Acupuncture modulates ovarian senescence through metabolic reprogramming: A multi-omics investigation in chemotherapy - induced POF model.

Motivation: The availability of modern sequencing techniques has led to a rapid increase in the amount of reconstructed metabolic networks. Using these models as a platform for the analysis of high throughput transcriptomic, proteomic and metabolomic data can provide valuable insight into conditional changes in the metabolic activity of an organism. While transcriptomics and proteomics provide important insights into the hierarchical regulation of metabolic flux, metabolomics shed light on the actual enzyme activity through metabolic regulation and mass action effects. Here we introduce a new method, termed integrative omics-metabolic analysis (IOMA) that quantitatively integrates proteomic and metabolomic data with genome-scale metabolic models, to more accurately predict metabolic flux distributions. The method is formulated as a quadratic programming (QP) problem that seeks a steady-state flux distribution in which flux through reactions with measured proteomic and metabolomic data, is as consistent as possible with kinetically derived flux estimations.Results: IOMA is shown to successfully predict the metabolic state of human erythrocytes (compared to kinetic model simulations), showing a significant advantage over the commonly used methods flux balance analysis and minimization of metabolic adjustment. Thereafter, IOMA is shown to correctly predict metabolic fluxes in Escherichia coli under different gene knockouts for which both metabolomic and proteomic data is available, achieving higher prediction accuracy over the extant methods. Considering the lack of high-throughput flux measurements, while high-throughput metabolomic and proteomic data are becoming readily available, we expect IOMA to significantly contribute to future research of cellular metabolism.Contacts: kerenyiz@post.tau.ac.il; tomersh@cs.technion.ac.il

Design and analysis of experiments with high throughput biological assay data

Turner syndrome (TS) is a chromosomal disorder that affects about 1 in 2500 female births and is characterized by the partial or complete absence of the second X chromosome. Depending on karyotype, TS is associated with primary ovarian insufficiency (POI). Approximately 50% of girls with a mosaic 45, X/46, XX karyotype may enter puberty spontaneously, but only 5-10% of women with TS achieve pregnancy without egg donation. In this review, we will evaluate the clinical use of markers of ovarian function in TS patients. Based on longitudinal studies of serum concentrations of reproductive hormones as well as ovarian morphology in healthy females and patients with TS, we will evaluate how they can be applied in a clinical setting. This is important when counseling patients and their families about future ovarian function essential for pubertal development and fertility. Furthermore, we will report on 20 years of experience of transition from pediatric to gynecological and adult endocrinological care in our center at Rigshospitalet, Copenhagen, Denmark.

The prostate-specific Pten-knockout (KO) mouse carcinogenesis model is highly desirable for prostate cancer chemoprevention studies due to its close resemblance of many histopathological features of human prostate cancer including disease progression from prostatic intraepithelial neoplasia (PIN) to invasive adenocarcinomas. Here, we profiled the prostate proteome, transcriptome and aqueous metabolome of Pten-KO mice to identify reference molecular signatures that can be used for designing chemopreventive and/or therapeutic intervention and for selection of molecular biomarkers of responses to intervention. For proteomics, 4 pairs of whole prostates from Pten-KO mice (12-15 weeks of age, corresponding to high grade PIN) and their wild type littermate housed in same cages were obtained from the NCI Mouse Model Repository and analyzed by 8-plex iTRAQTM. For transcriptomic/microarray and metabolomic analyses, 3 additional matched pairs of prostate/tumor specimens at older age (22-20 weeks) were used. Proteomic and transcriptomic analyses using manual annotation methods with references from PubMed revealed top signatures that were up- and down-regulated by Pten deletion, particularly those implicated in immune function, inflammatory response, cancer, drug metabolism, cellular functions, prostate functions, and endoplasmic stress regulation. Similar to the manual annotation approach, each network analysis of 203 genes and 22 proteins (≥ 2- fold changes) by a bioinformatics software, Ingenuity Pathway Analysis (IPA) showed that inflammatory response, cellular movement, immune cell trafficking, immunological disease, and cancer were top 5 disease and biological functions in Pten-KO mice. Using references from PubMed, we manually assigned the unmapped prostate metabolites to functional categories, which included altered methionine-cysteine cycle fluxes and purine metabolites, increased nucleotide pools, cholesterol and polyamine synthesis and suppressed pools of sugar and choline derivatives, glycolysis intermediates, and purine bases. IPA network analysis of 25 metabolites (≥ 2- fold changes) revealed the biological functions related to molecular transport, amino acid metabolism, and small molecule biochemistry. In addition, we integrated transcriptomic, proteomic, and metabolomic data sets to identify latent biological relationships and to gain a comprehensive understanding of Pten-deficient prostate carcinogenesis. The integrative analysis predicted activation of inflammatory response and several central signaling nodes, such as IRF7, NF-κB, and IL-6. Collectively, the integrative analyses identify both active and latent reference molecular signatures and provide more insight into Pten-deficient prostate cancer than single omic approaches. Citation Format: Jinhui Zhang, Li Li, Sangyub Kim, LeeAnn Higgins, Yibin Deng, Christopher J. Kemp, Cheng Jiang, Junxuan Lu. Integrative analysis of transcriptomic, proteomic, and metabolomic data of Pten-knockout carcinogenic mouse prostate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5249. doi:10.1158/1538-7445.AM2017-5249

Insight into the effect of a heavy metal mixture on neurological damage in rats through combined serum metabolomic and brain proteomic analyses

e14098 Background: Premature ovarian failure (POF) is an established adverse effect of chemotherapy for breast cancer (BC); little is known about the ovarian toxicity of newer treatments (e.g. PARPi, CDK4/6 inhibitors, immunotherapy, anti-HER2 therapy). This study examined whether contemporary BC (neo)adjuvant clinical trials will provide data on POF. Methods: Eligible studies were phase 3 (neo)adjuvant trials of pharmacologic agents aimed to reduce BC events, that enrolled premenopausal women between June 2008 - Sept 2019. MEDLINE and EMBASE were searched using MESH terms / keywords: “breast neoplasm” or “breast adj2 (cancer* or tumo?r* or carcinoma*)”, “adjuvant chemotherapy” or “neoadjuvant therapy” or “neoadjuvant” or “adjuvant”. Clinicaltrials.gov and EudraCT were searched using the filters “breast cancer”, “interventional studies”, “phase 3”. Data extracted from eligible trial publications, protocols and clinicaltrials.gov/EudraCT summaries, included whether each trial assessed the variables - post treatment pregnancy/birth, attempt at pregnancy, menstrual data, antral follicle count, FSH, LH, estrogen, progesterone and AMH levels. Results: Of 1665 trials screened, 141 were eligible. Investigational treatments included chemotherapy (71%), endocrine (20%), anti-HER2 (26%), immunotherapy (8%), CDK4/6 inhibitor (5%), PARPi (2%). Few trials (28%) assessed quality of life (QOL). Study chairs were from Nth America (19%), Europe (36%), Asia (43%); most (67%) were male. POF and fertility measures were prespecified endpoints in 9 (6%) and 0 trials respectively. Twenty (14%) trials collected POF and/or fertility data; post treatment pregnancy (11/20), amenorrhoea (6/20), estrogen (8/20), FSH (7/20), LH (5/20), AMH (3/20), and progesterone levels (1/20) and antral follicle count (3/20). Assessment of POF was associated with collection of QOL data (p = 0.02) but not with BC phenotype, timing, treatment type, or sex or nationality of study chair. Conclusions: POF measures are rarely prespecified endpoints and are infrequently collected in phase 3 BC (neo)adjuvant trials. Trialists should consider POF, given the potential for this data to enhance informed decision making for premenopausal patients.

Pathway analysis of metabolomic and proteomic data is critical for understanding the intricacies of biological processes and disease mechanisms. The challenging process of pathway analysis has become less burdensome thanks to online tools and databases that enable the comparison of pathways in intricate biological systems. Data analysis tools, like MetaboAnalyst 6.0, evaluate complex interactions and metabolic networks in biological pathways. Metabolomic and proteomic data integration is performed by utilizing databases, like KEGG, to analyze pathways and determine the interplay of proteins, metabolites, and other molecules in common pathways. Molecular interactions and similarities between pathways are explored to promote hypothesis generation and guide further research. This comprehensive chapter will enhance the understanding of utilizing pathway analysis of metabolic and proteomic data of various conditions, biomarkers, organisms, and therapeutic molecular targets, paving the way for future investigations and clinical applications, especially in pharmacology. Drugs like Metformin can be studied more extensively with pathway analysis, as demonstrated in this chapter, to enhance our understanding of how drugs affect patients.

Stem cell transplantation has been considered a promising therapeutic approach for premature ovarian failure (POF). However, to date, no quantitative data analysis of stem cell therapy for POF has been performed. Therefore, the present study performed a meta-analysis to assess the efficacy of stem cell transplantation in improving ovarian function in animal models of POF. In addition, a case report of a patient with POF subjected to stem cell treatment was included to demonstrate that stem cell therapy also contributes to the recovery of ovarian function in patients. Published studies were identified by a systematic review of the PubMed, Embase, and Cochrane's library databases, and references cited in associated reviews were also considered. Data regarding follicle-stimulating hormone (FSH), estradiol (E2), ovarian weight, follicle count, the number of pregnancies and other parameters, including delivery route and cell type, were extracted. Pooled analysis, sensitivity analyses, subgroup analyses and meta-regression were performed. In the case of POF, transvaginal ultrasound (TVS), abdominal ultrasound (TAS) and color Doppler flow imaging (CDFI) were performed to observe the endometrial morphology and blood flow signals in the patient. Overall, pooled results from 16 pre-clinical studies demonstrated that stem cell-based therapy significantly improved FSH levels [standardized mean difference (SMD)=−1.330; 95% confidence interval (CI), -(2.095–0.565); P=0.001], E2 levels (SMD=2.334; 95% CI, 1.350–3.319; P<0.001), ovarian weight (SMD=1.310; 95% CI, 0.157–2.463; P=0.026), follicle count (SMD=1.871; 95% CI, 1.226–2.516; P<0.001), and the number of pregnancies (risk ratio=1.715, 95% CI, 1.213–2.424; P=0.002). The results of TVS and TAS demonstrated improved ovarian size and endometrial thickness in the patient with POF after MSC treatment. Of note, a rich blood flow signal in the endometrium was observed on CDFI. It appeared that stem cell-based therapy may be an effective method for the resumption of ovarian function in a patient and in animal models of POF; however, large-scale and high-quality future studies are required to confirm the present findings due to heterogeneity.

Glioblastoma (GBM) is the most aggressive nervous system cancer, with median survival under 2 years. Understanding its molecular pathogenesis is crucial for improving diagnosis and treatment. We performed an integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs. We identified key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation as well as potential targets for EGFR-, TP53- and RB1-altered tumors. We detected immune subtypes, driven by the presence of distinct immune cell populations using bulk omics, validated by snRNA-seq, and they were correlated with specific expression and histone acetylation patterns. Acetylation of histone H2B in classical-like and immune-low GBM was driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identified specific lipid distributions across subtypes and distinct global metabolic changes in IDH mutated tumors. By comparing the adult GBM proteomics to the adolescent and young adult (AYA) GBM cohort from the HOPE study, we found downregulated IDH1 expression and up-regulated expression of genes in the NADH dehydrogenase family, including NDUFB1, and NDUFB3 among others, which may be related to high IDH1 mutation frequency in AYA. This work highlights biological relationships which could potentially aid GBM patient stratifications for more effective treatments. Citation Format: Liang-Bo Wang, Alla Karpova, Marina A. Gritsenko, Jennifer E. Kyle, Song Cao, Yize Li, Dmitry Rykunov, Antonio Colaprico, Joseph Rothstein, Runyu Hong, Vasileios Stathias, MacIntosh Cornwell, Francesca Petralia, Richard D. Smith, Antonio Iavarone, Milan G. Chheda, Jill S. Barnholtz-Sloan, Karin D. Rodland, Tao Liu, Li Ding, Clinical Proteomic Tumor Analysis Consortium. Proteogenomic and metabolomic characterization of human glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2170.

Proteogenomic and metabolomic characterization of human glioblastoma.

Glioblastoma (GBM) is the most aggressive nervous system cancer, with median survival under 2 years. Understanding its molecular pathogenesis is crucial for improving diagnosis and treatment. We performed an integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs. We identified key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation as well as potential targets for EGFR-, TP53- and RB1-altered tumors. We detected immune subtypes, driven by the presence of distinct immune cell populations using bulk omics, validated by single nulcei RNA sequencing (snRNA-seq), and they were correlated with specific expression and histone acetylation patterns. Acetylation of histone H2B in classical-like and immune-low GBM was driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identified specific lipid distributions across subtypes and distinct global metabolic changes in IDH mutated tumors. This work highlights biological relationships which could potentially aid GBM patient stratifications for more effective treatments.

Long-term ovarian function in women treated with CHOP or CHOP plus etoposide for aggressive lymphoma.

Background and objectiveThe effects of hormonal contraception (HC) use on ovarian reserve (OR) markers in individuals seeking an infertility evaluation and the success of assisted reproductive technology (ART) warrant further investigation. Therefore, the aim of this study was to determine if women seeking an evaluation for unexplained infertility who used long-term (≥2 years) HC have lower ovarian reserve (OR) markers and higher uptake of ART compared to short-term (<2 years) or never HC users.MethodsWe performed a cross-sectional patient survey involving a retrospective medical chart review of patients seeking an evaluation for unexplained infertility at the University of Colorado Advanced Reproductive Medicine (CU ARM) clinic.ResultsMost participants (87%; 107/123) reported a history of HC use with 98 (79.7%) reporting long-term continuous use for two or more years. Median OR markers were similar between long-term and short-term/never HC users [anti-Müllerian hormone (AMH): 2.4 vs. 3.2, p=0.20; antral follicle count (AFC): 18 vs. 26, p=0.10; follicle-stimulating hormone (FSH): 7.6 vs. 6.3, p=0.26] and remained so after adjusting for age and diagnosis of polycystic ovarian syndrome (PCOS) or primary ovarian insufficiency (POI) in linear regression models. However, among HC users aged less than 30 years (n=9), those who had discontinued HC between two and three years prior to the assessment of their OR markers had a 6.20 ng/mL increase in AMH level compared to those who had discontinued HC less than two years prior to the assessment (p=0.02). Additionally, there was a marginally increased use of ART overall among long-term HC users compared to short-term/never HC users (64.3% vs. 44.0%, p=0.06), specifically in the use of in vitro fertilization (IVF) (58.7% vs. 18.2%, p=0.01). Among long-term HC users, ovulation induction was less likely to result in live birth compared to short-term/never HC users (8.9% vs. 62.5%, p<0.001); however, after adjusting for age, PCOS, POI, and type of ART used, there was no difference in the odds of live birth after ART between long-term HC users and short-term/never users.ConclusionWhile long-term HC users report increased use of ART, in particular IVF, the overall conception rates and live birth outcomes among ART users do not appear to be significantly affected by a history of long-term HC use.

Study question Does a combination of thrombospondin 1 (THBS1), kit ligand (KITLG), and fibroblast growth factor 2 (FGF2) promote ovarian function in a POI mouse model? Summary answer Two consecutive intraovarian injections of THBS1, KITLG, and FGF2 rescue the reproductive outcomes in POI mice by improving oocyte yield and early embryo development. What is known already Different stem cell-based strategies have been explored to recover ovarian function in patients with impaired ovarian reserves, suggesting that beneficial effects are mainly mediated by the secretion of stem cell-soluble factors. Our previous findings suggest that the specific growth factors (GFs) THBS1, KITLG and FGF2 play a crucial role in these regenerative ovarian effects, observed in both mice and humans. Specifically, THBS1 and FGF2 seem to be related to the promotion of follicle development and ovarian niche vascularization, while KITLG associates with ovulation and ovarian fibrosis. Further research is still required to validate their potential to recover ovarian function. Study design, size, duration This experimental animal study involved 24 eight-week-old NOD/SCID female mice randomized into four groups: Young-healthy (as reference), POI-Sham, POI-GFs-low, and POI-GFs-high. Thirteen days after POI induction by chemotherapy, mice received a 10-µl intraovarian injection of either saline (POI-Sham), low- or high-dose of GFs, followed by a second injection 13 days later. After two weeks, mice underwent ovarian hyperstimulation and euthanasia for ovary/oocyte collection. Retrieved MII-oocytes were subjected to in vitro fertilization and embryo culture. Participants/materials, setting, methods POI condition was induced by a single intraperitoneal injection of cyclophosphamide (120mg/kg) and busulfan (12mg/kg). Intraovarian injections were performed using 30-gauge needles. The low-dose GFs treatment consisted of 2µg/ml THBS1 +400pg/ml KITLG +40pg/ml FGF2, while the high dose included 10µg/ml THBS1 +400ng/ml KITLG +10 ng/ml FGF2. Ovarian hyperstimulation was induced with 10 IU of PMSG, followed by 10 IU of hCG 48 hours later. Oocytes were released by tearing the ampulla of the oviducts. Main results and the role of chance Ovarian mass (POI-Sham: 6.0±2.4 mg vs. Young-healthy: 17.4±2.9 mg; p &amp;lt; 0.001), fibrotic area (14.8±3.5% vs. 4.9±0.6%; p = 0.005) and oocyte yield (1.6±1.8 vs. 25.0±13.7; p = 0.008) were significantly impaired by chemotherapy. Interestingly, POI females receiving a double intraovarian injection of a high dose of GFs showed improved ovarian mass (POI-GFs high: 10.2±2.7 mg vs. POI-Sham: 6.0±2.4 mg; p = 0.010) and fibrosis (9.5±3.2% vs. 14.8±3.5%; p = 0.049), and a 5-fold increase in the number of retrieved oocytes when compared to POI-Sham (7.9±4.9 vs. 1.6±1.8, p = 0.023), suggesting a promotion of gonadotropin-responsive follicles. This treatment with high GFs dose also increased the percentage of oocytes with optimal meiotic spindle assembly (78.6% vs. 60%) and chromosome alignment (71.4% vs. 60%) when compared to POI-Sham mice. In vitro fertilization rates were similar between groups, but the POI-Sham group showed impaired embryo development compared to Young-healthy females, with decreases in blastocyst formation (81.8% vs. 98.3%, p = 0.002) and hatching rates (63.6% vs. 92.5%, p = 0.002). These impairments were mitigated in both the low- and the high-dose GFs-treated mice (POI-GFs-low: 88.9% and 83.3%, respectively; POI-GFs-high: 93.0% and 70.0%, respectively). Limitations, reasons for caution Further validation using human ovarian samples is required as this study was conducted using an animal model of POI. The magnitude of the observed effects may vary due to interspecies differences. Wider implications of the findings These results suggest that the combination of THBS1, KITLG, and FGF2 has the potential to recover ovarian function in POI females, increasing the number of retrieved mature-oocytes and improving embryo development. Upon validation in humans, this approach could represent a promising therapeutic alternative to be developed for women with POI. Trial registration number No

To observe the effects of autologous blood injection and 0.9% NaCl at Zusanli (ST 36) on ovarian function in patients with primary ovarian insufficiency. Sixty patients with primary ovarian insufficiency were randomly divided into an observation group and a control group, 30 cases in each one. The patients in the observation group were treated with injection of autologous blood at Zusanli (ST 36); the patients in the control group were treated with 0.9% NaCl with identical volume at Zusanli (ST 36). Both the treatments were given once a week for 3 months. The ovarian function, including follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) were tested before treatment, 1 month, 2 months and 3 months after first acupoint injection; the endometrial thickness before and after treatment and clinical efficacy were compared in the two groups. Compared before treatment, FSH was lowered in the observation group after 1-month treatment (P<0.05), while FSH and LH were lowered and E2 was increased after 2-month treatment and 3-month treatment (all P<0.05). Compared with 1-month treatment, FSH and LH were lowered and E2 was increased in the observation group after 2-month treatment and 3-month treatment (all P<0.05). Compared with 2-month treatment, FSH was lowered and E2 was increased in the observation group after 3-month treatment (both P<0.05). The differences of all serum tests before and after treatment were insignificant in the control group (all P>0.05). The FSH after 1-month treatment, and FSH, LH and E2 after 2-month treatment and 3-month treatment in the observation group were significantly different from those in the control group (all P<0.05). The endometrial thickness after treatment in the observation group was higher than that before treatment (P<0.05), while the endometrial thickness after treatment in the control group was similar to that before treatment (P>0.05); the difference of endometrial thickness before and after treatment in the observation group was higher than that in the control group (P<0.05). The clinical effective rate was 83.3% (25/30) in the observation group, which was superior to 46.7% (14/30) in the control group (P<0.05). The autologous blood injection at Zusanli (ST 36) can significantly improve ovarian function, promote endometrial growth in patients with primary ovarian insufficiency.