving this phenotype of K19-positive HCC remain elusive. DESIGN: Clinicopathological value of K19 was compared with EpCAM, and α-fetoprotein, in a Caucasian cohort of 242 consecutive patients (167 surgical specimens, 75 needle biopsies) with different underlying aetiologies. Using microarrays and microRNA profiling the molecular phenotype of K19-positive HCCs was identified. Clinical primary HCC samples were submitted to in vitro invasion assays and to side population analysis. HCC cell lines were transfected with synthetic siRNAs against KRT19 and submitted to invasion and cytotoxicity assays. RESULTS: In the cohort of surgical specimens, K19 expression showed the strongest correlation with increased tumour size (p<0.01), decreased tumour differentiation (p<0.001), metastasis (p<0.05) and microvascular invasion (p<0.001). The prognostic value of K19 was also confirmed in a set of 75 needle biopsies. Profiling showed that K19-positive HCCs highly express invasion-related/metastasis-related markers (eg, VASP, TACSTD2, LAMB1, LAMC2, PDGFRA), biliary/HPC markers (eg, CD133, GSTP1, NOTCH2, JAG1) and members of the miRNA family 200 (eg, miR-141, miR-200c). In vitro, primary human K19-positive tumour cells showed increased invasiveness, and reside in the chemoresistant side population. Functionally, K19/KRT19 knockdown results in reduced invasion, loss of invadopodia formation and decreased resistance to doxorubicin, 5-fluorouracil and sorafenib. CONCLUSIONS: Giving the distinct invasive properties, the different molecular profile and the poor prognostic outcome, K19-positive HCCs should be considered as a seperate entity of HCCs.

A novel hypoxia-associated subset of FN1 high MITF low melanoma cells: identification, characterization, and prognostic value.

In many human cancers, the epithelial-to-mesenchymal transition has an important role in the induction of cancer stem-like cells, and hence, in the causation of intratumoral heterogeneity. This process, also referred to as mesenchymal mimicry, is, however, only poorly understood in melanoma and histological correlation is still lacking. In an immunohistochemical analysis of a large prospective series of 220 primary and metastatic melanomas for the well-known epithelial-to-mesenchymal transition marker FN1, we observed melanoma cells with high FN1 expression in metastases with ischemic necrosis, but rarely or not at all in samples lacking evidence of hypoxia. In a blinded, retrospective series of 82 melanoma metastases with 10-year follow-up, the presence of clusters of these FN1(high) melanoma cells correlated significantly with shortened melanoma-specific survival, highlighting the prognostic value of their presence. We describe in detail the unique light- and electron-microscopic features of these FN1(high) melanoma cells, enabling their identification in routinely hematoxylin-and-eosin-stained sections. In addition, by laser microdissection and subsequent gene expression analysis and immunohistochemistry, we highlight their distinctive, molecular phenotype that includes expression of various markers of the epithelial-to-mesenchymal transition (eg, ZEB1) and of melanoma stem-like cells (eg, NGFR), and lack of immunoreactivity for the melanocytic marker MITF. This phenotype could be reproduced in vitro by culturing melanoma cells under hypoxic conditions. Functionally, the hypoxic microenvironment was shown to induce a more migratory and invasive cell type. In conclusion, we identified a novel clinically relevant FN1(high)MITF(low) cell type in melanoma associated with ischemic necrosis, and propose that these cells reside at the crossroad of the epithelial-to-mesenchymal transition and stem-like cell induction, plausibly triggered by the hypoxic environment.

From Pluripotent Stem Cells to Organoids and Bioprinting: Recent Advances in Dental Epithelium and Ameloblast Models to Study Tooth Biology and Regeneration.

Ameloblasts are the specialized dental epithelial cell type responsible for enamel formation. Following completion of enamel development in humans, ameloblasts are lost and biological repair or regeneration of enamel is not possible. In the past, in vitro models to study dental epithelium and ameloblast biology were limited to freshly isolated primary cells or immortalized cell lines, both with limited translational potential. In recent years, large strides have been made with the development of induced pluripotent stem cell and organoid models of this essential dental lineage - both enabling modeling of human dental epithelium. Upon induction with several different signaling factors (such as transforming growth factor and bone morphogenetic proteins) these models display elevated expression of ameloblast markers and enamel matrix proteins. The advent of 3D bioprinting, and its potential combination with these advanced cellular tools, is poised to revolutionize the field - and its potential for tissue engineering, regenerative and personalized medicine. As the advancements in these technologies are rapidly evolving, we evaluate the current state-of-the-art regarding in vitro cell culture models of dental epithelium and ameloblast lineage with a particular focus toward their applicability for translational tissue engineering and regenerative/personalized medicine.

Ovarian ferroptosis induced by androgen is involved in pathogenesis of PCOS.

STUDY QUESTION: Does ovarian ferroptosis play an active role in the development of polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Increased ovarian ferroptosis was present in PCOS ovaries and the inhibition of ferroptosis with ferrostatin-1 (Fer-1) ameliorated polycystic ovary morphology and anovulation. WHAT IS KNOWN ALREADY: Programmed cell death plays a fundamental role in ovarian follicle development. However, the types and mechanisms of cell death involved in the ovary are yet to be elucidated. Ferroptosis is a recently discovered iron-dependent programmed cell death. Impaired iron metabolism and cell death have been observed in women with PCOS, the main cause of anovulatory infertility. Additionally, previous studies reported that an abnormal expression of noncoding RNA may promote ferroptosis in immortalized ovarian granulosa cell lines. However, little is known about whether ovarian ferroptosis is increased in PCOS, and there is insufficient direct evidence for a role of ferroptosis in PCOS, and the underlying mechanism. Moreover, the effect of the inhibition of ferroptosis with Fer-1 in PCOS remains unclear. STUDY DESIGN SIZE DURATION: Ferroptosis was evaluated in human granulosa cells (hGCs) from non-PCOS (n = 6-16) and PCOS (n = 7-18) patients. The experimental study was completed in vitro using primary hGCs from women undergoing IVF. Improvements in PCOS indicators following ferroptosis inhibition with Fer-1 were investigated in a dehydroepiandrosterone (DHEA)-induced PCOS rat model (n = 8 per group). PARTICIPANTS/MATERIALS SETTING METHODS: Ovarian ferroptosis was evaluated in the following ways: by detecting iron concentrations via ELISA and fluorescent probes; measuring malondialdehyde (MDA) concentrations via ELISA; assessing ferroptosis-related protein abundance with western blotting; observing mitochondrial morphology with transmission electron microscopy; and determining cell viability. Primary hGCs were collected from women undergoing IVF. They were treated with dihydrotestosterone (DHT) for 24 h. The effect of DHT on ferroptosis was examined in the presence or absence of small interfering RNA-mediated knockdown of the putative receptor coregulator for signaling molecules. The role of ovarian ferroptosis in PCOS progression was explored in vivo in rats. The DHEA-induced PCOS rat model was treated with the ferroptosis inhibitor, Fer-1, and the oocytes and metaphase II oocytes were counted after ovarian stimulation. Additionally, rats were treated with the ferroptosis inducer, RSL3, to further explore the effect of ferroptosis. The concentrations of testosterone, FSH, and LH were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Increased ferroptosis was detected in the ovaries of patients with PCOS and in rats with DHEA-induced PCOS. Increased concentrations of Fe2+ (P < 0.05) and MDA (P < 0.05), and upregulated nuclear receptor coactivator 4 protein levels, and downregulated ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) proteins were observed in the hGCs in patients with PCOS and ovaries of PCOS rats (P < 0.05 versus control). DHT was shown to induce ferroptosis via activation of NOCA4-dependent ferritinophagy. The inhibition of ferroptosis with Fer-1 in rats ameliorated a cluster of PCOS traits including impaired glucose tolerance, irregular estrous cycles, reproductive hormone dysfunction, hyperandrogenism, polycystic ovaries, anovulation, and oocyte quality (P < 0.05). Treating rats with RSL3 resulted in polycystic ovaries and hyperandrogenism (P < 0.05). LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Although ovarian-targeted ferroptosis inhibition may be a more targeted treatment for PCOS, the underlying mechanisms in the cycle between ferroptosis and hyperandrogenism require further exploration. Additionally, since PCOS shows high heterogeneity, it is important to investigate whether ferroptosis increases are present in all patients with PCOS. WIDER IMPLICATIONS OF THE FINDINGS: Androgen-induced ovarian ferroptosis appears to play a role in the pathogenesis of PCOS, which potentially makes it a promising treatment target in PCOS. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Key R&D Program of China (2023YFC2705500, 2023YFC2705505, 2019YFA0802604), National Natural Science Foundation of China (No. 82130046, 82320108009, 82101708, 82101747, and 82001517), Shanghai leading talent program, Innovative research team of high-level local universities in Shanghai (No. SHSMU-ZLCX20210201, No. SSMU-ZLCX20180401), Shanghai Jiaotong University School of Medicine, Affiliated Renji Hospital Clinical Research Innovation Cultivation Fund Program (RJPY-DZX-003) and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (No. 20161413), Shanghai's Top Priority Research Center Construction Project (2023ZZ02002), and Three-Year Action Plan for Strengthening the Construction of the Public Health System in Shanghai (GWVI-11.1-36). The authors report no competing interests.

Locationally activated PRP via an injectable dual-network hydrogel for endometrial regeneration.

Enhancing the effectiveness of platelet-rich plasma (PRP) for endometrial regeneration is challenging, due to its limited mechanical properties and burst release of growth factors. Here, we proposed an injectable interpenetrating dual-network hydrogel that can locationally activate PRP within the uterine cavity, sustained release growth factors and further address the insufficient therapeutic efficacy. Locational activation of PRP is achieved using the dual-network hydrogel. The phenylboronic acid (PBA) modified methacrylated hyaluronic acid (HAMA) dispersion chelates Ca2+ by carboxy groups and polyphenol groups, and in situ crosslinked with PRP-loaded polyvinyl alcohol (PVA) dispersion by dynamic borate ester bonds thus establishing the soft hydrogel. Subsequently, in situ photo-crosslinking technology is employed to enhance the mechanical performance of hydrogels by initiating free radical polymerization of carbon-carbon double bonds to form a dense network. The PRP-hydrogel significantly promoted the endometrial cell proliferation, exhibited strong pro-angiogenic effects, and down-regulated the expression of collagen deposition genes by inhibiting the TGF-ß1-SMAD2/3 pathway in vitro. In vivo experiments using a rat intrauterine adhesion (IUA) model showed that the PRP-hydrogel significantly promoted endometrial regeneration and restored uterine functionality. Furthermore, rats treated with the PRP-hydrogel displayed an increase in the number of embryos, litter size, and birth rate, which was similar to normal rats. Overall, this injectable interpenetrating dual-network hydrogel, capable of locational activation of PRP, suggests a new therapeutic approach for endometrial repair.

Organoid models of the pituitary gland in health and disease.

The pituitary gland represents the hub of our endocrine system. Its cells produce specific hormones that direct multiple vital physiological processes such as body growth, fertility, and stress. The gland also contains a population of stem cells which are still enigmatic in phenotype and function. Appropriate research models are needed to advance our knowledge on pituitary (stem cell) biology. Over the last decade, 3D organoid models have been established, either derived from the pituitary stem cells or from pluripotent stem cells, covering both healthy and diseased conditions. Here, we summarize the state-of-the-art of pituitary-allied organoid models and discuss applications of these powerful in vitro research and translational tools to study pituitary development, biology, and disease.

Matrix scaffolds for endometrium-derived organoid models.

The uterus-lining endometrium is essential to mammalian reproduction, receiving and accommodating the embryo for proper development. Despite its key role, mechanisms underlying endometrial biology (menstrual cycling, embryo interaction) and disease are not well understood. Its hidden location in the womb, and thereby-associated lack of suitable research models, contribute to this knowledge gap. Recently, 3D organoid models have been developed from both healthy and diseased endometrium. These organoids closely recapitulate the tissue's epithelium phenotype and (patho)biology, including in vitro reproduction of the menstrual cycle. Typically, organoids are grown in a scaffold made of surrogate tissue extracellular matrix (ECM), with mouse tumor basement membrane extracts being the most commonly used. However, important limitations apply including their lack of standardization and xeno-derivation which strongly hinder clinical translation. Therefore, researchers are actively seeking better alternatives including fully defined matrices for faithful and efficient growth of organoids. Here, we summarize the state-of-the-art regarding matrix scaffolds to grow endometrium-derived organoids as well as more advanced organoid-based 3D models. We discuss remaining shortcomings and challenges to advance endometrial organoids toward defined and standardized tools for applications in basic research and translational/clinical fields.