Ovarian Stromal Cells Research Articles

Conclusive evidence for the origin of the carcinoid component in strumal carcinoids and a new morphological pattern

Abstract Introduction/Objective There has been debate whether strumal carcinoids are carcinoids with areas resembling thyroid or arise from bonafide thyroid tissue in struma. While the thyroid nature has been proven by demonstrating positive TTF-1 in the struma component, evidence for the origin of the carcinoid component is sparse. Herein, we describe a case of strumal carcinoid and use a panel of markers to show definitive morphological and immunohistochemical evidence of the evolution of the neoplasm from struma ovarii to struma carcinoid, within the same lesion. We also report a unique component of a more sinister single cell pattern of invasion apart from the usual insular, trabecular, and mucinous patterns. Methods/Case Report A 40-year-old woman with an adnexal mass underwent bilateral salpingo- oophorectomy/hysterectomy. Microscopic analysis identified thyroid tissue consistent with struma ovarii, juxtaposed with a neuroendocrine neoplasm. The interface between the two showed strong PAX-8 and uniform TTF-1 staining in the struma but gradual loss of TTF-1 with retained/weak PAX-8 staining, moving away from the struma component and into the carcinoid. Additionally, a pattern of single cell invasion was identified, away from the main tumor and at the ovarian surface. These cells closely resemble luteinized ovarian stromal cells but are stained like the carcinoid tumor. Two months postoperatively, the patient had no evidence of recurrence or metastasis. Results (if a Case Study enter NA) NA Conclusion We have provided conclusive morphological and immunohistochemical evidence that strumal carcinoid develops from native neuroendocrine cells of thyroid tissue in struma ovarii. Specifically, retained/weak PAX-8 with interspersed TTF-1 in an adnexal carcinoid provides valuable insight when dealing with tumors of unknown etiology, strongly favoring an ovarian primary (specifically, from struma ovarii) and not metastasis from the gastrointestinal tract or other Mullerian tissue. Lastly, the single cell pattern of invasion that we elucidate here is something to be aware of in the context of metastases or frozen sections.

The senolytic drug ABT-263 accelerates ovarian aging in older female mice

Previous studies have reported that senolytic drugs can reverse obesity-mediated accumulation of senescent cells in the ovary and protect against cisplatin-induced ovarian injury by removing senescent cells. Early intervention with ABT-263 has been shown to mitigate ovarian aging. However, it remains unknown whether treatment with ABT-263 could rejuvenate the aged ovary in reproductively old females. Therefore, the current study was aimed to investigate whether advanced age intervention with ABT-263 could ameliorate age-related decline in ovarian function. Fourteen 16-month-old mice with a C57/BL6 background were treated with ABT-263 (N = 7) or vehicle (N = 7) for two weeks. Mice were initially treated with ABT-263 (60 mg/kg/d) or vehicle for 7 consecutive days. After a 7-day break, the treatment was repeated for another 7 consecutive days. Six 2-month-old mice with C57BL/6 were used as a young control. The hormonal levels, estrus cycles, ovarian reserve, ovarian cell proliferation and apoptosis, ovarian fibrosis, and steroidogenic gene expression of ovarian stromal cells were evaluated. ABT-263 treatment did not rescue abnormal estrus cycles and sex hormonal levels, or inhibit the formation of multinucleated giant cells and ovarian stromal cell apoptosis in aged ovaries. However, it reduced ovarian fibrosis and preserved the steroidogenic gene expression of ovarian stromal cells in aged ovaries. Importantly, ABT-263 treatment further depleted ovarian follicles in aged mice. In conclusion, ABT-263 treatment accelerated the depletion of ovarian follicles in aged mice, suggesting that senolytic drugs for reproductively old female may adversely affect female fertility.

8354 OVARIAN HYPERTHECOSIS: AN UNCOMMON CAUSE OF HYPERANDROGENISM IN A POSTMENOPAUSAL WOMAN

Abstract Disclosure: M.D. Severino: None. F.P. Silva: None. M.V. Lopes: None. C. Nunes: None. M.I. Alexandre: None. M.J. Bugalho: None. Introduction: Severe hirsutism in postmenopausal women demands a careful clinical evaluation to exclude a malignant cause, such as adrenal or ovarian androgen-secreting tumors. Ovarian hyperthecosis is an uncommon and benign cause of hyperandrogenism and, consequently, hirsutism. It is caused by persistent testosterone synthesis due to proliferation of ovarian stromal cells and nests of luteinized theca cells. It occurs more often in postmenopausal women, as aromatization of testosterone to estradiol decreases due to the loss of granulosa cells. It is also frequently associated with obesity and insulin resistance. Clinical case: We present the case of a 61-year-old woman, with a history of hypertension and dyslipidemia, referred to Endocrinology Clinic due to a year long history of hirsutism, most severe on her face, chest, and abdomen. She also mentioned slight clitoromegaly. She had no other virilization symptoms such as acne, male-pattern alopecia, voice deepening or mammary atrophy. Her menarche occurred at the age of 14 and she had regular menses until menopause, at the age of 51. She had a BMI of 32 kg/m2 and an uncharacteristic phenotype. At observation, she had a mild hirsutism (Ferriman-Gallwey score of 9). This contrasted with the biochemical findings of severe androgenism - total testosterone of 366 ng/dL (reference values [RV] for women between 5-48) with free testosterone 7.8 pg/mL and SHBG 74 nmol/L (RV 32-128), resulting in a Free Androgen Index of 17,16 (RV <6.6). DHEAS, androstenedione, 17-hydroxyprogesterone and prolactin were normal, and gonadotropins were in the menopausal range. Blood tests also showed hyperinsulinemia and pre-diabetes with HbA1c 6,2%. She denied exposure to steroids in the form of new medication, supplements, or creams/gels (including second-hand exposure through her partner) and she had no exposure to other drugs with androgenic effect. Abdominal and pelvic CT scan, as well as transvaginal ultrasound, showed no adrenal or ovarian lesions. An ovarian cause was deemed the most likely and laparoscopic diagnostic bilateral salpingo-oophorectomy was performed. Gross pathology revealed ovaries of regular size, while histology showed bilateral ovarian stromal hyperthecosis. Testosterone levels returned to normal one month after surgery (14,2 ng/dL). Conclusion: Benign, ovarian causes of hyperandrogenism are uncommon in postmenopausal women, requiring a high index of suspicion. The diagnosis is difficult, and laparoscopic bilateral salpingo-oophorectomy is essential as both a diagnostic and therapeutic tool. Presentation: 6/2/2024

5048 A Rare Cause of Hyperandrogenism in Postmenopausal Women: Ovarian Stromal Hyperplasia

Abstract Disclosure: S. Mark: None. M.H. Shanik: None. I.J. Romao: None. Ovarian stromal hyperplasia (OSH) is a rare cause of hyperandrogenism. It is a non-neoplastic proliferation of ovarian stromal cells that leads to the overproduction of testosterone. This case report highlights that OSH should be considered in patients who present with hyperandrogenism.A 49-year-old postmenopausal woman with a past medical history of polycystic ovary syndrome, type 2 diabetes and obesity complained of unwanted hair growth. On the physical exam her BMI was 51. She had an obese body habitus, hirsutism of her chin, chest and back and acanthosis nigricans on her neck. Blood work showed a total testosterone of 203 (2-45 ng/dL) and free testosterone 29 (0.1-6.pg/dL). FSH, LH, DHEAS, estradiol, SHBG and 17 hydroxyprogesterone (17 OHP) were in the normal range. She completed a 1 mg dexamethasone suppression test that was normal. She took spironolactone for hirsutism with minimal relief. Pelvic ultrasound was limited due to body habitus. CT of the abdomen and pelvis showed no adrenal or ovarian masses. The patient had a family history of pancreatic, ovarian, and breast cancer. Given her symptoms and her family history, the patient underwent genetic testing that was negative. She underwent bilateral salpingo-oophorectomy. Pathology revealed bilateral ovarian stromal hyperplasia. After surgery, her total testosterone normalized to 18 ng/dL and free testosterone was 1.6ng/dL with improvement in hair distribution. The patient continued to struggle with her weight and diabetes management.OSH is a non-malignant proliferation of ovarian stromal cells which leads to overproduction of testosterone. It occurs more in postmenopausal women and rarely in women of reproductive age. There is approximately 1 case of OSH in every 1000 cases of hirsutism. Clinical features include obesity, insulin resistance, acanthosis nigricans, acne, and hirsutism. Testosterone levels are usually greater than 150 ng/dL. Differential diagnosis includes PCOS, Cushing’s disease, adrenal or ovarian tumor and non-classic adrenal hyperplasia. Transvaginal ultrasound can be used to assess the ovaries for tumors or increase in size. If negative a pelvic MRI or CT can be done for further assessment. The gold standard for diagnosis would be histological evaluation following bilateral oophorectomy which is curative. Pathology usually shows stromal hyperplasia that is nodular or diffuse. Alternative treatment options include GNRH agonist therapy in patients who are not surgical candidates. Complications of this condition include insulin resistance, acanthosis nigricans and diabetes mellitus. After treatment hyperandrogenism skin changes improve. It is important that clinicians consider OSH as a cause of hyperandrogenism to ensure timely diagnosis and treatment. Presentation: 6/2/2024

8354 Ovarian Hyperthecosis: an Uncommon Cause of Hyperandrogenism in a Postmenopausal Woman

Abstract Disclosure: M.D. Severino: None. F.P. Silva: None. M.V. Lopes: None. C. Nunes: None. M.I. Alexandre: None. M.J. Bugalho: None. Introduction: Severe hirsutism in postmenopausal women demands a careful clinical evaluation to exclude a malignant cause, such as adrenal or ovarian androgen-secreting tumors. Ovarian hyperthecosis is an uncommon and benign cause of hyperandrogenism and, consequently, hirsutism. It is caused by persistent testosterone synthesis due to proliferation of ovarian stromal cells and nests of luteinized theca cells. It occurs more often in postmenopausal women, as aromatization of testosterone to estradiol decreases due to the loss of granulosa cells. It is also frequently associated with obesity and insulin resistance. Clinical case: We present the case of a 61-year-old woman, with a history of hypertension and dyslipidemia, referred to Endocrinology Clinic due to a year long history of hirsutism, most severe on her face, chest, and abdomen. She also mentioned slight clitoromegaly. She had no other virilization symptoms such as acne, male-pattern alopecia, voice deepening or mammary atrophy. Her menarche occurred at the age of 14 and she had regular menses until menopause, at the age of 51. She had a BMI of 32 kg/m2 and an uncharacteristic phenotype. At observation, she had a mild hirsutism (Ferriman-Gallwey score of 9). This contrasted with the biochemical findings of severe androgenism - total testosterone of 366 ng/dL (reference values [RV] for women between 5-48) with free testosterone 7.8 pg/mL and SHBG 74 nmol/L (RV 32-128), resulting in a Free Androgen Index of 17,16 (RV <6.6). DHEAS, androstenedione, 17-hydroxyprogesterone and prolactin were normal, and gonadotropins were in the menopausal range. Blood tests also showed hyperinsulinemia and pre-diabetes with HbA1c 6,2%. She denied exposure to steroids in the form of new medication, supplements, or creams/gels (including second-hand exposure through her partner) and she had no exposure to other drugs with androgenic effect. Abdominal and pelvic CT scan, as well as transvaginal ultrasound, showed no adrenal or ovarian lesions. An ovarian cause was deemed the most likely and laparoscopic diagnostic bilateral salpingo-oophorectomy was performed. Gross pathology revealed ovaries of regular size, while histology showed bilateral ovarian stromal hyperthecosis. Testosterone levels returned to normal one month after surgery (14,2 ng/dL). Conclusion: Benign, ovarian causes of hyperandrogenism are uncommon in postmenopausal women, requiring a high index of suspicion. The diagnosis is difficult, and laparoscopic bilateral salpingo-oophorectomy is essential as both a diagnostic and therapeutic tool. Presentation: 6/2/2024

Metallic-based phthalocyanine nanoemulsions for photodynamic purging of ovarian tissue in leukemia patients

For cancer patients with a high risk of ovarian tissue metastasis, ovarian autotransplantation is not advised due to the potential spread of malignant cells. Ex vivo purging of ovarian fragments may offer a more suitable alternative for fertility restoration. Eradicating malignant cells should be done selectively without affecting follicles or ovarian stromal cells (SCs). As a clinically licensed method, photodynamic therapy (PDT) is a minimally invasive treatment to destroy cancer cells. This study evaluates the effectiveness of nanoemulsions (NE) containing two phthalocyanine photosensitizers; aluminum (III) phthalocyanine (AlPc) and zinc (II) phthalocyanine (ZnPc) in eliminating cancer cells. Human leukemic malignant (HL-60) and ovarian stromal cells (SCs) were treated with AlPc/ZnPc loaded NEs with or without diode laser irradiation. HL-60 leukemia cells in 2D culture were eliminated by treatment with 10nM AlPc-NE or 0.1µM ZnPc-NE, while no toxicity was observed in SCs. In 3D culture models, although the cells showed more resistance to the NEs as a result of limited oxygen and photosensitizer penetration, the treatment remained selective for cancer cells. These approaches have the potential to eliminate malignant cells from ovarian tissue fragments.

Hyperplastic ovarian stromal cells express genes associated to tumor progression: a case study

The current study presents the analysis of stromal cells obtained from an hyperplastic left-ovary of a Holstein cow. Cultured hyperplastic stromal cells displayed a fibroblast-like morphology and ceased proliferation after the 8th passage. The non-cancerous nature of stromal cells was confirmed by in vitro cell proliferation and migration assays. Negligible amounts of E2 were detected in the spent media of cultured stromal cells, which suggests that stromal cells were non-estradiol synthesizing cells. As revealed in immunofluorescence and gene expression analysis, the hyperplastic stromal cells explicitly expressed vimentin in their cytoskeleton. Upon hematoxylin staining, a highly dense population of stromal cells was observed in the stromal tissue of the hyperplastic ovary. To explore genome-wide alterations, mRNA microarray analysis was performed using Affymetrix Bovine Gene 1.0ST Arrays compared to normal ovarian derived stromal cells. The microarray identified 1396 differentially expressed genes, of which 733 were up- and 663 down-regulated in hyperplastic stromal cells. Importantly, asporin (ASPN) and vascular cell adhesion molecule 1 (VCAM1) were among the highly up-regulated genes. Higher expression of ASPN was also confirmed by immunohistochemistry and RT-qPCR analysis. Ingenuity pathway analysis (IPA) identified about 98 significantly enriched (-log (p value ≥ 1.3) canonical pathways, importantly of which the “Sirutin Signaling Pathway” and “Mitochondrial Dysfunction” were highly activated while “Oxidative phosphorylation” was inhibited. Additionally, higher proportion of hyperplastic stromal cells in the S-phase of cell cycle, could be attributed to higher expression levels of cell proliferation genes such as CCND2 and CDK6.

Systemic low-dose anti-fibrotic treatment attenuates ovarian aging in the mouse.

The female reproductive system is one of the first to age in humans, resulting in infertility and endocrine disruptions. The aging ovary assumes a fibro-inflammatory milieu which negatively impacts gamete quantity and quality as well as ovulation. Here, we tested whether the systemic delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs attenuates ovarian aging in mice. We first evaluated the ability of these drugs to decrease the expression of fibro-inflammatory genes in primary ovarian stromal cellstreated with a pro-fibrotic or a pro-inflammatory stimulus. Whereas Etanercept did not block Tnf expression in ovarian stromal cells, Pirfenidone significantly reduced Col1a1 expression. We then tested Pirfenidone in vivo where the drug was delivered systemically via mini-osmotic pumps for 6weeks. Pirfenidone mitigated the age-dependent increase in ovarian fibrosis without impacting overall health parameters. Ovarian function was improved in Pirfenidone-treated mice as evidenced by increased follicle and corpora lutea number, AMH levels, and improved estrous cyclicity. Transcriptomic analysis revealed that Pirfenidone treatment resulted in an upregulation of reproductive function-related genes at 8.5months and a downregulation of inflammatory genes at 12months of age. These findings demonstrate that reducing the fibroinflammatory ovarian microenvironment improves ovarian function, thereby supporting modulating the ovarian environment as a therapeutic avenue to extend reproductive longevity.

A novel bioprinted microtumour model for studying acute-leukemia-cell- contaminated in ovarian tissue

Microtumor models, combining cancer and stromal cells within 3D hydrogels, are vital for testing anticancer therapies. Bioprinting hydrogel scaffolds allows tailored in vitro models. We created a 3D microtumor model using a bioprinter, with varying ratios of ovarian stromal cells and leukemia cells (HL-60). PEGylated fibrinogen and alginate hydrogel were used. Cell dynamics and proliferation were assessed via immunofluorescence staining. Microtumors with different HL-60 ratios (1:1, 1:10, 1:100) were cultured for 5 days. Results showed tumor development modulation by cell ratios and culture time. A significant cell density increase occurred in 1:1 ratio microtumors, indicating rapid cancer cell proliferation. No HL-60 cells were found in 1:100 ratio microtumors by day 5. The 1:10 ratio closely mimicked leukemia invasion in ovarian tissue, showing detectable cancer cells by days 3 and 5 without altering total cell density dynamics significantly. This bioprinted leukemia microtumor model offers better physiological relevance than 2D assays, promising applications in cellular analysis and drug screening.

COL6A3 Exosomes Promote Tumor Dissemination and Metastasis in Epithelial Ovarian Cancer.

Our study explores the role of cancer-derived extracellular exosomes (EXs), particularly focusing on collagen alpha-3 (VI; COL6A3), in facilitating tumor dissemination and metastasis in epithelial ovarian cancer (EOC). We found that COL6A3 is expressed in aggressive ES2 derivatives, SKOV3 overexpressing COL6A3 (SKOV3/COL6A3), and mesenchymal-type ovarian carcinoma stromal progenitor cells (MSC-OCSPCs), as well as their EXs, but not in less aggressive SKOV3 cells or ES2 cells with COL6A3 knockdown (ES2/shCOL6A3). High COL6A3 expression correlates with worse overall survival among EOC patients, as evidenced by TCGA and GEO data analysis. In vitro experiments showed that EXs from MSC-OCSPCs or SKOV3/COL6A3 cells significantly enhance invasion ability in ES2 or SKOV3/COL6A3 cells, respectively (both, p <0.001). In contrast, ES2 cells with ES2/shCOL6A3 EXs exhibited reduced invasion ability (p < 0.001). In vivo, the average disseminated tumor numbers in the peritoneal cavity were significantly greater in mice receiving intraperitoneally injected SKOV3/COL6A3 cells than in SKOV3 cells (p < 0.001). Furthermore, mice intravenously (IV) injected with SKOV3/COL6A3 cells and SKOV3/COL6A3-EXs showed increased lung colonization compared to mice injected with SKOV3 cells and PBS (p = 0.007) or SKOV3/COL6A3 cells and PBS (p = 0.039). Knockdown of COL6A3 or treatment with EX inhibitor GW4869 or rapamycin-abolished COL6A3-EXs may suppress the aggressiveness of EOC.

P-589 Effects of phthalates on the 3D spheroid cell culture as a model for the human ovarian extracellular matrix

Abstract Study question Do phthalates affect the ovary by impacting its extracellular matrix (ECM)? Summary answer Phthalate mixture (PM) negatively affects the components of ovarian ECM, as demonstrated by spheroids as a model. What is known already The widespread production and pervasive use of phthalates as plasticizers in everyday products significantly increases human exposure to these substances. Their documented toxic effects on reproductive and endocrine functions raise substantial public health concerns. Yet, a full understanding of how phthalates affect ovarian functions and especially the ECM structure in females—more exposed to these products than men—remains incomplete. Moreover, there has been limited investigation in identifying a suitable model capable of mimicking the 3D ovarian ECM structure in vitro for studying phthalate impacts. Study design, size, duration Stromal cells were isolated from the ovarian cortex of post-menopausal organ donors. Cells were seeded onto agarose micro-well molds to form the spheroids. After 6 days, spheroids were treated with a PM (0 and 100 µg/ml) for 4 days. Cell proliferation, spheroids diameter and protein levels of ECM components were measured for both groups. Participants/materials, setting, methods Ovaries from four deceased post-menopausal multi-organ donors (IRB reference 2018/19DEC/475) were used for cell isolation. After seeding of cells, their diameter was measured on days 6 (start of treatment) and 10 (end of treatment) using an inverted microscope to assess their development. The study measured the protein levels of collagen VI, Emilin, and Fibrilin using immunohistochemistry and Ki67 using immunofluorescence in ovarian spheroids. The picrosirius red staining (PSR) was used to measure the collagen content. Main results and the role of chance The evaluation of spheroid diameter indicated comparable sizes on day 6 for both groups (0 µg/ml PM: 109±1.3 µm; 100 µg/ml PM: 112.5±1.1 µm). By day 10, there were no notable differences in spheroid size for the group exposed to PM in comparison to the control group (0 µg/ml PM: 104±1.2 µm; 100 µg/ml PM: 107.2±1.2 µm). The assessment of proliferation revealed no significant difference in the expression of ki67-positive cells between the groups (0 µg/ml PM: 3.211±0.64 %; 100 µg/ml PM: 3.379±0.61%; Fig 1). On the other hand, immunostaining results displayed a decrease in EMILIN-1 (171.6±5.31 vs 201.9±4.71 in control, P &amp;lt; 0.0001) and fibrillin levels (53.1±3.34 vs. 88.3±3.68 in control, P &amp;lt; 0.0001) post-PM treatment. Conversely, collagen VI levels were significantly higher in spheroids treated with PM compared to the control group (122.8±4.99 vs 104.8±4.04, P = 0.005). Although PSR revealed an increase following treatment (9.62±0.75 vs 8.64±0.70 in control), this change did not reach statistical significance (P = 0.344). Limitations, reasons for caution This study exclusively focused on ovarian stromal cells from post-menopausal women. Further research is needed to explore the impacts of PM on ECM of prepubertal and reproductive-age women. Wider implications of the findings Our study highlights the detrimental impact of phthalates on the ECM of postmenopausal women. Findings suggest ovarian stiffness by decreased elastogenesis-associated proteins (EMILIN-1, fibrillin-1) and increased collagen VI (not I, III) post-phthalate exposure.This pioneering study prompts further exploration of phthalate-induced ECM changes and their potential influence on steroidogenesis and folliculogenesis. Trial registration number not applicable

P-786 Mesenchymal stem cells as a potential source of ovarian theca interna cell differentiation

Abstract Study question Are mesenchymal stem cells (MSCs) the cell source of origin during the differentiation of ovarian stromal cells into theca interna cells (TICs)? Summary answer MSC-like cells were observed during in vitro TIC differentiation, particularly on day 2, suggesting their potential role in the origin of TICs. What is known already Folliculogenesis is the process during which a follicle develops from its primordial towards its antral stage. Follicles in the antral stage consist of an oocyte, granulosa and theca cells. While the oocyte is surrounded by granulosa cells in the primordial stage, theca cells differentiate from their precursor cell from the secondary stage onwards. Recent studies identified the existence of theca stem cells (TSCs) in both humans and animals. These TSCs express both platelet-derived growth factor receptor (PDGFR) and nerve growth factor receptor (NGFR), and demonstrate the ability to differentiate into mesenchymal cell lineages, resembling characteristics of MSCs. Study design, size, duration Ovarian stromal cells were isolated from cryopreserved cortex fragments, and cultured in vitro for 13 days, using cell media enriched with various growth factors and hormones, as previously established by our group. Flow cytometry, immunocytochemistry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were employed to analyze the cells and their media at days 0, 2, 8 and 13. Participants/materials, setting, methods Whole ovaries from three postmenopausal organ donors were obtained (50, 54, and 64 years) and the cortex was cryopreserved in small 5x5 fragments prior to the isolation and differentiation into TICs. Main results and the role of chance Immunocytochemistry results showed an increase in the proportion of cells positive for TIC markers from day 0 to day 8 (12.13 ± 2.11% vs. 57.73 ± 49.99%), while the proportion of MSC-like cell markers was increased significantly from day 0 to day 2 (4.54 ± 1.71% vs. 57.34 ± 18.05%). This TIC proportion aligns with the rising androstenedione levels from day 2 to day 13 (81.31 ± 25.09 pg/ml vs. 384.29 ± 245.82 pg/ml). Flow cytometry analysis identified a CD73+ CD90+ CD105+ CD34- CD45- HLA-DR- cell population, expanding from less than 1% on day 0, to 15.30 ± 7.9% on day 2. A CD13+ cell population and its corresponding gene expression increased significantly from the beginning towards the end of the culture (13.52 ± 7.9% vs. 84.13 ± 11.08, and 1.38 ± 0.97 vs. 285.80 ± 27.43, flow cytometry and gene expression analysis respectively). Moreover, NGFR and PDGFRA expression was induced on day 0 and day 2, respectively, compared to day 13 (1.12 ± 0.60 vs. 0.08 ± 0.08, and 2.03 ± 1.11 vs. 0.24 ± 0.14). In conclusion, our study provides valuable insights into the differentiation potential of TICs, suggesting a possible origin from MSCs. Limitations, reasons for caution Due to the limited accessibility of a large quantity of human ovarian tissue from postmenopausal women, this study is restricted to three biological replicates. When examining statistical differences, it is crucial to bear in mind the considerable biological variabilities that exist among humans. Wider implications of the findings Characterizing the origin of TICs will allow for a comprehensive study of TICs, leading to a broader knowledge of these cells. The in vitro differentiation to TICs from easily accessible cells, like MSCs, holds potential significance in toxicity studies and investigating TIC-related disorders, such as polycystic ovary syndrome (PCOS). Trial registration number not applicable

Systemic low-dose anti-fibrotic treatment attenuates ovarian aging in the mouse.

The female reproductive system is one of the first to age in humans, resulting in infertility and endocrine disruptions. The aging ovary assumes a fibro-inflammatory milieu which negatively impacts gamete quantity and quality as well as ovulation. Here we tested whether the systemic delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs attenuates ovarian aging in mice. We first evaluated the ability of these drugs to decrease the expression of fibro-inflammatory genes in primary ovarian stromal cells. Whereas Etanercept did not block Tnf expression in ovarian stromal cells, Pirfenidone significantly reduced Col1a1 expression. We then tested Pirfenidone in vivo where the drug was delivered systemically via mini-osmotic pumps for 6-weeks. Pirfenidone mitigated the age-dependent increase in ovarian fibrosis without impacting overall health parameters. Ovarian function was improved in Pirfenidone-treated mice as evidenced by increased follicle and corpora lutea number, AMH levels, and improved estrous cyclicity. Transcriptomic analysis revealed that Pirfenidone treatment resulted in an upregulation of reproductive function-related genes at 8.5 months and a downregulation of inflammatory genes at 12 months of age. These findings demonstrate that reducing the fibroinflammatory ovarian microenvironment improves ovarian function, thereby supporting modulating the ovarian environment as a therapeutic avenue to extend reproductive longevity.

Ovarian neuroendocrine tumor metastases can induce estrogen production in postmenopausal patients.

Neuroendocrine tumors (NETs) are malignant neoplasms that can be associated with specific hormonal syndromes. We describe a novel syndrome of postmenopausal vaginal bleeding and ovarian estradiol overproduction due to ovarian NET localizations. An extensive workup was performed for 2 index patients with ovarian metastases of small bowel neuroendocrine tumors and symptoms of postmenopausal vaginal bleeding. Clinically significant ovarian estrogen production was demonstrated by a combination of ovarian vein sampling and normalization of circulating estrogen levels after oophorectomy. Immunohistochemical analyses revealed marked aromatase immunoactivity in the ovarian NET cells, while CYP17A1 and SF-1 were detected in the adjacent ovarian stromal cells but not the NET cells. Ex vivo and in vivo endocrine tests were unable to identify a paracrine mechanism of ovarian estradiol overproduction by NET cells. A retrospective search of electronic medical records revealed that 21% (14/66) of postmenopausal patients with an ovarian NET localization reported symptoms of vaginal blood loss. Together, these findings support the presence of a novel NET-associated hormonal syndrome.

Thymol increases primordial follicle activation, protects stromal cells, collagen fibers and down-regulates expression of mRNA for superoxide dismutase 1, catalase and periredoxin 6 in cultured bovine ovarian tissues

This study aims to investigate the influence of thymol on primordial follicle growth and survival, as well as on collagen fibers and stromal cells density in bovine ovarian tissues cultured in vitro. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the thiol levels and the expression of mRNAs for SOD1, CAT, periredoxin 6 (PRDX6) and GPX1 were also investigated. Ovarian cortical tissues were cultured in α-MEM+ alone or with thymol (400, 800, 1600 or 3200 μg/mL) for six days. Before and after culture, the tissues were processed for histological analysis to evaluate follicular activation, growth, morphology, ovarian stromal cell density and collagen fibers. The levels of mRNA for SOD1, CAT, GPX1 and PRDX6 were evaluated by real-time PCR. The results show that tissues cultured with thymol (400 and 800 µg/mL) had increased percentages of normal follicles, when compared to tissues cultured in other treatments. At concentrations of 400 and 800 µg/mL, thymol maintained the rate of normal follicles similar to the uncultured control. In addition, 400 µg/mL thymol increased follicle activation, collagen fibers and stromal cell density of when compared to tissues cultured in control medium. The presence of 800 µg/mL thymol in culture medium increased CAT activity, while 400 or 800 µg/mL thymol reduced mRNA levels for SOD1, CAT and PRDX6, but did not alter GPX1 expression. In conclusion, 400 µg/mL thymol increases primordial follicle activation, preserves stromal cells, collagen fibers, and down-regulates expression of mRNA for SOD1, CAT and PRDX6 in cultured bovine ovarian tissues.

Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries

Background and aimsOvum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions. MethodsMSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4). ResultsCharacterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions. ConclusionsMSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo.

Extract of Actaea racemosa Protects Mice Ovarian Follicles Against Doxorubicin-induced Toxicity

Background: Given the cytotoxicity of chemotherapy drugs used in cancer treatment, there is a need to develop alternative agents to protect female fertility. This study investigated the effect of Actaea racemosa (A. racemosa) extract on mice ovarian cells and the damage caused by doxorubicin (DOX) to the mice ovaries. Methods: We evaluated the effects of A. racemosa extract on mice ovaries (n=42) after DOX treatment. The mice were pre-treated with saline solution (controls) or with 0.5 or 5 mg/kg A. Racemosa extract. Afterward, during a period of 10 days, they were treated daily with one of the six protocols: (i) saline solution (control), (ii) 10 mg/kg DOX, (iii) 0.5 mg/kg A. racemosa extract, (iv) both DOX and 5 mg/kg A. racemosa extract, (v) A. racemosa extract (5 mg/kg), and (vi) both DOX and 0.5 mg/kg A. racemosa extract. At the end of these treatments, the ovaries were fixed for histopathological examinations. Ovarian follicular morphology, stromal cell density, collagen fibers, and TNF-α expression were evaluated. Some ovaries were fixed for transmission electron microscopy or stored at -80oC to study the mRNA expression for Caspase-3 and TNF-α. Results: The Mice treated with A. racemosa extract had reduced follicular degeneration and cell death after exposure to DOX. Ovaries of mice treated with 0.5 mg/kg A. racemosa extract had granulosa cells and oocytes with preserved ultrastructure, decreased immunostaining for TNF-α, and reduced Caspase-3 mRNA. Conclusion: The A. racemosa extract supported follicular survival and protected the ovarian follicles and stromal cells against DOX-induced cytotoxicity.

Bioprinting of a Liposomal Oxygen-Releasing Scaffold for Ovary Tissue Engineering.

This study addresses a critical challenge in bioprinting for regenerative medicine, specifically the issue of hypoxia compromising cell viability in engineered tissues. To overcome this hurdle, a novel approach using a microfluidic bioprinter is used to create a two-layer structure resembling the human ovary. This structure incorporates a liposomal oxygen-releasing system to enhance cell viability. The bioprinting technique enables the simultaneous extrusion of two distinct bioinks, namely, bioink A (comprising alginate 1% and 5 mg/mL PEGylated fibrinogen in a 20:1 molar ratio) and bioink B (containing alginate 0.5%). In addition, liposomal catalase and hydrogen peroxide (H2O2) are synthesized and incorporated into bioinks A and B, respectively. The liposomes are prepared using thin film hydration with a monodisperse size (140-160 nm) and high encapsulation efficiency. To assess construct functionality, isolated human ovarian cells are added to bioink A. The bioprinted constructs, with or without liposomal oxygen-releasing systems, are cultured under hypoxic and normoxic conditions for 3 days. Live/Dead assay results demonstrate that liposomal oxygen-releasing systems effectively preserve cell viability in hypoxic conditions, resembling viability under normoxic conditions without liposomes. PrestoBlue assay reveals significantly higher mitochondrial activity in constructs with liposomal oxygen delivery systems under both hypoxic and normoxic conditions. The evaluation of apoptosis status through annexin V immunostaining shows that liposomal oxygen-releasing scaffolds successfully protect cells from hypoxic stress, exhibiting a proportion of apoptotic cells similar to normoxic conditions. In contrast, constructs lacking liposomes in hypoxic conditions exhibit a higher incidence of cells in early-stage apoptosis. In conclusion, the study demonstrates the promising potential of bioprinted oxygen-releasing liposomal scaffolds to protect ovarian stromal cells in hypoxic environments. These innovative scaffolds not only offer protection but also recapitulate the mechanical differences between the medulla and the cortex in the normal ovary structure. This opens new avenues for advanced ovary tissue engineering and transplantation strategies.

Therapeutic efficacy and anti-inflammatory mechanism of baicalein on endometriosis progression in patient-derived cell line and mouse model

BackgroundBaicalein is a flavonoid extracted from the roots of Scutellaria baicalensis G. that has anti-inflammatory and antitumor effects. However, therapeutic mechanisms of baicalein in patients with endometriosis in vivo have yet to be elucidated. As a chronic inflammatory gynecological disease, endometriosis causes pain and infertility, and has no complete treatment to date. Current treatment strategies cause several side effects and have high recurrence rates. PurposeThis study aimed to identify the in vivo therapeutic effects of baicalein on endometriosis and verify the action mechanisms of baicalein, focusing on regulating inflammation. MethodsIn this study, an autologous transplant mouse model and patient-derived immortalized human ovarian endometriotic stromal cells (ihOESCs) were used to investigate the therapeutic activities of baicalein. The mouse model was administered with 40 mg/kg baicalein by oral gavage for 4 weeks, and the treatment outcomes of baicalein-treated mice were compared with vehicle- and dienogest-treated groups. ihOESCs were treated with 0–5 μg/ml baicalein for in vitro studies. ResultsBaicalein significantly alleviated the progression of endometriosis in mouse models. Baicalein reduced the expression of proinflammatory cytokines in endometriotic lesions and ihOESCs, and cytokine expression and T cell proportions in mouse spleen. in vitro results showed that baicalein increased mitochondrial calcium flux and induced mitochondrial depolarization and ROS generation in ihOESCs. Ultimately, baicalein inactivated the MAPK/PI3K signaling and induced cell death in ihOESCs. ConclusionIn conclusion, baicalein effectively attenuated the progression of endometriosis through its anti-inflammatory activities. Baicalein can be an alternative or supplemental treatment for endometriosis to ameliorate the side effects of hormonal therapy.

Evaluation of the morphology and development of preantral ovarian follicles in mice submitted to a chronic diet of dietary supplementation with Pereskia aculeata Miller leaves.

This study aimed to investigate the effect of including mouse feed with different concentrations (5, 10, or 20%) of Pereskia aculeata Miller (PAM) leaves on the morphology and development of preantral ovarian follicles and ovarian stromal cell density. The oral toxicity was performed using repeated dose toxicity assays subdivided into experiments of 30 days and 90 days of treatment. After the experiments, the ovaries of each animal were collected and submitted to classical histology. At 30 and 90 days, there was an equivalent percentage of normal, primordial, and developing follicles (P > 0.05) between PAM treatments compared to the control. Regarding the different stages of follicular development, after 90 days, there was a higher percentage (P < 0.05) of developing follicles only in the control group compared to day 30. The PAM 5% treatment was the only one that affected the cell density in the stroma after 90 days of treatment. Thus, we observed that supplementing the diet with P. aculeata did not pose any risk concerning animal consumption; specifically, there were no toxic reproductive effects observed from adding Pereskia aculeata Miller to the mouse diet.