Unusual radiologic imaging in juvenile granulosa cell tumor with precocious puberty: A unilocular cyst.

Juvenile Granulosa Cell Tumor (JGCT) represents 5% of all granulosa cell cancers. Precocious puberty is a frequent feature of this tumor. A 2-year and 2-month-old girl was referred with a diagnosis of suspected ovarian cancer, dysfunctional uterine bleeding, and precocious puberty. Radiologic examination revealed the following: Abdominal ultrasonography showed a solitary anechoic cystic lesion in the pelvic cavity. MRI confirmed the existence of solid components on its walls. JGCT was then confirmed using immunohistochemistry (IHC) markers. JGCT, along with adult granulosa cell tumors (AGCT) are subgroups of granulosa cell tumors (GCTs), which are part of pure sex cord tumors. The 2 forms share imaging findings due to their comparable gross appearance. GCTs require diagnostic imaging tests to distinguish them from other ovarian tumors. Two ultrasound patterns can be identified GCTs, and MRI showed that GCTs are more heterogeneous than other sex-cord stromal tumors (OSCs). In our case, the imaging characteristics for juvenile granulosa cell tumors were nonspecific and these tumors cannot be reliably distinguished from other ovarian neoplasms based on imaging alone. Although GCTs have imaging characteristics that can help to distinguish them from other tumors, confirmation by histopathology and IHC is still mandatory, especially in cases with nonspecific radiological features.

The Protective Role of Quercetin Against Copper-Induced Female Reproductive Toxicity: Insights from Transcriptome Analysis.

Quercetin has been shown to mitigate the cytotoxic effects of heavy metals. While copper is an essential trace element for bodily functions, excessive intake has been linked to impaired female reproductive function. Transcriptome analysis was employed to identify genes that are differentially expressed in response to high copper and were validated through qRT-PCR and WB. ATP content and Tunel were used to identify the damage of mitochondrial and cell apoptosis. PPI analysis revealed that MKI67, TOPII, ASPM, CASP3, PLK1, and TTK are central proteins within the network. Additionally, exposure to elevated levels of copper resulted in the dysregulation of 86 genes associated with mitochondria. Conversely, treatment with quercetin (QUE) in combination with high copper led to the normalization of 42 mitochondria-related genes previously affected by high copper levels. Furthermore, CuSO4 decreases ATP content and induces cell apoptosis, which can be reversed by QUE. Results suggest that elevated copper levels can lead to oxidative stress and apoptosis by inducing mitochondrial damage, while QUE has the potential to mitigate these effects, ultimately safeguarding granulosa cells and halting the progression of cell death. This study provides novel insights into the molecular pathways involved in female reproductive toxicity caused by excessive copper exposure.

A Rare Ovarian Mixed Sex Cord Stromal Tumor in a Patient with Ollier Disease: A Case Report.

This is a case report of a 10-year-old with Ollier disease and an ovarian mass. Ollier disease, a rare disorder characterized by multiple enchondromas resulting in bone deformities, has been occasionally associated with ovarian juvenile granulosa cell tumor. This patient developed signs of precocious puberty and was found to have an ovarian tumor; however, pathology revealed a mixed sex-cord stromal tumor with components of juvenile granulosa and Sertoli-Leydig cell tumor. Tumor genomic testing revealed an IDH1 mutation. Mixed sex-cord stromal tumors of this type, also called "gynandroblastomas," have been associated with DICER1 mutations and DICER1 tumor predisposition syndrome but never with Ollier disease. Our findings expand the known spectrum of syndromic associations with this tumor type, with implications for tumor screening.

Telomerase activity, telomere length, and the euploidy rate of human embryos.

BACKGROUND: Telomeres maintain chromosome stability, while telomerase counteracts their progressive shortening. Telomere length varies between cell types, with leukocyte telomere length (LTL) decreasing with age. Reduced telomerase activity has been linked to reproductive issues in females, such as low pregnancy rates and premature ovarian failure, with recent studies indicating correlations between telomere length in granulosa cells and IVF outcomes. OBJECTIVES: The study aims to explore the relationship between telomere length, telomerase activity, and euploid blastocyst rate in infertile women undergoing IVF/ICSI PGT-A cycles. METHODS: This prospective study involves 108 patients undergoing controlled ovarian stimulation and PGT-A. Telomere length and telomerase activity were measured in peripheral mononuclear cells and granulosa cells (GC), respectively. RESULTS: The telomere repeat copy number to single gene copy number ratio (T/S) results respectively 0.6 ± 0.8 in leukocytes and 0.7 ± 0.9 in GC. An inverse relationship was found between LTL and the patient's age (p < .01). A higher aneuploid rate was noticed in patients with short LTL, with no differences in ovarian reserve markers (p = .15), number of oocytes retrieved (p = .33), and number of MII (p = 0.42). No significant association was noticed between telomere length in GC and patients' age (p = 0.95), in ovarian reserve markers (p = 0.32), number of oocytes retrieved (p = .58), number of MII (p = .74) and aneuploidy rate (p = .65). CONCLUSION: LTL shows a significant inverse correlation with patient age and higher aneuploidy rates. Telomere length in GCs does not correlate with patient age or reproductive outcomes, indicating differential telomere dynamics between leukocytes and granulosa cells.

Granulosa Cell-Layer Stiffening Prevents Escape of Mural Granulosa Cells from the Post-Ovulatory Follicle.

Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post-ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC-layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post-ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)-cAMP-PKA-CREB signaling cascade, is necessary for mGC-layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of "mGC-layer stiffening", shedding light on the mechanism that prevents mGC escape from the post-ovulatory follicle.

Protease Expression in the Human and Rat Cumulus Oocyte Complex (COC) During the Periovulatory Period: A Role in COC Migration.

The migratory and matrix-invading capacities of the cumulus oocyte complex (COC) have been shown to be important for the ovulatory process. In metastatic cancers, these capacities are due to increased expression of proteases, however, there is limited information on protease expression in the COCs. The present study examined COC expression of plasmins, matrix metalloproteases (MMP) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family members in the rat and human. In the rat, hCG administration increased COC expression of Mmp2, Mmp9, Mmp13, Mmp14, Mmp16, Adamts1, and the protease inhibitors Timp1, Timp3 and Serpine1 by 8-12 hours. This ovulatory induction of proteases in vivo could be mimicked by forskolin and ampiregulin treatment of cultured rat COCs with increases observed in Mmp2, Mmp13, Mmp14, Mmp16, Mmp19, Plat, and the protease inhibitors Timp1, Timp3 and Serpine1. Comparison of expression between rat COCs and granulosa cells at the time of ovulation showed decreased Mmp9 and increased Mmp13, Mmp14, Mmp16, Adamts1, Timp1 and Timp3 expression in the COCs. In human, comparison of expression between cumulus and granulosa cells at the time of IVF retrieval showed decreased MMP1, MMP2, MMP9, and ADAMTS1, while expression of MMP16, TIMP1, and TIMP3 were increased. Treatment of expanding rat COCs with a broad spectrum MMP inhibitor, GM6001, significantly reduced the migration of cumulus cells in vitro. These data provide evidence that multiple proteases and their inhibitors are expressed in the COCs and play an important role in imparting the migratory phenotype of the COCs at the time of ovulation.

Single-cell transcriptomic profiling unveils insights into ovarian fibrosis in obese mice.

BACKGROUND: Adiposity profoundly impacts reproductive health in both humans and animals. However, the precise subpopulations contributing to infertility under obese conditions remain elusive. RESULTS: In this study, we established an obese mouse model through an eighteen-week high-fat diet regimen in adult female mice. Employing single-cell RNA sequencing (scRNA-seq), we constructed a comprehensive single-cell atlas of ovarian tissues from these mice to scrutinize the impact of obesity on the ovarian microenvironment. ScRNA-seq revealed notable alterations in the microenvironment of ovarian tissues in obese mice. Granulosa cells, stromal cells, T cells, and macrophages exhibited functional imbalances compared to the control group. We observed heightened interaction strength in the SPP1-CD44 pairing within lgfbp7+ granulosa cell subtypes and Il1bhigh monocyte subtypes in the ovarian tissues of obese mice. Moreover, the interaction strength between Il1bhigh monocyte subtypes and Pdgfrb+ stromal cell subtypes in the form of TNF - TNFrsf1α interaction was also enhanced subsequently to obesity, potentially contributing to ovarian fibrosis pathogenesis. CONCLUSIONS: We propose a model wherein granulosa cells secrete SPP1 to activate monocytes, subsequently triggering TNF-α secretion by monocytes, thereby activating stromal cells and ultimately leading to the development of ovarian fibrosis. Intervening in this process may represent a promising avenue for improving clinical outcomes in fertility treatments for obese women.

Basket study of oral progesterone antagonist onapristone extended release in progesterone receptor-positive recurrent granulosa cell, low-grade serous ovarian cancer, or endometrioid endometrial cancer.

OBJECTIVE: To determine the safety and efficacy of the oral progesterone antagonist onapristone extended release (onapristone-XR) in patients with recurrent progesterone receptor (PR)-positive adult-type granulosa cell tumor (aGCT), low-grade serous ovarian cancer (LGSOC), or endometrioid endometrial cancer (EEC). METHODS: This single-institution phase II study included patients with PR-positive aGCT, LGSOC, or EEC who received ≥1 prior line of chemotherapy. Patients were enrolled from 5/2019-5/2020. PR status was evaluated via immunohistochemistry. Eligible patients had PR expression ≥1% on tissue collected within 3 years of enrollment. Patients received 50 mg of onapristone-XR twice daily until disease progression or treatment discontinuation. Adverse events were graded by Common Terminology Criteria for Adverse Events version 5.0. The primary endpoint was overall response rate (ORR) by Response Evaluation Criteria in Solid Tumors 1.1. Secondary endpoints were response duration, clinical benefit rate (CBR), and safety. RESULTS: Five patients with LGSOC and 1 with EEC enrolled, but both cohorts closed early due to slow accrual. Fourteen patients with aGCT enrolled and completed stage 1 accrual. No responses were observed. Four patients with LGSOC were evaluable, with median PFS of 4.4 months (range, 1.8-NE) and CBR of 50% (range, 6.8%-93.2%). All 14 patients with aGCT were evaluable, with median PFS of 2.8 months (range, 1.6-4.9), 6-month PFS rate of 21.4% (range, 5.2%-44.8%), 12-month PFS rate of 14.3% (range, 2.3%-36.6%), and a CBR of 35.7% (range, 12.8%-64.9%). CONCLUSIONS: The study did not meet its primary endpoint. While onapristone-XR was well tolerated in all 3 arms, no objective responses were observed.

Transcriptome Dynamics and Cell Dialogs Between Oocytes and Granulosa Cells in Mouse Follicle Development.

The development and maturation of follicles is a sophisticated and multistage process. The dynamic gene expression of oocytes and their surrounding somatic cells and the dialogs between these cells are critical to this process. In this study, we accurately classified the oocyte and follicle development into nine stages and profiled the gene expression of mouse oocytes and their surrounding granulosa cells and cumulus cells. The clustering of the transcriptomes showed the trajectories of two distinct development courses of oocytes and their surrounding somatic cells. Gene expression changes precipitously increased at Type 4 stage and drastically dropped afterward within both oocytes and granulosa cells. Moreover, the number of differentially expressed genes between oocytes and granulosa cells dramatically increased at Type 4 stage, most of which persistently passed on to the later stages. Strikingly, cell communications within and between oocytes and granulosa cells became active from Type 4 stage onward. Cell dialogs connected oocytes and granulosa cells in both unidirectional and bidirectional manners. TGFB2/3, TGFBR2/3, INHBA/B, and ACVR1/1B/2B of TGF-ß signaling pathway functioned in the follicle development. NOTCH signaling pathway regulated the development of granulosa cells. Additionally, many maternally DNA methylation- or H3K27me3-imprinted genes remained active in granulosa cells but silent in oocytes during oogenesis. Collectively, Type 4 stage is the key turning point when significant transcription changes diverge the fate of oocytes and granulosa cells, and the cell dialogs become active to assure follicle development. These findings shed new insights on the transcriptome dynamics and cell dialogs facilitating the development and maturation of oocytes and follicles.

Collision Tumor of the Ovary: Adult Granulosa Cell Tumor and Mesonephric-like Adenocarcinoma.

Collision tumors of the ovaries are rare, with only a few reports in the literature. Adult granulosa cell tumors are a relatively common primary tumor component of previously reported collision tumors. The combination of serous and mucinous tumors with adult granulosa cell tumors has been reported in several cases. On the other hand, mesonephric-like adenocarcinomas are rare neoplasms that commonly arise in the uterine corpus and ovaries. In this report, we present the case of a collision tumor composed of an adult granulosa cell tumor and mesonephric-like adenocarcinoma of the ovary in a 63-year-old woman. The initial magnetic resonance imaging findings showed a cystic mass with an internal hemorrhage, which suggested an adult granulosa cell tumor, and a solid mass with different enhancements. Microscopically, the tumor had two distinct components: An adult granulosa cell tumor and a mesonephric-like adenocarcinoma. Recognizing collision tumors consisting of slow-growing and aggressive tumors may prove beneficial in future diagnostic and treatment strategies.

CircRNAs involved in the red light of effect on follicle selection in pigeons.

Red light (RL) can enhance egg production in poultry. CircRNAs play a crucial role by serving as transcriptional regulators. However, their role in influencing follicle development in White King pigeons remains unexplored. In this study, 54 paired White King pigeons were chosen and divided into RL and white light (WL) groups, each with 3 subgroups. The egg production of paired pigeons in each replicate was recorded for 45 d, and the characteristics of follicle development were monitored during the laying interval (LI). The granulosa cell layer from follicles of the second-largest follicle (F2) was collected, and high-throughput sequencing was performed to elucidate the molecular mechanism of follicle development in pigeons. The study confirmed that RL enhances egg production in pigeons. Additionally, under RL, the F2 follicle was selected, while under WL, small follicles were kept on the third day (LI3). A total of 5,510 circRNAs were identified across all samples, revealing differentially expressed circRNAs (DECs) in various comparisons: 627 in RF1 vs. WF1, 900 in RF2 vs. WF2, 606 in RF1 vs. RF2, and 937 in WF1 vs. WF2. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that host genes of DECs were enriched in pathways like steroid hormone biosynthesis, oocyte meiosis, GnRH signaling pathway, and apoptosis pathway. Moreover, circRNA_5497, circRNA_2016, and circRNA_3328 were common DECs across 4 groups, sharing miRNA binding sites with follicle selection-associated genes. In conclusion, our findings suggest that RL promotes egg production by stimulating follicle selection during LI, offering insights into the regulatory mechanisms of circRNAs in follicle selection under RL. This knowledge can help enhance the reproductive performance of pigeons.

Expression levels of the selenium-uptake receptor LRP8, the antioxidant selenoprotein GPX1 and steroidogenic enzymes correlate in granulosa cells.

Abstract: Reactive oxygen species (ROS) are a by-product of the activity of cytochrome P450 steroidogenic enzymes. Antioxidant enzymes protect against ROS damage. To identify if any particular antioxidant enzyme is used to protect against ROS produced by granulosa cells as follicles enlarge and produce oestradiol, we measured in the bovine granulosa cells the expression of two steroidogenic enzymes (CYP11A1, CYP19A1), important for progesterone and oestradiol production. We also measured the expression of the members (FDXR, FDX1, POR) of their electron transport chains (ETC). We measured antioxidant enzymes (GPXs 1-8, CAT, SODs 1 and 2, PRDXs 1-6, GSR, TXN, TXNRDs 1-3). Since selenium is an active component of GPXs, the selenium-uptake receptors (LRPs 2 and 8) were measured. Only the selenium-dependent GPX1 showed the same increase in expression as the steroidogenic enzymes did with increasing follicle size. GPX4 and PRDX2/6 decreased with follicle size, whereas SOD1/2, CAT, GSR, and TXNRD3 were lowest at the intermediate sizes. The other antioxidant enzymes were unchanged or expressed at low levels. The expression of the selenium-uptake receptor LRP8 also increased significantly with follicle size. Correlation analysis revealed statistically significant and strongly positive correlations of the steroidogenic enzymes and their ETCs with both GPX1 and LRP8. These results demonstrate a relationship between the expression of genes involved in steroidogenesis and selenium-containing antioxidant defence mechanisms. They suggest that during the late stages of folliculogenesis, granulosa cells are dependent on sufficient expression of GPX1 and the selenium transporter LRP8 to counteract increasing ROS levels caused by the production of steroid hormones. Lay summary: In the ovary, eggs are housed in follicles which contain the cells that produce oestrogen in the days leading up to ovulation of the egg. Oestrogen is produced by the action of enzymes. However, some of these enzymes also produce by-products called reactive oxygen species (ROS). These are harmful to eggs. Fortunately, cells have protective antioxidant enzymes that can neutralise ROS. This study was interested in which particular antioxidant enzyme(s) might be involved in neutralising the ROS in follicle cells. It was found that only one antioxidant enzyme, GPX1, appeared to be co-regulated with the enzymes that produce oestrogen and progesterone in the follicular cells. GPX1 contains the essential mineral selenium. In summary, this study has identified which antioxidant appears to be involved in neutralising ROS in the days leading to ovulation. It highlights the importance of selenium in the diet.

An Approach to Improve Endometrial Receptivity: Is It Beneficial to Flush The Uterine Cavity with Follicular Fluid and Granulosa Cells? A Phase III Randomised Clinical Trial.

BACKGROUND: The follicular fluid (FF) of mature oocytes contains a high concentration of growth factors and cytokines that have the potential to influence implantation in either a paracrine or autocrine manner. During the physiological processes of ovulation, FF enters the fallopian tubes in conjunction with the oocyte. The purpose of this study is to evaluate implantation and clinical pregnancy rates following uterine flushing with FF and granulosa cells in infertile women with moderate male factor infertility after ovum retrieval for intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: This phase III randomised clinical trial enrolled 140 women with moderate male factor infertility who intended to undergo ICSI at Royan Infertility Clinic (Tehran, Iran). A computer-generated program and opaque sealed envelopes were used to randomly allocate patients to either an intervention group (n=70) or a control group (n=70). Participants in the intervention group received 2 ml of clear FF (without blood contamination) from 2 to 3 dominant follicles after oocyte retrieval. The control group only underwent uterine cavity catheterisation. RESULTS: The intervention group had a clinical pregnancy rate of 38.5% (25/65) compared to the control group [42.9% (27/63); P=0.719] and an implantation rate of 24.1% compared to the control group (27%; P=0.408). These rates did not differ between the groups. There were no statistically significant differences between the intervention and control groups in terms of pregnancy-related complications-ectopic pregnancy, blighted ovum or anembryonic pregnancy, and abortion. CONCLUSION: Uterine cavity flushing with FF from mature follicles following oocyte retrieval had no effect, either positively or negatively, on clinical pregnancy or implantation rates in women with moderate male factor infertility (registration number: NCT04077970).

An extensive immunohistochemical analysis of 290 ovarian adult granulosa cell tumors with 29 markers.

The current knowledge about the immunohistochemical features of adult granulosa cell tumor (AGCT) is mostly limited to the "traditional" immunohistochemical markers of sex cord differentiation, such as inhibin, calretinin, FOXL2, SF1, and CD99. Knowledge about the immunohistochemical markers possibly used for predictive purpose is limited. In our study, we focused on the immunohistochemical examination of 290 cases of AGCT classified based on strict diagnostic criteria, including molecular testing. The antibodies used included 12 of the "diagnostic" antibodies already examined in previous studies, 10 antibodies whose expression has not yet been examined in AGCT, and 7 antibodies with possible predictive significance, including the expression of HER2, PD-L1, CTLA4, and 4 mismatch repair (MMR) proteins. The results of our study showed expression of FOXL2, SF1, CD99, inhibin A, calretinin, ER, PR, AR, CKAE1/3, and CAIX in 98%, 100%, 90%, 78%, 45%, 41%, 94%, 82%, 26%, and 9% of AGCT, respectively. GATA3, SATB2, napsin A, MUC4, TTF1, and CD44 were all negative. PTEN showed a loss of expression in 71% of cases and DPC4 in 4% of cases. The aberrant staining pattern (overexpression) of p53 was found in 1% (3/268) of cases, 2 primary tumors, and 1 recurrent case. Concerning the predictive markers, the results of our study showed that AGCT is microsatellite stable, do not express PD-L1, and are HER2 negative. The CTLA4 expression was found in almost 70% of AGCT tumor cells.

The experiance of tertiary center for adult granulosa cell tumor: which factors predict survival?

BACKGROUND: This retrospective study aims to evaluate the clinical course and long-term outcomes of patients diagnosed with adult granulosa cell tumors (AGCT). METHODS: The study analyzed a cohort of 112 AGCT patients with a median follow-up of 87 months. Data regarding disease-free survival (DFS), overall survival (OS), recurrence rates, and prognostic factors were collected and analyzed. Surgical interventions, including lymphadenectomy and cytoreductive surgery, were assessed for their impact on outcomes. RESULTS: The study revealed favorable long-term outcomes, with a 5-year DFS of 85% and a 10-year DFS of 83%. Additionally, a 5-year OS of 100% and a 10-year OS of 96% were observed. Recurrence occurred in 13.4% of cases, with advanced stage and positive peritoneal cytology identified as independent poor prognostic factors for DFS. Lymph node involvement was rare, and routine lymphadenectomy did not improve outcomes. Conservative surgery showed comparable DFS rates to definitive surgery in early-stage disease. However, cytoreductive surgery was crucial for advanced and recurrent tumors, with complete tumor resection enhancing survival outcomes. CONCLUSION: The study underscores the importance of vigilant follow-up and individualized treatment strategies for AGCT patients. Despite the retrospective nature of the analysis, the substantial patient cohort and meticulous surgical interventions contribute valuable insights into AGCT management. Prospective multicenter studies are warranted to further elucidate prognostic factors and optimize treatment approaches for this rare malignancy.

Epigenetic age acceleration in follicular fluid and markers of ovarian response among women undergoing IVF.

STUDY QUESTION: Are markers of epigenetic age acceleration in follicular fluid associated with outcomes of ovarian stimulation? SUMMARY ANSWER: Increased epigenetic age acceleration of follicular fluid using the Horvath clock, but not other epigenetic clocks (GrimAge and Granulosa Cell), was associated with lower peak estradiol levels and decreased number of total and mature oocytes. WHAT IS KNOWN ALREADY: In granulosa cells, there are inconsistent findings between epigenetic age acceleration and ovarian response outcomes. STUDY DESIGN, SIZE, DURATION: Our study included 61 women undergoing IVF at an academic fertility clinic in the New England area who were part of the Environment and Reproductive Health Study (2006-2016). PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants provided a follicular fluid sample during oocyte retrieval. DNA methylation of follicular fluid was assessed using a genome-wide methylation screening tool. Three established epigenetic clocks (Horvath, GrimAge, and Granulosa Cell) were used to predict DNA-methylation-based epigenetic age. To calculate the age acceleration, we regressed epigenetic age on chronological age and extracted the residuals. The association between epigenetic age acceleration and ovarian response outcomes (peak estradiol levels, follicle stimulation hormone, number of total, and mature oocytes) was assessed using linear and Poisson regression adjusted for chronological age, three surrogate variables (to account for cellular heterogeneity), race, smoking status, initial infertility diagnosis, and stimulation protocol. MAIN RESULTS AND ROLE OF CHANCE: Compared to the median chronological age of our participants (34 years), the Horvath clock predicted, on an average, a younger epigenetic age (median: 24.2 years) while the GrimAge (median: 38.6 years) and Granulosa Cell (median: 39.0 years) clocks predicted, on an average, an older epigenetic age. Age acceleration based on the Horvath clock was associated with lower peak estradiol levels (-819.4 unit decrease in peak estradiol levels per standard deviation increase; 95% CI: -1265.7, -373.1) and fewer total (% change in total oocytes retrieved per standard deviation increase: -21.8%; 95% CI: -37.1%, -2.8%) and mature oocytes retrieved (% change in mature oocytes retrieved per standard deviation increase: -23.8%; 95% CI: -39.9%, -3.4%). The age acceleration based on the two other epigenetic clocks was not associated with markers of ovarian response. LIMITATIONS, REASONS FOR CAUTION: Our sample size was small and we did not specifically isolate granulosa cells from follicular fluid samples so our samples could have included mixed cell types. WIDER IMPLICATIONS OF THE FINDINGS: Our results highlight that certain epigenetic clocks may be predictive of ovarian stimulation outcomes when applied to follicular fluid; however, the inconsistent findings for specific clocks across studies indicate a need for further research to better understand the clinical utility of epigenetic clocks to improve IVF treatment. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants ES009718, ES022955, ES000002, and ES026648 from the National Institute of Environmental Health Sciences (NIEHS) and a pilot grant from the NIEHS-funded HERCULES Center at Emory University (P30 ES019776). RBH was supported by the Emory University NIH Training Grant (T32-ES012870). TRIAL REGISTRATION NUMBER: N/A.

Autophagy is involved in granulosa cell death and follicular atresia in ewe ovaries.

In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.

Follicular fluid-derived exosomal HMOX1 promotes granulosa cell ferroptosis involved in follicular atresia in geese (Anser cygnoides).

The proliferation and death of granulosa cells (GCs) in poultry play a decisive role in follicular fate and egg production. The follicular fluid (FF) contains a variety of nutrients and genetic substances to ensure the communication between follicular cells. Exosomes, as a new intercellular communication, could carry and transport the proteins, RNA, and lipids to react on GCs, which had been found in FF of various domestic animals. Whether exosomes of FF in poultry play a similar role is unclear. In this study, geese, a poultry with low egg production, were chosen, and the effect of FF exosomes on the proliferation and death of GCs was investigated. Firstly, there were not only a large number of healthy small yellow follicles (HSYFs) but also some atresia small yellow follicles (ASYFs) in the egg-laying stage. Also, the GC layers of ASYFs became loose interconnections, inward detachment, and diminished survival rate than that of HSYFs. Besides, compared to HSYFs, the contents of E2, P4, and the mRNA expression levels of ferroptosis-related genes GPX4, FPN1, and FTH1 were significantly decreased, while COX2, NCOA4, VDAC3 mRNA were significantly increased, and the structure of mitochondrial cristae disappeared and the outer membrane broke in the GC layers of ASYFs. Moreover, the ROS, MDA, and oxidation levels in the GC layers of ASYFs were significantly higher than those of HSYFs. All these hinted that ferroptosis might result in a large number of GCs death and involvement in follicle atresia. Secondly, FF exosomes were isolated from HSYFs and ASYFs, respectively, and identified by TEM, NTA, and detection of exosome marker proteins. Also, we found the exosomes were phagocytic by GCs by tracking CM-Dil. Moreover, the addition of ASYF-FF exosomes significantly elevated the MDA content, Fe2+ levels, and the mitochondrial membrane potential (MMP) in GCs, thus significantly inhibiting the proliferation of GCs, which was restored by the ferroptosis inhibitor ferrostatin-1. Thirdly, the proteomic sequencing was performed between FF-derived exosomes of HSYFs and ASYFs. We obtained 1615 differentially expressed proteins, which were mainly enriched in the protein transport and ferroptosis pathways. Among them, HMOX1 was enriched in the ferroptosis pathway based on differential protein-protein interaction network analysis. Finally, the role of HMOX1 in regulating ferroptosis in GCs was further explored. The highly expressed HMOX1 was observed in the exosomes of ASYF-FF than that in HSYF-FF. Overexpression of HMOX1 increased ATG5, LC3II, and NCOA4 expression and reduced the expression of FTH1, GPX4, PCBP2, FPN1 in the ferroptosis pathway, also promoted intracellular Fe2+ accumulation and MDA surge, which drove ferroptosis in GCs. The effects of HMOX1 on ferroptosis could be blocked by its inhibitor Znpp. Taken together, the important protein HMOX1 was identified in FF, which could be delivered to GCs via exosomes, triggering ferroptosis and thus determining the fate of follicles.

Changes in adiponectin levels of subclinical ketosis cows and their effects on steroid hormone secretion and proliferation in follicular granulosa cells.

In dairy cows, the occurrence of subclinical ketosis (SCK) is particularly high during early lactation. Previously, we documented alterations in the abundance of adiponectin (ADPN) in anestrus cows with SCK in comparison to cows in estrus. In the present study, 60 cows were divided into two groups: control (C, n = 30) and SCK (n = 30). Based on cow's estrus situation in two group at 55-60 days postpartum, 15 anestrus SCK cows and estrus cows were designated the SCK-A group and C-E group, respectively. The SCK-A group had downregulated serum and follicular fluid ADPN levels compared with the C-E group. The serum ADPN level was positively correlated with the insulin level and follicle growth rate, and there was a positive correlation between ADPN and glucose in the follicular fluid. Primary culture of dairy cow granulosa cells (GCs) was established to observe the effect of low glucose (Glu) and/or ADPN on GCs cyclins and proteins important for steroid synthesis. The results showed that the addition of 1 µg/mL ADPN alleviated the negative effects of low Glu treatment on the proliferation of GCs and the expression of steroid secretion related protein proteins. Treatment with LY294002 (PI3K inhibitor) four experimental GCs groups: control (0 µg/mL ADPN), 1 µg/mL ADPN, LY294002 inhibitor, and 1 µg/mL ADPN+LY294002. The results showed that ADPN promotes the secretion of steroid hormones by GCs through the PI3K-AKT. In summary, ADPN plays a crucial role in ameliorating postpartum anestrus in dairy cows with SCK.