Oocytes Research Articles

Association of Preoperative and Postoperative Plasma Syndecan-1 and Colorectal Cancer Outcome.

A three-dimensional network constructed using glycocalyx (GCX) extends throughout the cancer cell nest in human colorectal cancer (CRC). GCX was found to be closely related to cancer. We examined the prognostic correlation and potential of syndecan-1 (SDC1), a representative proteoglycan of GCX, as a biomarker. We analyzed SDC1 in the transcriptomic profiles of a major publicly available CRC cohort from The Cancer Genome Atlas (TCGA) using a computational algorithm. We investigated serum SDC1 levels preoperatively and on postoperative day seven in 48 patients with stage I-III CRC who underwent surgery during July-December 2019 at Gifu University Hospital. For TCGA, no significant differences existed between the high and low SDC1 expression groups regarding disease-free, disease-specific, and overall survival for stage I-III, and only overall survival for stage IV was significantly different. In our study, among the 48 patients, 17 (no recurrence), 13 (1 recurrence), and 18 (10 recurrences) had stage I-III, respectively. Preoperative and postoperative day 7 SDC1 levels for patients with stage I-III were 10.7±2.3 and 9.9±3.1 ng/ml (p=0.40), 11.1±1.7 and 10.1±0.8 ng/ml (p=0.07), and 10.3±2.0 and 9.5±1.4 ng/ml (p=0.15), respectively. In stage II and III, patients were divided into two groups according to differences between preoperative and postoperative SDC1 levels (SDC1pre-pro). SDC1pre-pro ≤0 group significantly prolonged disease-free survival compared with SDC1pre-pro >0 group (p=0.048). Dynamic change in serum SDC1 levels serves as a prognostic biomarker for stage II and III colorectal cancer.

Fast and furious: successful survival and resumption of meiosis in immature human oocytes vitrified and warmed using a short protocol

RESEARCH QUESTIONCan immature oocytes vitrified and warmed using a short protocol survive and resume meiosis? DESIGNThis study examined modifications of oocyte vitrification and warming protocols that shorten the time of exposure to vitrification and warming solutions. A total of 561 germinal vesicles (GV), and 218 metaphase I (MI) oocytes that were immature at oocyte retrieval were vitrified at room temperature in 2 minutes. Warming was done at 37°C in 2 minutes. Resumption of meiotic activity was evaluated after 24 and 48 hours of culture. Two different commercially available vitrification and warming kits were used for comparison. RESULTSNinety five percent of GV oocytes survived with no difference observed between kits. The survival of MI oocytes was on average 95.4% and not significantly different between the two kits. Of the 533 GV that survived, 491 converted to MI (92.1%). After culture for 48 hours, 54.4% converted to MII oocytes. In addition, of the 208 MI that survived the warming, 84.1% converted to MII oocytes after culture for 24 hours. CONCLUSIONSVitrification and warming of oocytes at different nuclear maturation stages can be done with a 2-minute exposure time to hypertonic and a 2-minute exposure time to hypotonic solutions. With this approach, the oocytes are less exposed to room temperature during dehydration and rehydration. Warming in 0.5M sucrose helps to maintain and support the potential to resume nuclear meiotic activity of oocytes and conversion from GV to MI and to MII oocytes.

Effect of Moderate Static Magnetic Fields on Mice Oocyte Vitrification: Calcium-Related Genes Expression.

The ability to cryopreserve oocytes without ultrastructural injury has been a concern in the development and use of methods to preserve female reproduction. The stability of the cell membrane must be preserved to reduce the damage caused by ice crystals during vitrification. One approach that has been explored is the use of static magnetic fields (SMFs), which are believed to influence cell membrane stability. In this study, the in vitro effects of SMF that range between 20-80 mT on the vitrification of mice germinal vesicle (GV) oocytes were studied. The viability and mitochondrial (Mt) membrane potential of both vitrified and nonvitrified oocytes were assessed using Trypan blue and JC1 staining. The high in vitro maturation (IVM) rate and high Mt membrane potential in metaphase II (MII) oocytes were taken into account to determine the optimal magnetic field intensity, that is, 20 mT. None of the SMF conditions resulted in intact spindles in MII oocytes. The study also explored the expression of store-operated calcium entry (Stim1, Orai1, and Ip3r) and meiosis resumption (Ccnb, Cdk) genes in GV and MII oocytes of both vitrified and control groups. The results show that the expressions of Orai1 and Ccnb genes in Vit-MII-SMF oocytes were considerably increased. However, no significant difference in Stim1 expression was observed between the groups. The Vit-MII-SMF group exhibited a significantly higher Ccnb expression compared to other groups. In vitro fertilization (IVF) was performed to evaluate the 2 pronuclear (2PN) rates. The findings demonstrated that using 20 mT SMF improved 2PN rates compared to the nonvitrified groups. This study provides a deeper understanding of the effects of moderate SMF and vitrification on the expression of calcium channel genes in GV and MII oocytes. The results suggest that applying a 20 mT SMF can help prevent cryoinjury and enhance the characteristics of vitrified-warmed oocytes.

Abstract 1091: Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma

Abstract Goal: Evaluate αvβ3 and HER2 as antigens for a bispecific chimeric antigen receptor T (CAR T) cell therapy in pediatric rhabdomyosarcoma. Background: Current treatments are ineffective against relapsed/refractory or metastatic rhabdomyosarcoma (RMS). Antigen loss or modulation on tumor cells commonly leads to tumor escape across diverse cancer types after CAR T therapy targeting different antigens. We hypothesize that simultaneously targeting two RMS antigens with a bispecific CAR T will reduce the likelihood of tumor escape. The integrin alphavbeta3 (αvβ3) plays a role in cancer progression, metastasis and neovascularization in several malignancies. HER2 also contributes to cancer progression and metastasis. Methods: Control of intramuscular RMS tumors by αvβ3 or HER2 CAR Ts was assessed in NSG mice, with non-specific CD19 CAR Ts as controls. Tumor burden was assessed weekly by bioluminescence imaging. Persistence and phenotype of CAR Ts were evaluated by flow cytometry. CAR T cell infiltration, tumor antigen expression and residual disease were determined by immunohistochemistry. Results: Six of six pediatric patient-derived RMS cell lines express αvβ3 and/or HER2. αvβ3 and HER2 CAR Ts kill RMS cells in vitro at low effector-to-target ratios. αvβ3 and HER2 CAR Ts independently control—and nearly eliminate—orthotopic RMS tumors. Fewer HER2 CAR Ts (5 × 106) are needed to control tumors than αvβ3 CAR Ts (10 × 106), but this is not due to lack of persistence or exhaustion of αvβ3 CAR Ts. Tumor control by αvβ3 CAR Ts is delayed (>21 days) compared to that by HER2 CAR Ts (7 days), and is robust, despite initial tumor progression following treatment. Importantly, despite strong tumor control by both CAR Ts, at 7 weeks post-treatment residual tumor nests are evident by histology. At this time, CAR Ts are still abundant, are not exhausted and express memory markers, suggesting that the remaining tumor cells are no longer susceptible to CAR T killing. We are currently evaluating the hypothesis that the remaining tumor cells have down-regulated antigen expression. Delayed tumor control by αvβ3 CAR Ts was ameliorated by engineering αvβ3 overexpression in RMS tumors. Conclusions: αvβ3 and HER2 CAR Ts control orthotopic RMS tumors by distinct mechanisms with changes in αvβ3 antigen expression, likely due to tumor progression, one prevalent factor. This is consistent with our prior reports of αvβ3 CAR Ts preventing breast cancer metastases, which naturally upregulate αvβ3 compared to the primary tumor. Despite near elimination of tumors, residual nests of tumor cells are likely to rebound as CAR T resistant tumors, similar to what is seen in patients treated with monospecific CAR Ts. These data support the incorporation of αvβ3 and HER2 CAR Ts into a bispecific CAR T approach to mitigate antigen escape. Our data also support investigation into the role of αvβ3 in RMS. Citation Format: Amanda M. Lulu, Nagyeong Park, Joseph J. Caruso, Dustin A. Cobb, Daniel W. Lee. Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1091.

FOXL2 interaction with different binding partners regulates the dynamics of ovarian development.

The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing of Foxl2 deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes, suggesting different roles across development. Here, we comprehensively investigated FOXL2's role through a multi-omics approach to characterize gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in somatic cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle formation and steroidogenesis. Deletion of one interactor, ubiquitin-specific protease 7 (Usp7), results in impairment of somatic cell differentiation, germ cell nest breakdown, and ovarian development, leading to sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.

The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

Comparative transcriptomic characterization of the ovary in the spawning process of the mud crab Scylla paramamosain.

Oviposition is induced upon mating in most insects. Spawning is a physiological process that is fundamental for the reproduction of Scylla paramamosain. However, the molecular mechanisms underlying the spawning process in this species are poorly understood. Herein, comprehensive ovary transcriptomic analysis was conducted at the germinal vesicle breakdown stage (GVBD), spawning stage, 0.5 h post-spawning stage, and 24 h post-spawning stage of S. paramamosain for gene discovery. A total of 67,230 unigenes were generated, and 27,975 (41.61%) unigenes were annotated. Meanwhile, the differentially expressed genes (DEGs) between the different groups were identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was subsequently conducted. These results suggested that octopamine (OA) and tyramine (TA) could induce oviposition, while dopamine (DA) and serotonin (5-hydroxytryptamine [5-HT]) inhibit oviposition. The 20-hydroxyecdysone (20E) and methyl farnesoate (MF) signal pathways might be positively associated with oviposition. Furthermore, numerous transcripts that encode neuropeptides and their G-protein-coupled receptors (GPCRs), such as CNMamide, RYamide, ecdysis-triggering hormone (ETH), GPA2/GPB5 receptor, and Moody receptor, appear to be differentially expressed during the spawning process. Eleven unigenes were selected for qRT-PCR and the pattern was found to be consistent with the transcriptome expression pattern. Our work is the first spawning-related investigation of S. paramamosain focusing on the ovary at the whole transcriptome level. These findings assist in improving our understanding of spawning regulation in S. paramamosain and provide information for oviposition studies in other crustaceans.

Administration of Arginine Vasotocin and modified Isotocin improve artificial propagation and post-spawning survival of female yellow catfish

Low rate of spawning and high percentage of death after artificial propagation are frequently observed in catfish, which do harms to the long-term population stability and genetic breeding programs in aquaculture. In this study, yellow catfish (Pelteobagrus fulvidraco), an important freshwater species in China was used as a research model, since the farmed female yellow catfish had poor reproduction performance and a high death rate after treatment with the traditional exogenous hormones, luteinizing hormone releasing hormone analogues2 (LHRH-A2) and human chorionic gonadotropin (HCG). We tried to investigate the roles of the Arginine Vasotocin (AVT)/ Isotocin (IT) neuropeptide family in reproduction and design an optimal strategy for inducing artificial propagation in yellow catfish. Two copies of pro-avt and pro-it were detected in hypothalamus and ovaries of yellow catfish, and their mRNA levels gradually increased during spring-summer season. Furthermore, the transcript levels of pro-avt and pro-it in hypothalamus significantly increased after injection of LHRH-A2 and HCG. Although AVT could not induce germinal vesicle breakdown (GVBD) in female yellow catfish, administration of AVT significantly stimulated premature ovulation in a dose-dependent manner and induced the expression of ovulation-related genes, ptgs2a and acvr1. Furthermore, there is a significant up-regulation of serum prostaglandin E2 (PGE2) and luteinizing hormone (LH) in female fish after AVT treatment, whereas no difference was observed in follicle stimulating hormone (FSH). Since IT of yellow catfish was insoluble in saline, a rigid peptide segment was added at the end of IT (the modified IT was named IT-M), which could greatly improve its solubility and induce ovulation in female yellow catfish. Subsequently, we demonstrated that adding carp pituitary extraction (CPE) to the second injection of traditional methods could efficiently improve the spawned fish, eggs production, fertilization rate, hatching rate and post-spawning survival of female yellow catfish, despite at a relatively high cost in hormones. We further discovered that IT-M + AVT could substitute for HCG + CPE in the second injection and had high efficiency in artificial propagation, reducing the hormone costs to less than one-third. Altogether, we provide an effective and affordable combination of exogenous hormones to improve reproduction performances and post-spawning survival rate in yellow catfish.

The severity of meiotic aneuploidy is associated with altered morphokinetic variables of mouse oocyte maturation.

Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse? The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI). Morphokinetic variables measured using time-lapse technology allow for the non-invasive evaluation of preimplantation embryo development within clinical assisted reproductive technology (ART). We recently applied this technology to monitor meiotic progression during IVM of mouse gametes. Whether there is a relationship between morphokinetic variables of meiotic progression and aneuploidy in the resulting egg has not been systematically examined at the resolution of specific chromosomes. Next-generation sequencing (NGS) is a robust clinical tool for determining aneuploidy status and has been reverse-translated in mouse blastocysts and oocytes. Therefore, we harnessed the technologies of time-lapse imaging and NGS to determine the relationship between the morphokinetics of meiotic progression and egg aneuploidy. Cumulus-oocyte complexes were collected from large antral follicles from hyperstimulated CD-1 mice. Cumulus cells were removed, and spontaneous IVM was performed in the absence or presence of two doses of Nocodazole (25 or 50 nM) to induce a spectrum of spindle abnormalities and chromosome segregation errors during oocyte meiosis. Comprehensive chromosome screening was then performed in the resulting eggs, and morphokinetic variables and ploidy status were compared across experimental groups (control, n = 11; 25 nM Nocodazole, n = 13; 50 nM Nocodazole, n = 23). We monitored IVM in mouse oocytes using time-lapse microscopy for 16 h, and time to germinal vesicle breakdown (tGVBD), tPB1, and dMI were analyzed. Following IVM, comprehensive chromosome screening was performed on the eggs and their matched first polar bodies via adaptation of an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) assay. Bioinformatics analysis was performed to align reads to the mouse genome and determine copy number-based predictions of aneuploidy. The concordance of each polar body-egg pair (reciprocal errors) was used to validate the results. Ploidy status was categorized as euploid, 1-3 chromosomal segregation errors, or ≥4 chromosomal segregation errors. Additionally, aneuploidy due to premature separation of sister chromatids (PSSC) versus non-disjunction (NDJ) was distinguished. We applied and validated state-of-the-art NGS technology to screen aneuploidy in individual mouse eggs and matched polar bodies at the chromosome-specific level. By performing IVM in the presence of different doses of Nocodazole, we induced a range of aneuploidy. No aneuploidy was observed in the absence of Nocodazole (0/11), whereas IVM in the presence of 25 and 50 nM Nocodazole resulted in an aneuploidy incidence of 7.69% (1/13) and 82.61% (19/23), respectively. Of the aneuploid eggs, 5% (1/20) was due to PSSC, 65% (13/20) to NDJ, and the remainder to a combination of both. There was no relationship between ploidy status and tGVBD, but tPB1 and the dMI were both significantly prolonged in eggs with reciprocal aneuploidy events compared to the euploid eggs, and this scaled with the severity of aneuploidy. Eggs with ≥4 aneuploid chromosomes had the longest tPB1 and dMI (P < 0.0001), whereas eggs with one to three aneuploid chromosomes exhibited intermediate lengths of time (P < 0.0001). N/A. We used Nocodazole in this study to disrupt the meiotic spindle and induce aneuploidy in mouse oocytes. Whether the association between morphokinetic variables of meiotic progression and the severity of aneuploidy occurs with other compounds that induce chromosome segregation errors remain to be investigated. In addition, unlike mouse oocytes, human IVM requires the presence of cumulus cells, which precludes visualization of morphokinetic variables of meiotic progression. Thus, our study may have limited direct clinical translatability. We validated NGS in mouse eggs to detect aneuploidy at a chromosome-specific resolution which greatly improves the utility of the mouse model. With a tractable and validated model system for characterizing meiotic aneuploidy, investigations into the molecular mechanisms and factors which may influence aneuploidy can be further elaborated. Time-lapse analyses of morphokinetic variables of meiotic progression may be a useful non-invasive predictor of aneuploidy severity. This work was supported by the Bill & Melinda Gates Foundation (INV-003385). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.

Meiotic and developmental competence of growing pig oocytes derived from small antral follicles is enhanced in culture medium containing FGF2, LIF, and IGF1 (FLI medium)

BackgroundOocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles.MethodsThe growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed.ResultsThe maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF.ConclusionsThis work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

Deterministic nuclear reprogramming of mammalian nuclei to a totipotency-like state by Amphibian meiotic oocytes for stem cell therapy in humans.

The ultimate aim of nuclear reprogramming is to provide stem cells or differentiated cells from unrelated cell types as a cell source for regenerative medicine. A popular route towards this is transcription factor induction, and an alternative way is an original procedure of transplanting a single somatic cell nucleus to an unfertilized egg. A third route is to transplant hundreds of cell nuclei into the germinal vesicle (GV) of a non-dividing Amphibian meiotic oocyte, which leads to the activation of silent genes in 24 h and robustly induces a totipotency-like state in almost all transplanted cells. We apply this third route for potential therapeutic use and describe a procedure by which the differentiated states of cells can be reversed so that totipotency and pluripotency gene expression are regained. Differentiated cells are exposed to GV extracts and are reprogrammed to form embryoid bodies, which shows the maintenance of stemness and could be induced to follow new directions of differentiation. We conclude that much of the reprogramming effect of eggs is already present in meiotic oocytes and does not require cell division or selection of dividing cells. Reprogrammed cells by oocytes could serve as replacements for defective adult cells in humans.

Lymph node metastasis regulation by peritumoral tonsillar tissue mitochondria-related pathway activation in oropharyngeal cancer.

Immune-related gene expression profiles of peritumoral tonsillar tissues are modified by oropharyngeal cancer (OPC) nodal status. This study explored immunometabolism and immune cell count alterations in peritumoral tonsillar tissue according to OPC nodal status. Microarray data analysis of 27 peritumoral tonsillar tissue samples, using a newly generated mitochondrial metabolism-related gene set comprised of 948 genes, detected 228 differentially expressed genes (DEGs) (206 up- and 22 downregulated) in metastasis-negative cases compared to metastasis-positive ones. REACTOME pathway analysis of the 206 upregulated genes revealed the Toll-like receptor 4 cascade were most enriched. Immune cell proportion analysis using the CIBERSORTx algorithm revealed a significantly higher rate of naïve B cells, but lower rates of regulatory T cells and resting natural killer cells in metastasis-negative cases. Digital spatial profiling of the 6 OPC tissues detected 9 DEGs in the lymphoid regions, in contrast, no DEGs were identified in tumor regions according to nodal status. Cancer cell nests and pair matched normal epithelia mitochondrial DNA (mtDNA) from 5 OPC tissues were analyzed by next generation sequencing for variant detection. However, no significant mtDNA variation was found. This study identified mitochondria-related immune cell transcriptional programs and immune cell profiles associated with OPC lymphatic spread in peritumoral tonsil tissue, further evaluation of which will elucidate targetable immune mechanisms associated with OPC lymphatic dissemination.

Reassessing the impact of letrozole co-administration in controlled ovarian hyperstimulation: findings from a single-center repeated measures study.

To explore whether letrozole improved outcomes in subsequent controlled ovarian hyperstimulation (COH) cycles. This was a retrospective repeated measures cohort study examining COH cycles. Patients were included if they underwent two cycles for unexplained infertility, male factor infertility, or planned oocyte/embryo cryopreservation. The first cycles for all patients implemented a non-letrozole, conventional gonadotropin protocol. Second cycles for the study group included letrozole (2.5-7.5 mg for 5 days) with no medication change to second cycles amongst controls. Our primary objective was to compare oocyte yield. Cohorts were then subdivided by pursuit of oocyte (OC) or embryo (IVF) cryopreservation. Secondary outcome amongst the OC subgroup was oocyte maturation index (metaphase II (MII)/total oocytes). Secondary outcomes amongst the IVF subgroup were normal fertilization rate (2-pronuclear zygotes (2PN)/oocytes exposed to sperm), blastocyst formation rate (blastocysts/2PNs), and embryo ploidy (%euploid and aneuploid). Fifty-four cycles (n = 27) were included in letrozole and 108 cycles (n = 54) were included in control. Oocyte yield was higher in second cycles (p < 0.008) in the letrozole group but similar in second cycles (p = 0.26) amongst controls. Addition of letrozole did not impact MII index (p = 0.90); however, MII index improved in second cycles amongst controls (p < 0.001). Both groups had similar rates of normal fertilization (letrozole: p = 0.52; control: p = 0.61), blast formation (letrozole: p = 0.61; control: p = 0.84), euploid (letrozole: p = 0.29; control: p = 0.47), and aneuploid embryos (letrozole: p = 0.17; control: p = 0.78) between cycles. Despite improved oocyte yield, letrozole did not yield any difference in oocyte maturation or embryo outcomes.

The possible regulatory role of miR-514 and miR-642b in cumulus cells on the oocyte maturation in patients with polycystic ovary syndrome

Polycystic ovary syndrome is a common endocrine disorder in reproductive-age women. Accordingly, abnormal microenvironment may negatively influence oocyte developmental competence as a result of the altered expression profile of cumulus cells (CCs), mainly the key players of oocyte maturation, such as epidermal growth factor receptor (EGFR) and prostaglandin E receptor-2 (PTGER2). This study aimed to examine the expression levels of miR-514, miR-642b, and their candidate target genes (EGFR and PTGER2, respectively) in CCs of immature and mature oocytes in patients with PCOS. A total of 40 oocytes at germinal vesicle (GV) and 40 oocytes at metaphase II (MII) stages were retrieved from 30 PCOS women. Quantitative real-time PCR was performed to analyze the expression level of miR-514, miR-642b, EGFR, and PTGER2 in cumulus cells (CCS) of each oocyte. The expression level of miRNAs and their candidate target genes were compared between CCs of GV and MII oocytes. Our study suggests an inverse relationship exists between the expression levels of miR-514 and EGFR, and miR-642b and PTGER2. Furthermore, we observed that CCs of GV oocytes had higher levels of EGFR and PTGER2 mRNA and lower levels of miR-514 and miR-642b expression compared to those of MII oocytes. The present study demonstrated that miR-514 and miR-642b can regulate oocyte development by targeting EGFR and PTGER2, respectively. Therefore, examination of these miRNAs in CCs could be promising parameters to predict oocyte competence in PCOS patients.

Mapping Spatiotemporal Heterogeneity in Multifocal Breast Tumor Progression by Noninvasive Ultrasound Elastography-Guided Mass Spectrometry Imaging Strategy.

Spatiotemporal heterogeneity of tumors provides an escape mechanism for breast cancer cells, which can obstruct the investigation of tumor progression. While molecular profiling obtained from mass spectrometry imaging (MSI) is rich in biochemical information, it lacks the capacity for in vivo analysis. Ultrasound diagnosis has a high diagnostic accuracy but low chemical specificity. Here, we describe a noninvasive ultrasound elastography (UE)-guided MSI strategy (UEg-MSI) that integrates physical and biochemical characteristics of tumors acquired from both in vivo and in vitro imaging. Using UEg-MSI, both elasticity histopathology metabolism "fingerprints" and reciprocal crosstalk are revealed, indicating the intact, multifocal spatiotemporal heterogeneity of spontaneous tumorigenesis of the breast from early, middle, and late stages. Our results demonstrate a gradual increase in malignant degree of primary focus in cervical and thoracic mammary glands. This progression is characterized by increased stiffness according to elasticity scores, histopathological changes from hyperplasia to increased nests of neoplastic cells and necrotic areas, and regional metabolic heterogeneity and reprogramming at the spatiotemporal level. De novo fatty acid (FA) synthesis focused on independent (such as ω-9 FAs) and dependent (such as ω-6 FAs) dietary FA intake in the core cancerous nest areas in the middle and late stages of tumor or in the peripheral microareas in the early stage of the tumor. SM-Cer signaling pathway and GPs biosynthesis and degradation, as well as glycerophosphoinositol intensity, changed in multiple characteristic microareas. The UEg-MSI strategy holds the potential to expand MSI applications and enhance ultrasound-mediated cancer diagnosis. It offers new insight into early cancer discovery and the occurrence of metastasis.

Review: Are moles senescent?

Melanocytic nevi (skin moles) have been regarded as a valuable example of cell senescence occurring invivo. However, a study of induced nevi in a mouse model reported that the nevi were arrested by cell interactions rather than a cell-autonomous process like senescence, and that size distributions of cell nests within nevi could not be accounted for by a stochastic model of oncogene-induced senescence. Moreover, others reported that some molecular markers used to identify cell senescence in human nevi are also found in melanoma cells-not senescent. It has thus been questioned whether nevi really are senescent, with potential implications for melanoma diagnosis and therapy. Here I review these areas, along with the genetic, biological, and molecular evidence supporting senescence in nevi. In conclusion, there is strong evidence that cells of acquired human benign (banal) nevi are very largely senescent, though some must contain a minor non-senescent cell subpopulation. There is also persuasive evidence that this senescence is primarily induced by dysfunctional telomeres rather than directly oncogene-induced.

The effects of prenatal azithromycin exposure on offspring ovarian development at different stages, doses, and courses

Azithromycin, a commonly used macrolide antibiotic for treating chlamydial infections during pregnancy, has sparked investigations into its potential effects on offspring development. Despite these inquiries, there remains uncertainty about the specific impact of prenatal azithromycin exposure (PAzE) on offspring ovarian development and the precise "effect window". Pregnant mice, following clinical guidelines for azithromycin dosing, were orally administered azithromycin at different gestational stages [(gestational day, GD) 10–12 or GD 15–17], doses (50, 100, or 200 mg/kg·d), and courses (single or multiple). On GD 18, we collected offspring blood and ovaries to examine changes in fetal serum estradiol (E2) levels, fetal ovarian morphology, pre-granulosa cell function, and oocyte development. Multiple courses of PAzE resulted in abnormal fetal ovarian morphological development, disorganized germ cell nests, enhanced ovarian cell proliferation, and reduced apoptosis. Simultaneously, multiple courses of PAzE significantly increased fetal serum E2 levels, elevated ovarian steroidogenic function (indicated by Star, 3β-hsd, and Cyp19 expression), disrupted oocyte development (indicated by Figlα and Nobox expression), and led to alterations in the MAPK signal pathway in fetal ovaries, particularly in the high-dose treatment group. In contrast, a single course of PAzE reduced fetal ovarian cell proliferation, decreased steroidogenic function, and inhibited oocyte development, particularly through the downregulation of Mek2 expression in the MAPK signal pathway. These findings suggest that PAzE can influence various aspects of fetal mouse ovarian cell development. Multiple courses enhance pre-granulosa cell estrogen synthesis function and advance germ cell development, while a single terminal gestation dose inhibits germ cell development. These differential effects may be associated with changes in the MAPK signal pathway.

Terato-Neuroendocrine Tumour of Testis: Review and Update

Neuro-endocrine tumours commonly emanate from intestinal and respiratory epithelium. Neuro-endocrine tumours could also on rare occasions afflict various organs of the human body. Neuro-endocrine tumour afflicting the testis is an uncommon tumour which does account for far less than one percent (&lt;1%) of all tumours of testis. To the knowledge of the author, less than 100 cases of primary neuro-endocrine tumour of the testis had been reported so far in the global literature. The reported cases of neuro-endocrine tumour of testis have ranged between 10 years and 83 years of age and the reported incidence rate had been noted to be higher within the fifth and sixth decade of life. Majority of patients who are afflicted by neuro-endocrine tumour of testis do tend to manifest with unilateral painless testicular mass. Sixteen percent (16%) of patients who are afflicted by neuro-endocrine tumour of testis do manifest with symptoms of neuroendocrine tumour syndrome. Eleven percent (11%) of primary neuroendocrine tumours of testis tend to be diagnosed initially with a contemporaneously-associated metastasis. Neuro-endocrine tumours of the testis occur as a primary testicular neuroendocrine tumour, or they may be metastatic neuroendocrine tumour to the testis with the primary tumour originating from elsewhere in the body especially from the gastrointestinal or cardiorespiratory tract system. Primary neuro-endocrine tumour of testis, could be further sub-divided into: (a) primary pure neuroendocrine tumour of testis; and (b) neuro-endocrine tumour of testis contemporaneously associated with teratoma or dermoid/epidermoid cysts. Once a neuro-endocrine tumour of testis is diagnosed, it is pivotal that clinicians should exclude a metastatic neuroendocrine tumour to the testis with the primary tumour originating from elsewhere within the human body. It has been iterated that the presence of teratomas elements within the neuro-endocrine tumour of testis does sufficiently rule out the primary site outside the testis. In order to accurately diagnose the neuro-endocrine tumour of the testis, it has been advised that it is pertinent to submit the tumour mass entirely and to look for any additional lineage of differentiation. Twenty five percent of primary neuro-endocrine tumours of testis are associated with teratoma. Histogenesis of pure neuroendocrine tumour of the testis is yet to be clarified. Two postulates had been propounded in relation to the mode origin of neuroendocrine tumour of testis including: (1) Emanation from a teratoma which is the germ cell origin postulate; (2) Emanating from argentaffin cells that are located within crypts of Lieberkühn .A study related to three pure testicular neuroendocrine tumours of testis had indicated that pure neuroendocrine tumour of testis might have different genetic background other than germ cell tumour.[11] Histopathology examination of specimens of neuro-endocrine tumour of testis has tended to demonstrate tumour cells that tend to be typified by the presence of nests of small round cells with uniform nuclei forming small acini and rosettes or sheets. The cells tend to contain eosinophilic granules within the cytoplasm and granular chromatin within the nuclei. The tumour cells do exhibit positive staining for neuroendocrine markers like chromogranin and synaptophysin as illustrated in detail in the article. Radical orchidectomy is the most adopted option of treatment; nevertheless, necessitation for the undertaking of provision of adjuvant treatment depending upon histological grading has not been clarified. Because of the possibility of development of local recurrence and or distant metastases after a long time, it is pivotal for all clinicians to carefully follow-up their patients over a long-period of time with clinical, laboratory testing and radiology-imaging testing follow-ups in order to establish early diagnosis of any local recurrence or distant metastasis to be able provide further treatment of curative intent early. There is also the possibility that neuro-endocrine tumours of testis had so far been under-reported due to mis diagnosis of the tumour upon pathology examination of the testicular tumour or the correct set of monoclonal and polyclonal antibodies had not been utilized in the immunohistochemistry study assessment of the tumours. It would be advised that all clinicians and pathologists globally should have a high index of suspicion for a neuro-endocrine tumour of testis in order to establish prompt diagnosis of the tumour and to provide appropriate assessment, treatment and follow-up of all their patients.

Bio‐Inspired Porous Microneedles Dwelled Stem Cells for Diabetic Wound Treatment

AbstractDiabetic wound healing is a serious, complex, and chronic process; one current promising and focusing technology in this area is stem cell treatment. Here, novel porous microneedle (MN) arrays is fabricated, which can highly mimetic stem cell niches, through template filling, and particle etching method. The human adipose‐derived stem cells (ADSCs) are encapsulated in Matrigel then loaded into the porous MN arrays by post‐perfusion. Because of the extracellular matrix‐mimicking, the biocompatible Matrigel offers a bionic microenvironment of stem cell nest suitable for growth. Benefiting from the numerous pore structures of MNs, the loaded ADSCs have enough space to fully absorb nutrients, proliferate greatly and exhibit prompted function. In addition, the cell‐loaded MN arrays have enough mechanical strength to penetrate the skin, allowing the ADSCs to get into the deep wound areas. Based on these features, the performance of the resultant MN arrays in promoting tissue regeneration is demonstrated, collagen deposition and angiogenesis in diabetes wounds of rat models. Thus, it is believe that the bioinspired porous MNs can act as excellent stem cell scaffolds and will find many practical values in clinic wound healing.

Characterization of insulin-like growth factor 3 and its potential role in the spotted steed Hemibarbus maculatus ovary development

As a new member of the insulin-like growth factors (Igfs), Igf3 was reported to play a vital role in fish reproduction. However, in spotted steed, the function of Igf3 remains largely unknown. In the present study, we identified and characterized Igf3 gene in spotted steed. Structural analysis showed that Igf3 contained five domains (B, C, A, D, E) and six conserved cysteine residues. The expression of Igf3 mRNA and protein were increased during ovary development and peaked in the maturation stage. The subcellular localization of IGF3 was highly expressed in granulosa cells and theca cells. Furthermore, recombinant IGF3 protein was produced and in vitro treatment with ovarian follicles significantly promoted the germinal vesicle breakdown (GVBD) rates of spotted steed follicles. The mRNA expression of cdc2 and cyclinB genes were significantly increased after IGF3 treatment, which were main components of maturation promoting factor (MPF). In addition, transcription levels of 3β-hsd, 20β-hsd, Cyp17a and Cyp19a1a were also changed. Taken together, these findings suggest that Igf3 is essential for ovary steroidogenesis and maturation in spotted steed.