BackgroundPolycystic ovary syndrome (PCOS) is characterized by altered follicular development and metabolic dysfunction, frequently exacerbated by obesity. The follicular fluid (FF) microenvironment plays a critical role in supporting oocyte maturation and granulosa cell function; however, the extent to which FF from women with PCOS and obesity is associated with alterations in granulosa cell metabolism remains unclear. This study aimed to evaluate how does the FF from women with PCOS and/or obesity shapes granulosa cell glycolytic and mitochondrial activity.ResultsFF from women with PCOS showed significantly increased concentrations of total testosterone and Δ4-androstenedione compared with controls, irrespective of BMI (p < 0.05 and p < 0.01, respectively). Exposure of human granulosa cell line (HGrC1) to FF from women with PCOS and obesity was associated with a marked reduction in glycolytic capacity (p < 0.05) and decreased mRNA expression of key glycolytic regulators, including GLUT1, HK2 and LDHA (p < 0.05). Mitochondrial function was also altered, as evidenced by reduced maximal respiration and mitochondrial membrane potential (p < 0.05), while reactive oxygen species levels remained unchanged. Metabolomic profiling revealed elevated glucose concentrations in FF from women with PCOS and obesity compared with normal-weight controls, consistent with potential alterations in glucose metabolism within the follicular environment.ConclusionGranulosa cells depict metabolic dysregulation, with reduced glycolytic activity and impaired mitochondrial function, when exposed to FF from women with PCOS, which is further exacerbated in the presence of obesity. These findings from a pilot hypothesis-generating study suggest that the intrafollicular environment may be associated with granulosa cell metabolic disturbances, which warrant mechanistic studies to establish causality and elucidate the downstream consequences for follicular maturation and ovulatory function.

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental pollutants that can accumulate in the human body, including follicular fluid (FF), potentially affecting the function of reproductive cells. We used the mixture of these chemicals at concentrations previously reported in human FF to assess their effects on in vitro maturation, DNA integrity, and mitochondrial function of mouse oocytes. Exposure to PFAS led to meiotic abnormalities, manifested by an increase in the percentage of oocytes arrested at the germinal vesicle breakdown (GVBD) stage and a decrease in the number of oocytes reaching the metaphase II (MII) stage. We demonstrated that arrest at GVBD is associated with Spindle Assembly Checkpoint (SAC) activation as experimental inhibition of SAC restores normal maturation to MII. In addition, we observed increased DNA damage and changes in oxidative balance, including an increase in reduced glutathione (GSH) and a decrease in reactive oxygen species (ROS) levels. However, we found an increase in mitochondrial activity, basal oxygen consumption (OCR), and mitochondrial respiration intensity in immature GV stage oocytes, indicating disturbances in bioenergetic homeostasis. These findings show for the first time that PFAS mixtures, at concentrations relevant to human exposure, interfere with oocyte maturation, genome integrity, and mitochondrial function. The data obtained highlight the need for further research on the toxicity of PFAS mixtures, especially at doses mirroring their environmental presence, and their potential impact on female fertility.

Wound healing is a complex biological process that restores the integrity and strength of the skin. Keratinocytes and fibroblasts play pivotal roles in tissue repair through their interactions and signaling pathways. Keratinocytes proliferate to re-epithelialize the wound, while fibroblasts contribute to the formation of new extracellular matrix. Although cell proliferation is essential for wound healing, excessive proliferation can lead to scar formation. Therefore, strategies that balance proliferation and migration, along with proper mechanotransduction, are crucial for efficient wound repair. Sheep and camel milk contain numerous bioactive substances, such as insulin, lactoferrin, proline, and conjugated linoleic acid, that have demonstrated promising effects on wound healing. While direct evidence on skin cell proliferation and migration is limited, the properties of these bioactive substances suggest beneficial effects in these areas. Therefore, incorporating these milks into hydrogel dressings can promote wound healing. Moreover, improving the cell-material mechano-interaction in such hydrogels could lead to the development of novel regenerative medical therapies.

Melanoma is an aggressive cancer characterized by metabolic reprogramming that supports invasion and metastasis. Receptor-interacting protein kinase 4 (RIPK4) has been linked to tumor progression, but its role in melanoma metabolism remains unclear. This study examined whether RIPK4 is associated with melanoma aggressiveness through modulation of cellular bioenergetics. Previously generated RIPK4-knockout A375 and WM266.4 melanoma cell lines (CRISPR/Cas9) were used, and RIPK4 expression was restored by plasmid transfection. Cellular metabolism was assessed using the Seahorse XF Mito Stress Test in 2D monolayer cultures. In 3D spheroid models, ATP assays, qRT-PCR, and Western blotting were performed, together with functional analyses using 3D matrix invasion and CCID assays. In vivo relevance was evaluated by immunohistochemical analysis of RIPK4 and GLUT1 in lung metastases from NOD/SCID mouse xenografts. RIPK4 knockout induced a metabolically compromised state, characterized by reduced mitochondrial respiration and glycolysis, decreased HK2 and GLUT1 expression, and increased SDHB levels also impaired 3D invasive behavior, including reduced formation of invasive protrusions and decreased intravascular invasion. GLUT1 expression was detected in lung metastases but was reduced in RIPK4-deficient tumors. RIPK4 re-expression restored AKT phosphorylation and partially rescued HK2 and GLUT1 levels; however, metabolic flux (OCR/ECAR) was not recovered. These findings suggest that RIPK4 is associated with melanoma metabolic regulation and invasion, potentially involving AKT-linked signaling, but also indicate that additional mechanisms beyond AKT-GLUT1 contribute to the observed metabolic phenotype.

Orotic acid (OA) is a natural component of milk and is found in many biological fluids such as human ovarian follicular fluid. However, its effect on ovarian cells is unknown. Some studies suggest that OA may alter lipid metabolism and energy production in cells. In the present study, we determine the effect of OA on mitochondrial function and lipid droplet content in the human granulosa cell line. The effect of OA on in vitro mouse oocyte maturation and mitochondrial activity was also investigated. We found that repeated exposure to OA (0.01–1000 µM) did not alter the viability of human epithelial (HOSEpiC) and granulosa (HGrC1) ovarian cells. HGrC1 cells treated with a high dose of OA (500 µM) showed a more aerobic and energetic phenotype than control cells, whereas this effect was not observed after treatment with lower doses (0.01 and 100 µM) of OA. In addition, OA at a high dose (500 µM) reduced lipid droplet (LD) content without altering glucose (GLUT1, GLUT4) and fatty acid transporter (SLC27A1) gene expression in HGrC1 cells. At the same time, OA at 100 µM did not disrupt mouse in vitro oocyte maturation, whereas OA at 500 µM inhibited this process by arresting oocytes at the germinal vesicle (GV) stage with a reduction in mitochondrial activity. Our results show that OA at high doses can disrupt female reproduction, but normal dietary orotate intake does not have a negative effect on ovarian function.

The new generation of bisphenols, including bisphenol AF (BPAF), B (BPB), P (BPP) and Z (BPZ), has been detected within the environment of human ovarian follicles. It is therefore hypothesized that these bisphenols may interact directly with ovarian granulosa cells (GCs), which have high metabolic and proliferative activity that is crucial for oocyte development. Therefore, this study aimed to evaluate the effects of environmentally relevant concentrations (0.1–10 nM) of BPB, BPAF, BPP and BPZ on the mitochondrial function, metabolic fuel utilization and proliferation capacity of human GCs. While all bisphenols significantly increased GCs’ proliferation after a single (24 h) treatment, only BPZ enhanced its effect after subsequent (48 h) exposure. Simultaneously, BPZ increased ATP production, and this effect was accompanied by significant disruptions to substrate oxidation. Exposure to BPZ notably shifted metabolism from glucose and glutamine toward fatty acids and increased mRNA expression of peroxisome proliferator‑activated receptors α and δ. Additionally, glutamine and glucose were identified as the primary metabolic fuels that support basal and BPZ-induced proliferation of GCs. The results of this study demonstrate that BPZ disrupts the metabolic flexibility of GCs and stimulates their extensive proliferation. This can have a detrimental effect on follicular development, which in turn can affect ovulation and fertility.

Premature ovarian insufficiency (POI), affecting approximately 1

The accumulation of a number of per- and polyfluoroalkyl substances (PFASs) in ovarian follicular fluid (FF) has been documented, raising serious questions about their impact on female fertility. Here, we tested the hypothesis that a mixture of PFASs acts in a paracrine manner on granulosa cells (GCs) as a metabolism-disrupting chemical. We selected perfluorooctane sulfonate (PFOS; 22.4ng/mL), perfluorooctanoic acid (PFOA; 14.5ng/mL), perfluorohexane sulfonate (PFHxS; 21.3ng/mL), perfluorodecanoic acid (PFDA; 0.9ng/mL), perfluoroheptane sulphonate (PFHpA; 0.6ng/mL), perfluoroundecanoic acid (PFUnDA; 0.4ng/mL), and perfluorononanoic acid (PFNA; 2ng/mL), which were the most commonly detected PFASs in FF of women undergoing assisted reproductive technology treatment. Exposure of mouse GCs to the PFAS mixture decreased the amount of active mitochondria and the mitochondrial membrane potential, which correlated with a reduction in ATP production and inhibition of oxidative phosphorylation (OXPHOS). At the same time, expression of the mitochondrial membrane-associated steroidogenic enzyme 3-beta-hydroxysteroid dehydrogenase (3βHSD) and production of the major steroids progesterone and estradiol were decreased. In addition, expression and activity of superoxide dismutase 1 (SOD1), an enzyme that neutralizes reactive oxygen species (ROS), were decreased while ROS levels and lipid peroxidation were increased without cell death, indicating that the PFAS mixture had subtoxic effects. Our results show that PFAS mixtures, at concentrations similar to those found in human FF led to GC dysfunction by impairing mitochondrial function and steroid secretions and therefore may have implications for reproductive health.

Bisphenols (BPs) are a group of environmental pollutants mainly represented by bisphenol S (BPS) and F (BPF). In ovaries, BPs can accumulate in follicular fluid (FF), changing the follicular microenvironment and simultaneously affecting ovarian granulosa cells (GCs) function. In the present study, we determined the effects of BPS and BPF on oxidative stress and mitochondrial function in human ovarian GCs. Single, short-term treatment with BPs at doses reflecting their concentrations in FF (10nM) did not affect reactive oxygen species (ROS) levels but induced mitochondrial membrane depolarization. BPF-induced mitophagy decreased the number of active mitochondria and consequently reduced the ATP production rate. The observed changes did not translate into lowered viability of GCs, but long-term treatment with BPF influenced the intrinsic apoptosis pathway by increasing caspase 9 activity without affecting apoptosis. GCs are crucial for ovarian function as they produce primary steroid hormones and regulate oocyte maturation and follicle growth. Mitochondrial dysfunction caused by BPs, manifesting as reduced ATP production in GCs, can directly cause ovarian disorders such as infertility. Therefore, this study highlights the significance of investigating the effects of BPs on reproductive health.

Bisphenols (BPs) are a group of organic compounds used extensively in plastics, coatings, and epoxy resins; they have been of concern recently due to their endocrine-disrupting effects. Among these, bisphenol A (BPA) is the most studied. Regulatory measures, such as the ban on BPA use in baby bottles by the European Union and its restricted use in thermal paper, reflect the growing awareness of the health risks of BPA. To mitigate these risks, analogs such as bisphenol S (BPS), bisphenol F (BPF), and others (BPAF, BPAP, BPB, BPP, BPZ) have been developed as alternatives. Despite their intended safety, these analogs have been detected in environmental media, including indoor dust and thermal receipt paper, as well as in human biological samples. Studies report their presence in urine at levels comparable to BPA, with BPS and BPF found in 78% and 55% of samples, respectively. In addition, BPs have been found in human follicular fluid (FF) at concentrations that could exert some paracrine effects on ovarian function and reproductive health. With the increased global production of BPs, occupational exposure and environmental contamination also increase. This review summarizes what is currently known about the effects of BPs on the ovary and the mechanisms by which PBs exert ovarian toxicity, with a particular focus on oogenesis, folliculogenesis, and steroidogenesis. Further, this review emphasizes their influence on reproductive functions and the need for further biosafety evaluations.

This review looks at the causes of the association between thyroid dysfunction (hyperthyroidism and hypothyroidism) and ovarian cancer (OC) risk. Epidemiological data have revealed that thyroid dysfunction, particularly hyperthyroidism, is associated with increased risk, progression, and mortality in patients with OC. In addition, research studies and databases have demonstrated that both the expression and localization of thyroid hormone receptors alpha (TRα) and beta (TRβ) and membrane thyroid hormone receptor integrin alpha V beta 3 (αvβ3) affect OC progression and survival in OC patients. Furthermore, this review described the levels of the thyroid hormones (THs) thyroxine (T4) and 3,5,3′-triiodo-L-thyronine (T3) in the blood of OC patients and their role in OC progression. Moreover, we present studies that reported the relationship between hyperthyroidism and hypothyroidism and the levels of metabolic hormones in the blood and the possible effects on metabolic reprogramming in OC cells. We also report data indicating the relationship between the treatment of thyroid dysfunction and OC progression. Finally, the cited case studies described the essential case of struma ovarii, which is OC, including thyroid tissue. This review describes the link between thyroid dysfunction and OC risk and progression, which may be important in treating OC patients with thyroid dysfunction.

Materials engineering has become an important tool in the field of hydrogel dressings used to treat difficult-to-heal wounds. Hydrogels filled with bioactive substances used as a targeted healing system are worthy of attention. Vitamin C has healing and supporting effects in the treatment of many skin problems. The aim of the research was to produce a hydrogel biomaterial enriched with ascorbic acid for use as a dressing for difficult-to-heal wounds. A total of four different dressings were developed, each with different modifications in each layer. The dressing with vitamin C in the third layer was shown to release vitamin C ions more slowly than the dressing with vitamin C in the first layer. The studies conducted have shown that the dressings containing vitamin C have, among other things, a higher compressive strength, are characterised by a lower relative shortening after the application of force and shorten without damage at a lower force than in the case of a dressing without vitamin C. The dressings designed have a very good stability in the temperature range of 18 °C to 60 °C. It was found that the higher the vitamin C content in the dressing, the greater the increase in the specific heat value of the transformations. Therefore, hydrogel dressings containing vitamin C may be candidates for local delivery of vitamin C to the skin and protection of the wound area.

Introduction/Background Bisphenols (BPs) are endocrine disrupting chemicals identified as contaminants of concern for reproductive toxicity. Currently, commonly used are bisphenol S (BPS) and F (BPF) that can be detected in human body fluids. We have established that both BPS and BPF affect cell cycle distribution and steroidogenic activity of human ovarian granulosa cells. Another process that can be affected by BPs is cell death. Apoptosis plays a crucial role in the pathogenesis of ovarian cancer, polycystic ovary syndrome (PCOS) and other ovarian diseases. In granulosa cells apoptosis predominantly occurs via caspase-dependent signaling pathways. Therefore, here we studied impact of BPs on cell death by researching how BPS and BPF would affect effector caspases activity. Methodology Human non-luteinised granulosa cell line (HGrC1) was used as in vitro model to investigate the effects of BPs. The cells were treated with 0.1, 1, 10 and 100 nM BPS or BPF for 24h prior to analysis of DNA content and caspase activity and with 10 nM for 6h prior to flow cytometry analysis. For real-time apoptotic progression test cells were treated with BPs (10 nM) and monitored up to 24h. Results We have established that BPs do not have an effect on DNA content and apoptosis but do have impact on caspases, mainly by lowering caspase-9 activity in HGrC1 cells. Conclusion Caspase-9 is directly linked to mitochondrial-centered cell death, therefore, we suggest that BPs by lowering caspase-9 desensitize ovarian granulosa cells to apoptosis and trigger non-apoptotic pathways. Caspase-9 is often connected to the execution of cell differentiation, and its non-apoptotic functions are increasingly being linked to cancer and other ovarian diseases pathogenesis. These studies highlight the importance of caspase 9 activity in granulosa cells functions and suggest that research into regulation of cell death may be the key to understanding development of female reproductive system dysfunctions. Disclosures This research has been funded by the program 'Excellence Initiative – Research University' at the Jagiellonian University.

The treatment of ovarian cancer (OC) remains one of the greatest challenges in gynaecological oncology. The presence of classic steroid receptors in OC makes hormone therapy an attractive option; however, the response of OC to hormone therapy is modest. Here, we compared the expression patterns of progesterone (PGR), androgen (AR) and oestrogen alpha (ERα) receptors between serous OC cell lines and non-cancer ovarian cells. These data were analysed in relation to steroid receptor expression profiles from patient tumour samples and survival outcomes using a bioinformatics approach. The results showed that ERα, PGR and AR were co-expressed in OC cell lines, and patient samples from high-grade and low-grade OC co-expressed at least two steroid receptors. High AR expression was negatively correlated, whereas ERα and PGR expression was positively correlated with patient survival. AR showed the opposite expression pattern to that of ERα and PGR in type 1 (SKOV-3) and 2 (OVCAR-3) OC cell lines compared with non-cancer (HOSEpiC) ovarian cells, with AR downregulated in type 1 and upregulated in type 2 OC. A low AR/PGR ratio and a high ESR1/AR ratio were associated with favourable survival outcomes in OC compared with other receptor ratios. Although the results must be interpreted with caution because of the small number of primary tumour samples analysed, they nevertheless suggest that the evaluation of ERα, AR and PGR by immunohistochemistry should be performed in patient biological material to plan future clinical trials.

The heterogeneity of ovarian cancer (OC) has made developing effective treatments difficult. Nowadays, hormone therapy plays a growing role in the treatment of OC; however, hormone modulators have had only limited success so far. To provide a more rigorous foundation for hormonal therapy for different OC subtypes, the current study used a series of bioinformatics approaches to analyse the expression profiles of genes encoding membrane progesterone (PGRMC1, progestins and the adipoQ receptor [PAQR] family), and androgen (zinc transporter member 9 [ZIP9], OXER1) receptors. Our work investigated also their prognostic value in the context of OC. We found differences in expression of ZIP9 and OXER1 between different OC subtypes, as well as between patient tumour and normal tissues. Expression of mRNA encoding PAQR7 and PAQR8 in a panel of OC cell lines was below the qPCR detection limit and was downregulated in tumour tissue samples, whereas high expression of PGRMC1 and PAQR4 mRNA was observed in rare subtypes of OC cell lines. In addition, chemical inhibition of PGRMC1 reduced the viability of rare OCs represented by COV434 cells. In conclusion, PGRMC1 and PAQR4 are promising targets for anticancer therapy, particularly for rare subtypes of OC. These findings may reflect differences in the observed responses of various OC subtypes to hormone therapy.