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We report a novel method to profile intrcellular oxygen concentration (icO 2 ) during in vitro mammalian oocyte and preimplantation embryo development using a commercially available multimodal phosphorescent nanosensor (MM2). Abattoir-derived bovine oocytes and embryos were incubated with MM2 in vitro. A series of inhibitors were applied during live-cell multiphoton imaging to record changes in icO 2 associated with mitochondrial processes. The uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) uncouples mitochondrial oxygen consumption to its maximum, while antimycin inhibits complex III to ablate mitochondrial oxygen consumption. Increasing oxygen consumption was expected to reduce icO 2 and decreasing oxygen consumption to increase icO 2 . Use of these inhibitors quantifies how much oxygen is consumed at basal in comparison to the upper and lower limits of mitochondrial function. icO 2 measurements were compared to mitochondrial DNA copy number analysed by qPCR. Antimycin treatment increased icO 2 for all stages tested, suggesting significant mitochondrial oxygen consumption at basal. icO 2 of oocytes and preimplantation embryos were unaffected by FCCP treatment. Inner cell mass icO 2 was lower than trophectoderm, perhaps reflecting limitations of diffusion. Mitochondrial DNA copy numbers were similar between stages in the range 0.9–4 × 10 6 copies and did not correlate with icO 2 . These results validate the MM2 probe as a sensitive, non-toxic probe of intracellular oxygen concentration in mammalian oocytes and preimplantation embryos.

ObjectiveTo assess the utilisation of and funding structure for fertility preservation for children diagnosed with cancer in the UK.DesignSurvey of paediatric oncologists/haematologists. Questionnaires were sent electronically with reminder notifications to non-responders.SettingUK Paediatric Oncology Principal Treatment Centres (PTCs).ParticipantsPaediatric oncologists/haematologists with an interest in the effects of treatment on fertility representing the 20 PTCs across the UK.Main outcome measuresReferral practices, sources and length of funding for storage of gametes or gonadal tissue for children diagnosed with cancer in the preceding 12 months.ResultsResponses were received from 18 PTCs (90%) with responses to 98.3% of questions. All centres had referred patients for fertility preservation: ovarian tissue collection/storage 100% (n=18 centres), sperm banking 100% (n=17; one centre was excluded due to the age range of their patients), testicular tissue storage 83% (n=15), mature oocyte collection 35% (n=6; one centre was excluded due to the age range of their patients). All centres with knowledge of their funding source reported sperm cryopreservation was NHS funded. Only 60% (n=9) centres reported the same for mature oocyte storage. Of the centres aware of their funding source, half reported that ovarian and testicular tissue storage was funded by charitable sources; this increased in England compared with the rest of the UK.ConclusionsInequality exists in provision of fertility preservation for children with cancer across the UK. There is lack of formalised government funding to support international guidelines, with resultant geographical variation in care. Centralised funding of fertility preservation for children and young adults is needed alongside establishment of a national advisory panel to support all PTCs.

Ovarian aging profoundly impacts reproductive health and accelerates the overall aging process, yet the development of effective therapeutic strategies remains a formidable challenge. In this study, we report the rejuvenating effects of HEP14, a natural activator of protein kinase C (PKC) pathway, on aged ovarian function by inducing mitophagy and effectively clearing reactive oxygen species. To ensure controlled and sustained delivery of HEP14 in vivo, we develop HEP14-loaded PLGA microspheres. Transcriptomic analysis reveals a significant overlap between the transcriptional profiles of HEP14-treated aged ovaries and those of adult ovaries, suggesting molecular rejuvenation process closely associated to HEP14-induced mitophagy. Histopathological evaluations further substantiate these findings, showing that HEP14 enhances mitophagy, exhibits antioxidative properties and promotes follicular regeneration. Consequently, ovarian endocrine function in aged mice is substantially restored. Using transmission electron microscopy, confocal microscopy, and western blot analysis alongside pharmocological inhibitors and PKC-specific siRNA, in vitro studies further demonstrate the restorative effect of HEP14 on mitophagy, leading to improved mitochondrial function and subsequent alleviation of oxidative stress in senescent ovarian granulosa cells. This effect is mediated through the activation of the PKC-ERK1/2 pathway, which plays an pivotal role in the action mechanism in HEP14. These discoveries offer new therapeutic hope for ovarian aging. PLGA microspheres ensure controlled delivery of HEP14, which restores ovarian function in aged mice by inducing mitophagy and clearing reactive oxygen species via molecular rejuvenation.

There is evidence that expression and methylation of the imprinted paternally expressed gene 1/mesoderm-specific transcript homologue (PEG1/MEST) gene may be affected by assisted reproductive technologies (ARTs) and infertility. In this study, we sought to assess the imprinting status of the MEST gene in a large cohort of in vitro-derived human preimplantation embryos, in order to characterise potentially adverse effects of ART and infertility on this locus in early human development. Embryonic genomic DNA from morula or blastocyst stage embryos was screened for a transcribed AflIII polymorphism in MEST and imprinting analysis was then performed in cDNA libraries derived from these embryos. In 10 heterozygous embryos, MEST expression was monoallelic in seven embryos, predominantly monoallelic in two embryos, and biallelic in one embryo. Screening of cDNA derived from 61 additional human preimplantation embryos, for which DNA for genotyping was unavailable, identified eight embryos with expression originating from both alleles (biallelic or predominantly monoallelic). In some embryos, therefore, the onset of imprinted MEST expression occurs during late preimplantation development. Variability in MEST imprinting was observed in both in vitro fertilization and intracytoplasmic sperm injection-derived embryos. Biallelic or predominantly monoallelic MEST expression was not associated with any one cause of infertility. Characterisation of the main MEST isoforms revealed that isoform 2 was detected in early development and was itself variably imprinted between embryos. To our knowledge, this report constitutes the largest expression study to date of genomic imprinting in human preimplantation embryos and reveals that for some imprinted genes, contrasting imprinting states exist between embryos.

Here we report the use of a microfluidic system to assess the differential metabolomics of murine embryos cultured with endometrial cells-conditioned media (CM). Groups of 10, 1-cell murine B6C3F1 × B6D2F1 embryos were cultured in the microfluidic device. To produce CM, mouse uterine epithelial cells were cultured in potassium simplex optimized medium (KSOM) for 24 h. Media samples were collected from devices after 5 days of culture with KSOM (control) and CM, analyzed by reverse phase liquid chromatography and untargeted positive ion mode mass spectrometry analysis. Blastocyst rates were significantly higher (p < 0.05) in CM (71.8%) compared to control media (54.6%). We observed significant upregulation of 341 compounds and downregulation of 214 compounds in spent media from CM devices when compared to control. Out of these, 353 compounds were identified showing a significant increased abundance of metabolites involved in key metabolic pathways (e.g., arginine, proline and pyrimidine metabolism) in the CM group, suggesting a beneficial effect of CM on embryo development. The metabolomic study carried out in a microfluidic environment confirms our hypothesis on the potential of uterine epithelial cells to enhance blastocyst development. Further investigations are required to highlight specific pathways involved in embryo development and implantation.

Premature ovarian insufficiency (POI) and age-related natural-aging ovarian insufficiency (ARNA-OI) pose pressing global health challenges, necessitating effective therapeutic strategies and a deep understanding of their underlying mechanisms. This study investigates how HEP14, a PKC pathway activator, boosts the regenerative potential of human adipose-derived stem cells (hADSCs) for ovarian regeneration. Transcriptome analysis reveals that HEP14 modulates gene expression profile in hADSCs, enhancing their regenerative capacity. In mouse models of POI and ARNA-OI, co-administration of HEP14-empowered hADSCs (h-hADSCs) with HEP14/PLGA microspheres significantly improves ovarian regeneration and function. These effects are attributed to increased h-hADSC retention and transdifferentiation, enhanced antifibrotic and proangiogenic capability, along with an optimized dosing strategy. The upregulation of MMP1, PDGFD, and STC1 through the HEP14-activated PKC-ERK1/2 signaling pathway is crucial for these effects. Our findings highlight the pivotal role of h-hADSCs and the HEP14-activated PKC-ERK1/2 pathway in ovarian regeneration and provide a promising advancement in treating ovarian insufficiency. HEP14, a PKC pathway activator, enhances ovarian regeneration of human adipose-derived stem cells for regenerative potential by activating the PKC-ERK1/2 pathway to upregulate MMP1, PDGFD, and STC1 in premature ovarian insufficiency and aging mice.

Purpose Gametocyte-specific factor 1 has been shown in other species to be required for the silencing of retrotransposons via the Piwi-interacting RNA (piRNA) pathway. In this study, we aimed to isolate and assess expression of transcripts of the gametocyte-specific factor 1 ( GTSF1 ) gene in the human female germline and in preimplantation embryos. Methods Complementary DNA (cDNA) libraries from human fetal ovaries and testes, human oocytes and preimplantation embryos and ovarian follicles isolated from an adult ovarian cortex biopsy were used to as templates for PCR, cloning and sequencing, and real time PCR experiments of GTSF1 expression. Results GTSF1 cDNA clones that covered the entire coding region were isolated from human oocytes and preimplantation embryos. GTSF1 mRNA expression was detected in archived cDNAs from staged human ovarian follicles, germinal vesicle (GV) stage oocytes, metaphase II oocytes, and morula and blastocyst stage preimplantation embryos. Within the adult female germline, expression was highest in GV oocytes. GTSF1 mRNA expression was also assessed in human fetal ovary and was observed to increase during gestation, from 8 to 21 weeks, during which time oogonia enter meiosis and primordial follicle formation first occurs. In human fetal testis, GTSF1 expression also increased from 8 to 19 weeks. Conclusions To our knowledge, this report is the first to describe the expression of the human GTSF1 gene in human gametes and preimplantation embryos.

A detailed understanding of the cryobiology of gametes and complex tissues has led to the development of methods that facilitate the successful low temperature banking of isolated mature human oocytes, or immature oocytes within fragments of human ovarian cortex. Although many outstanding research challenges remain to be addressed, the successful development of new treatments to preserve female fertility for a range of clinical indications has largely been underpinned by the conduct of extensive, fundamental research on oocytes and ovarian tissues from a number of laboratory and commercially important farm species. Indeed, the most recent evidence from large animals suggests that it is also possible to cryopreserve intact whole ovaries along with their supporting vasculature for later auto-transplantation and restoration of natural fertility. This review will explore how the methods developed to preserve human oocytes and ovarian tissues can now be used strategically to support the development of conservation strategies aimed at safeguarding the genetic diversity of commercially important domestic animals and also of preserving the female germplasm for wild animals and endangered species.

The in vitro maturation (IVM) of human oocytes represents a valuable assisted reproductive technology that bypasses the need for full ovarian stimulation, offering safer alternatives for patients with polycystic ovary syndrome (PCOS), resistant ovary syndrome, or those requiring fertility preservation before oncological treatment. Despite its potential, IVM efficiency remains lower than that of conventional in vitro fertilization (IVF) due to incomplete understanding of the molecular and metabolic mechanisms underpinning oocyte maturation. This review summarizes recent advances in IVM, including biphasic or simulated physiological oocyte maturation (SPOM) systems, optimization of culture media through hormones, growth factors, and antioxidants, and the influence of cumulus–oocyte communication on developmental competence. We also discuss the biochemical regulation of meiosis, metabolic interactions, and gene expression patterns associated with oocyte quality. Furthermore, we examine the translational and clinical applications of IVM in human fertility treatment, highlighting its efficacy in PCOS and oncofertility cases, and the limitations that persist in replicating in vivo conditions. Emerging technologies such as microfluidic and three-dimensional culture systems show promise in enhancing oocyte competence and embryo yield. Continued research into the molecular mechanisms governing oocyte maturation will be key to improving IVM outcomes and integrating this approach as a mainstream option in reproductive medicine.