Towards Sulphite-Free Winemaking: A New Horizon of Vinification and Maturation.

The complex dynamics between oxygen exposure, sulphur dioxide (SO2) utilization, and wine quality are of the utmost importance in wine sector, and this study aims to explore their fine balance in winemaking. As a common additive, SO2 works as an antiseptic and antioxidant. However, its excessive use has raised health concerns. Regulatory guidelines, including Council Regulation (EC) N° 1493/1999 and Commission Regulation (EC) No 1622/2000, dictate SO2 concentrations in wines. The increasing demand for natural preservatives is driving the search for alternatives, with natural plant extracts, rich in phenolic compounds, emerging as promising substitutes. In this context, Bioma Company has proposed alternative additives deriving from vineyard waste to replace SO2 during winemaking. Thus, the aim of the present work was to compare the compositional characteristics between the product obtained with the alternative vinification and the traditional one during the winemaking, as well as the aroma compositions of the final wines. After a year of experimentation, the wines produced with Bioma products showed compositional characteristics comparable to their traditional counterparts. Notably, these wines comply with current legislation, with significantly reduced total sulphur content, allowing their designation as "without added sulphites". Bioma products emerge as potential catalysts for sustainable and health-conscious winemaking practices, reshaping the landscape of the industry.

The role of metabolism in cardiac development.

Metabolism is the fundamental process that sustains life. The heart, in particular, is an organ of high energy demand, and its energy substrates have been studied for more than a century. In recent years, there has been a growing interest in understanding the role of metabolism in the early differentiation of pluripotent stem cells and in cancer research. Studies have revealed that metabolic intermediates from glycolysis and the tricarboxylic acid cycle act as co-factors for intracellular signal transduction, playing crucial roles in regulating cell behaviors. Mitochondria, as the central hub of metabolism, are also under intensive investigation regarding the regulation of their dynamics. The metabolic environment of the fetus is intricately linked to the maternal metabolic status, and the impact of the mother's nutrition and metabolic health on fetal development is significant. For instance, it is well known that maternal diabetes increases the risk of cardiac and nervous system malformations in the fetus. Another notable example is the decrease in the risk of neural tube defects when pregnant women are supplemented with folic acid. These examples highlight the profound influence of the maternal metabolic environment on the fetal organ development program. Therefore, gaining insights into the metabolic environment within developing fetal organs is critical for deepening our understanding of normal organ development. This review aims to summarize recent findings that build upon the historical recognition of the environmental and metabolic factors involved in the developing embryo.

Yinxieling decoction ameliorates psoriasis by regulating the differentiation and functions of Langerhans cells via the TGF-ß1/PU.1/IL-23 signal axis.

Langerhans cells (LCs) play a critical role in skin immune responses and the development of psoriasis. Yinxieling (YXL) is a representative Chinese herbal medicine for the treatment of psoriasis in South China. It was found to improve psoriasis without obvious side effects in the clinic. Here we attempted to clarify whether and how YXL regulates the differentiation and functions of LCs in Imiquimod (IMQ)-induced psoriasis in vivo and induced LCs in vitro. The Psoriasis Area Severity Index (PASI) score was used to evaluate the efficacy of YXL for IMQ-induced psoriasis-like mice. Flow cytometry was utilized to analyze the effects of YXL, to regulate the differentiation, migration, maturation, and antigen presentation of LCs. The results show that YXL significantly alleviated skin inflammation, as reduced in PASI score and classic psoriasis characteristics in pathological sections. Although there was no effect on the proportion of total DCs in the skin-draining lymph nodes, the expression of epidermal LCs and its transcription factor PU.1 were both markedly inhibited. LCs were also prevented from migrating from epidermal to skin-draining lymph nodes and mature. In addition, the number of LCs carrying antigens in the epidermis increased, which suggested that YXL could effectively prevent LCs from presenting antigens. In vitro, YXL had a significant impact on inhibiting the differentiation of LCs. Further data showed that YXL decreased the relative expression of transforming growth factor-ß (TGFß) messenger RNA (mRNA) and interleukin-23 (IL-23) mRNAs. Thus, YXL alleviates psoriasis by regulating differentiation, migration, maturation, and antigen presentation via the TGFß/PU.1/IL-23 signal axis.

The effect of L-carnitine supplementation during in vitro maturation on oocyte maturation and somatic cloned embryo development.

The quality of the recipient cytoplasm was reported as a crucial factor in maintaining the vitality of SCNT embryos and SCNT efficiency for dairy cows. Compared with oocytes matured in vivo, oocytes matured in vitro showed abnormal accumulation and metabolism of cytoplasmic lipids. L-carnitine treatment was found to control fatty acid transport into the mitochondrial ß-oxidation pathway, which improved the process of lipid metabolism. The results of this study show that 0.5 mg/ml L-carnitine significantly reduced the cytoplasmic lipid content relative to control. No significant difference was observed in the rate of oocyte nuclear maturation, but the in vitro developmental competence of SCNT embryos was improved in terms of increased blastocyst production and lower apoptotic index in the L-carnitine treatment group. In addition, the pregnancy rate with SCNT embryos in the treatment group was significantly higher than in the control group. In conclusion, the present study demonstrated that adding L-carnitine to the maturation culture medium could improve the developmental competence of SCNT embryos both in vitro and in vivo by reducing the lipid content of the recipient cytoplasm.

Does ethnicity influence bone health index in children? A pilot study.

BACKGROUND: Several pathological conditions can lead to variations in bone mineral content during growth. When assessing bone age, bone mineral content can be estimated without supplementary cost and irradiation. Manual assessment of bone quality using the Exton-Smith index (ESI) and automated assessment of the bone health index (BHI) provided by the BoneXpert® software are available but still not validated in different ethnic groups. OBJECTIVE: Our aim is to provide normative values of the ESI and BHI for healthy European Caucasian and first-generation children of North Africans living in Europe. MATERIALS AND METHODS: A sex- and aged-match population of 214 girls (107 European-Caucasian and 107 North African) and 220 boys (111 European-Caucasian and 109 North African) were retrospectively and consecutively included in the study. Normal radiographs of the left hand and wrist from healthy children were retrieved from those performed in a single institution from 2008 to 2017 to rule out a left-hand fracture. Radiographs were processed by BoneXpert® to obtain the BHI and BHI standard deviation score (SDS). One radiologist, blinded to BHI values, manually calculated ESI for each patient. The variability for both methods was assessed and compared using the standard deviation (SD) of the median (%) for each class of age and sex, and ESI and BHI trends were compared by sex and ethnic group. RESULTS: The final population comprised 434 children ages 3 to 15 years (214 girls). Overall, BHI was lower in North African children (mean = 4.23 for girls and 4.17 in boys) than in European Caucasians (mean = 4.50 for girls and 4.68 in boys) (P < 0.001). Regardless of ethnicity, 29 girls (13.6%) and 34 boys (15.5%) had BHI more than 2 SD from the mean. While correlated to BHI, ESI has a higher variability than BHI and is more pronounced from 8-12 years for both sexes (mean ESI in European Caucasian girls and boys 17.47 and 20.87, respectively) (P < 0.001). ESI showed more than 15% variability in European girls from 8-12 years and a plateau in North African boys from 12 years to 16 years. However, the BHI has less than 15% variability regardless of age and ethnic group. CONCLUSION: BHI may be a reliable tool to detect children with abnormal bone mineral content, with lower variability compared to ESI and with specific trends depending on sex and ethnicity.

Synergistic Glutathione Depletion and STING Activation to Potentiate Dendritic Cell Maturation and Cancer Vaccine Efficacy.

Dendritic cell (DC) maturation and antigen presentation are key factors for successful vaccine-based cancer immunotherapy. This study developed manganese-based layered double hydroxide (Mn-LDH) nanoparticles as a self-adjuvanted vaccine carrier that not only promoted DC maturation through synergistically depleting endogenous glutathione (GSH) and activating STING signaling pathway, but also facilitated the delivery of model antigen ovalbumin (OVA) into lymph nodes and subsequent antigen presentation in DCs. Significant therapeutic-prophylactic efficacy of the OVA-loaded Mn-LDH (OVA/Mn-LDH) nanovaccine was determined by the tumor growth inhibition in the mice bearing B16-OVA tumor. Our results showed that the OVA/Mn-LDH nanoparticles could be a potent delivery system for cancer vaccine development without the need of adjuvant. Therefore, the combination of GSH exhaustion and STING pathway activation might be an advisable approach for promoting DC maturation and antigen presentation, finally improving cancer vaccine efficacy.

Herbal Treatment of Female Urogenital Atrophy: A Retrospective Cohort Analysis.

INTRODUCTION: Urogenital atrophy and its sequelae, particularly genital dryness, urological problems, and pain on genital touching, are common medical problems for menopausal women and women undergoing antihormonal cancer treatment. To meet the requirements for a nonhormonal local treatment, a compounded herbal preparation was developed as a vaginal ovule (Dioscorea comp. ovulum), and the efficacy and applicability of this herbal treatment were investigated. METHODS: This was a retrospective chart review of patients' records. The study was approved by the Ethics Committee of the Canton of Zurich (project number BASEC 2016-01982). Between 2007 and 2011, patients with urogenital atrophy and related symptoms, who wanted to initiate herbal treatment, were asked for consent to be interviewed (4-point rating scale) and examined gynecologically with photo documentation of their vaginal discharge. A total of 26 patients met the enrollment criteria and consented to the procedure. The first 8 weeks consisted of a daily application of low-dose Dioscorea comp. ovulum followed by high-dose Dioscorea comp. ovule twice weekly for at least 3 months. RESULT: A total of 23 patients completed the trial. Of the 19 patients in the subgroup with an atrophic vaginal maturation index (VMI), 16 achieved a eutrophic VMI. Four patients began therapy with hypotrophy. There was a 96% decrease in complaints (22/23). The genital dryness score decreased from 1.80 to 0.25 points, urological problems from 2.38 to 0.85 points, and pain on genital touching from 1.70 to 0.60 points. Application, tolerability, and medical safety of the formula were good. CONCLUSION: The phytotherapeutic compounded preparation Dioscorea comp. ovule (Dioscorea villosa, Glycine max, Salvia officinalis) is suitable for the treatment of urogenital atrophy and its sequelae.Urogenitale Atrophie und ihre Folgeerscheinungen, insbesondere genitale Trockenheit, urologische Probleme und schmerzhafte genitale Berührungen, sind ein häufiges medizinisches Problem bei menopausalen Frauen und Frauen unter antihormoneller Krebstherapie. Um den Bedarf an einer nicht-hormonellen lokalen Behandlung zu decken, wurde ein pflanzliches Präparat in Form eines Vaginal-Ovulum (Dioscorea comp. Ovulum) entwickelt. Wirksamkeit und Anwendbarkeit dieser pflanzlichen Behandlung werden untersucht.Es handelt sich um eine retrospektive Analyse. Das Studienprotokoll wurde von der Ethikkommission des Kantons Zürich genehmigt (Projekt Nummer BASEC 2016-01982). Zwischen 2007 und 2011 wurden geeignete Patientinnen mit urogenitaler Atrophie und assoziierten Beschwerden, die mit einer pflanzlichen Behandlung beginnen wollten, um ihr Einverständnis zu einer Befragung (4-Punkte-Score) und gynäkologischen Untersuchungen mit Fotodokumentation des Vaginalausflusses (Nativpräparat) gebeten. 26 Patientinnen erfüllten die Kriterien und erklärten sich mit dem Verfahren einverstanden.Nach einer täglichen Anwendung eines niederdosierten Dioscorea comp. Ovulum folgte eine zweimal wöchentliche Anwendung des höher dosierten Dioscorea comp. Ovulum für mindestens 3 Monate.Insgesamt 23 Patientinnen schlossen die Studie ab. 16 von 19 Patientinnen in der Untergruppe mit einem atrophen Vaginalen Maturations-Index (VMI) erreichten ein eutrophes Vaginalepithel. Vier Patientinnen begannen mit einer Hypotrophie. Ein Rückgang der Beschwerden wurde bei 96% (22 von 23) erreicht: Trockenheit im Genitalbereich ging von 1,80 auf 0,25 Score-Punkte zurück, urologische Probleme von 2,38 auf 0,85 Score-Punkte und schmerzhafte Berührungen im Genitalbereich von 1,70 auf 0,60 Score-Punkte. Anwendung, Verträglichkeit und medizinische Sicherheit der Magistralrezeptur sind gut.Das phytotherapeutische Kombinationspräparat Dioscorea comp. Ovulum (Dioscorea villosa L., Glycine Max Merr., Salvia officinalis L.) eignet sich zur Behandlung der urogenitalen Atrophie und ihrer Folgeerscheinungen.

Anethole supplementation during in vitro maturation increases in vitro goat embryo production in a concentration-dependent manner.

During in vitro maturation (IVM) cumulus-oocyte complexes (COCs) are exposed to conditions that can trigger oxidative stress, thus, reducing oocyte maturation and viability. Aiming to mitigate these detrimental conditions, the effects of IVM medium supplementation with anethole have been tested. Anethole, also known as trans-anethole (1-methoxy-4 [1-propenyl]-benzene), is a naturally occurring phenylpropanoid with various pharmacological properties, including antioxidant effects. However, no study has examined anethole effect on goat COCs during IVM. Thus, the aim of this study was to evaluate the effects of different anethole concentrations on oocyte maturation, oxidative stress, and in vitro development of caprine embryos after parthenogenetic activation. Goat COCs were selected and randomly distributed into the following treatments: TCM-199+ medium (control), or TCM-199+ medium supplemented with 30 µg/mL (AN30); 300 µg/mL (AN300) or 2000 µg/mL (AN2000) of anethole. After IVM, part of the COCs was chosen for oocyte viability and chromatin configuration, intracellular reactive oxygen species levels, and mitochondrial membrane potential assessment. Another part of COCs was parthenogenetically activated, and presumptive zygotes were cultured for 7 days. Results demonstrated that anethole at 30 µg/mL increased oocyte maturation and cleavage rates when compared to the other treatments (P < 0.05), as well as oocyte viability and in vitro embryo production when compared to the control treatment (P < 0.05). Additionally, treatment with anethole at 2000 µg/mL decreased oocyte nuclear maturation and cleavage rates when compared to other treatments (P < 0.05) and embryo production if compared to control and AN30 treatments (P < 0.05). Moreover, anethole at 2000 µg/mL increased mitochondrial membrane potential when compared to the other treatments (P < 0.05). In conclusion, anethole exerts a concentration-dependent effect during goat COCs IVM. For a more desirable outcome of oocyte viability and maturation, and in vitro embryo production, the use of anethole at 30 µg/mL is recommended.

Ameliorating Effect of Sodium Selenite on Developmental and Molecular Response of Bovine Cumulus-Oocyte Complexes Matured in Vitro Under Heat Stress Condition.

Selenium (Se), an essential trace element, plays an important role in the antioxidative defense mechanism, and it has been proven to improve fertility and reproductive efficiency in dairy cattle. The present study evaluated the potential protective action of Se supplement of in vitro maturation (IVM) media on the maturation and subsequent development of bovine cumulus-oocyte complexes (COCs) exposed to heat stress (HS). The treatment with Se improved the viability of cumulus cells (CCs) and oocytes (P < 0.05). The proportion of oocytes reached metaphase II (MII) and those arrested at metaphase I (MI) was greater and lower in treatment than control respectively (P < 0.05). Supplementation with Se increased the percentage of cleaved embryos, total blastocysts, and blastocyst/cleavage ratio (P < 0.05). Moreover, the upregulation of CCND1, SEPP1, GPX-4, SOD, CAT, and downregulation of GRP78, CHOP, and BAX in both Se-treated CCs and oocytes were recorded. The upregulation of NRF2 was detected in Se-treated CCs other than in oocytes, which showed upregulation of IGF2R and SOX-2 as the markers of quality as well. Se supplement in IVM media improved the viability, maturation, and the level of transcripts related to antioxidant defense and quality of heat-treated oocytes, which coincided with greater subsequent development outcomes. Se ameliorated the viability of CCs along with upregulation of antioxidative candidate gene expression and downregulation of apoptosis-related ones to support their protective role on restoring the quality of oocytes against compromising effects of HS.

Synergistic impact of -linolenic acid and -tocopherol on in vitro maturation and culture of buffalo oocytes

Abstract The objective of the current study was to investigate the synergistic impact of -Tocopherol and -Linolenic acid (100 µM) on IVM and IVC of Nili Ravi buffalo oocytes. Oocytes were obtained from the ovaries of slaughtered buffaloes within two hours after slaughter and brought to laboratory. Buffalo cumulus oocyte complexes were placed randomly in the five experimental groups included; GROUP 1: Maturation media (MM) + 100 µM ALA (control), GROUP 2: MM + 100 µM ALA + 50M -Tocopherol, GROUP 3: MM + 100 µM ALA + 100M -Tocopherol, GROUP 4: MM + 100 µM ALA + 200 M -Tocopherol and GROUP 5: MM + 100 µM ALA + 300 M -Tocopherol under an atmosphere of 5% CO2 in air at 38.5 °C for 22-24 h. Cumulus expansion and nuclear maturation status was determined (Experiment 1). In experiment 2, oocytes were matured as in experiment 1. The matured oocytes were then fertilized in Tyrodes Albumin Lactate Pyruvate (TALP) medium for about 20 h and cultured in synthetic oviductal fluid (SOF) medium to determine effect of -Linolenic acid (100 µM) and -Tocopherol in IVM medium on IVC of presumptive zygotes. To study the effect of -Linolenic acid (100 µM) in IVM media and increasing concentration of -tocopherol in the culture media on early embryo development (Experiment 3), the presumptive zygotes were randomly distributed into the five experimental groups with increasing concentration of -tocopherol in culture media. Higher percentage of MII stage oocytes in experiment 1(65.2±2.0), embryos at morula stage in experiment 2 (30.4±1.5) and experiment 3 (22.2±2.0) were obtained. However, overall results for cumulus cell expansion, maturation of oocyte to MII stage and subsequent embryo development among treatments remain statistically similar (P > 0.05). Supplementation of -tocopherol in maturation media having -Linolenic acid and/or in embryo culture media did not further enhance in vitro maturation of oocyte or embryo production.

Resumo O objetivo do presente estudo foi investigar o impacto sinérgico do -tocoferol e do ácido -linolênico (100 µM) na MIV e CIV de oócitos de búfala Nili Ravi. Os oócitos foram obtidos dos ovários de búfalos abatidos duas horas após o abate e levados ao laboratório. Complexos de oócitos cumulus de búfalo foram colocados aleatoriamente nos cinco grupos experimentais incluídos; GRUPO 1: Meio de maturação (MM) + 100 µM ALA (controle), GRUPO 2: MM + 100 µM ALA + 50 µM -tocoferol, GRUPO 3: MM + 100 µM ALA + 100 µM -tocoferol, GRUPO 4: MM + 100 µM ALA + 200 M -tocoferol e GRUPO 5: MM + 100 µM ALA + 300 M -tocoferol sob uma atmosfera de 5% de CO2 em ar a 38,5 °C por 22-24 h. A expansão cumulus e o estado de maturação nuclear foram determinados (Experimento 1). No experimento 2, os oócitos foram maturados como no experimento 1. Os oócitos maturados foram então fertilizados em meio de Tyrode's Albumina Lactato Piruvato (TALP) por cerca de 20 h e cultivados em meio de fluido oviductal sintético (SOF) para determinar o efeito do ácido -linolênico (100 µM) e -tocoferol em meio IVM em IVC de presumíveis zigotos. Para estudar o efeito do ácido -linolênico (100 µM) em meio IVM e aumentar a concentração de -tocoferol no meio de cultura no desenvolvimento inicial do embrião (Experimento 3), os presumíveis zigotos foram distribuídos aleatoriamente nos cinco grupos experimentais com concentração crescente de -tocoferol em meios de cultura. Maior porcentagem de oócitos em estágio MII no experimento 1 (65,2 ± 2,0), embriões em estágio de mórula no experimento 2 (30,4 ± 1,5) e experimento 3 (22,2 ± 2,0) foram obtidos. No entanto, os resultados gerais para a expansão das células do cumulus, maturação do oócito para o estágio MII e desenvolvimento embrionário subsequente entre os tratamentos permanecem estatisticamente semelhantes (P> 0,05). A suplementação de -tocoferol em meios de maturação com ácido -linolênico e / ou em meios de cultura de embriões não aumentou ainda mais a maturação in vitro de oócitos ou a produção de embriões.

Exploring the mechanism of Lycium barbarum fruit cell wall polysaccharide remodeling reveals potential pectin accumulation contributors.

The level of polysaccharides in the mature Lycium barbarum fruit (LBF) cell wall depends on their metabolism, trafficking, and reassembly within the cell. In this study, we examined the composition, content, and ultrastructure of the cell wall polysaccharides of LBF during maturation, and further analyzed cell wall polysaccharide remodeling using isotope tagging with relative and absolute quantification (iTRAQ)-based proteomics. The results showed that the contents of cellulose and hemicellulose tended to increase in the pre-maturation stage and decrease in the later stage, while pectin level increased before fruit maturing. The differential expression of the 54 proteins involved in the metabolic pathways for glucose, fructose, galactose, galacturonic acid and arabinose was found to be responsible for these alterations. The work provides a biological framework for the reorganization of polysaccharides in the LBF cell wall, and supports the hypothesis that pectic polysaccharide glycosyl donors come from starch, cellulose, hemicellulose and isomorphic pectin.

Metabolomics analysis of quality components metabolism during the growth process of pepino (Solanum muricatum) fruit.

Pepino (Solanum muricatum), a horticultural crop that has experienced significant growth in the highlands of China over the past two decades, is widely embraced by consumers due to its distinctive taste and nutritional advantages. This study focused on the cultivar 'Qingcanxiang' of pepino grown on the Qinghai-Tibetan Plateau was analyzed using UPLC-QTOF-MS and RNA-seq transcriptome sequencing. Fruit samples were collected at three distinct stages of development, and the results of the metabolomics and transcriptomics were compared and correlated. The study's findings indicate that the 'Qingcanxiang' fruit contained a total of 187 metabolites, comprising 12 distinct categories of compounds, including amino acids and their derivatives, organic acids, sugars and alcohols, phenols and phenolic acids. Of these metabolites, 94 were identified as differential. Significant variations in nutrient composition were observed across the three growth stages of the fruit. Specifically, the stage spanning from the growth to the maturation was identified as the critical stages for nutrient accumulation and flavor development. Transcriptome sequencing analysis revealed a set of highly associated genes between aspartate and quinic acid, namely SIR2, IRAK4, RP-L29, and CCNH. These genes are potentially involved in the regulation of both amino acid and phenolic acid synthesis. Through the application of metabolomics and transcriptomics, this investigation elucidates the alterations in metabolites and the underlying molecular regulatory mechanisms of pepino fruits during three growth stages. The findings furnish a theoretical foundation for the evaluation of nutritional quality and the enhancement of breeding strategies for pepino.

Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation.

STUDY QUESTION: Can in vitro maturation (IVM) and developmental competence of human oocytes be improved by co-culture with ovarian support cells (OSCs) derived from human-induced pluripotent stem cells (hiPSCs)? SUMMARY ANSWER: OSC-IVM significantly improves the rates of metaphase II (MII) formation and euploid Day 5 or 6 blastocyst formation, when compared to a commercially available IVM system. WHAT IS KNOWN ALREADY: IVM has historically shown highly variable performance in maturing oocytes and generating oocytes with strong developmental capacity, while limited studies have shown a positive benefit of primary granulosa cell co-culture for IVM. We recently reported the development of OSCs generated from hiPSCs that recapitulate dynamic ovarian function in vitro. STUDY DESIGN, SIZE, DURATION: The study was designed as a basic science study, using randomized sibling oocyte specimen allocation. Using pilot study data, a prospective sample size of 20 donors or at least 65 oocytes per condition were used for subsequent experiments. A total of 67 oocyte donors were recruited to undergo abbreviated gonadotropin stimulation with or without hCG triggers and retrieved cumulus-oocyte complexes (COCs) were allocated between the OSC-IVM or control conditions (fetal-like OSC (FOSC)-IVM or media-only IVM) in three independent experimental design formats. The total study duration was 1 April 2022 to 1 July 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: Oocyte donors between the ages of 19 and 37 years were recruited for retrieval after informed consent, with assessment of anti-Mullerian hormone, antral follicle count, age, BMI and ovarian pathology used for inclusion and exclusion criteria. In experiment 1, 27 oocyte donors were recruited, in experiment 2, 23 oocyte donors were recruited, and in experiment 3, 17 oocyte donors and 3 sperm donors were recruited. The OSC-IVM culture condition was composed of 100 000 OSCs in suspension culture with hCG, recombinant FSH, androstenedione, and doxycycline supplementation. IVM controls lacked OSCs and contained either the same supplementation, FSH and hCG only (a commercial IVM control), or FOSCs with the same supplementation (Media control). Experiment 1 compared OSC-IVM, FOSC-IVM, and a Media control, while experiments 2 and 3 compared OSC-IVM and a commercial IVM control. Primary endpoints in the first two experiments were the MII formation (i.e. maturation) rate and morphological quality assessment. In the third experiment, the fertilization and embryo formation rates were assessed with genetic testing for aneuploidy and epigenetic quality in blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE: We observed a statistically significant improvement (∼1.5×) in maturation outcomes for oocytes that underwent IVM with OSCs compared to control Media-IVM and FOSC-IVM in experiment 1. More specifically, the OSC-IVM group yielded a MII formation rate of 68% ± 6.83% SEM versus 46% ± 8.51% SEM in the Media control (P = 0.02592, unpaired t-test). FOSC-IVM yielded a 51% ± 9.23% SEM MII formation rate which did not significantly differ from the media control (P = 0.77 unpaired t-test). Additionally, OSC-IVM yielded a statistically significant ∼1.6× higher average MII formation rate at 68% ± 6.74% when compared to 43% ± 7.90% in the commercially available IVM control condition (P = 0.0349, paired t-test) in experiment 2. Oocyte morphological quality between OSC-IVM and the controls did not significantly differ. In experiment 3, OSC-IVM oocytes demonstrated a statistically significant improvement in Day 5 or 6 euploid blastocyst formation per COC compared to the commercial IVM control (25% ± 7.47% vs 11% ± 3.82%, P = 0.0349 logistic regression). Also in experiment 3, the OSC-treated oocytes generated blastocysts with similar global and germline differentially methylated region epigenetic profiles compared commercial IVM controls or blastocysts after either conventional ovarian stimulation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: While the findings of this study are compelling, the cohort size remains limited and was powered on preliminary pilot studies, and the basic research nature of the study limits generalizability compared to randomized control trials. Additionally, use of hCG-triggered cycles results in a heterogenous oocyte cohort, and potential differences in the underlying maturation state of oocytes pre-IVM may limit or bias findings. Further research is needed to clarify and characterize the precise mechanism of action of the OSC-IVM system. Further research is also needed to establish whether these embryos are capable of implantation and further development, a key indication of their clinical utility. WIDER IMPLICATIONS OF THE FINDINGS: Together, these findings demonstrate a novel approach to IVM with broad applicability to modern ART practice. The controls used in this study are in line with and have produced similar to findings to those in the literature, and the outcome of this study supports findings from previous co-culture studies that found benefits of primary granulosa cells on IVM outcomes. The OSC-IVM system shows promise as a highly flexible IVM approach that can complement a broad range of stimulation styles and patient populations. Particularly for patients who cannot or prefer not to undergo conventional gonadotropin stimulation, OSC-IVM may present a viable path for obtaining developmentally competent, mature oocytes. STUDY FUNDING/COMPETING INTEREST(S): A.D.N., A.B.F., A.G., B.P., C.A., C.C.K., F.B., G.R., K.S.P., K.W., M.M., P.C., S.P., and M.-J.F.-G. are shareholders in the for-profit biotechnology company Gameto Inc. P.R.J.F. declares paid consultancy for Gameto Inc. P.C. also declares paid consultancy for the Scientific Advisory Board for Gameto Inc. D.H.M. has received consulting services from Granata Bio, Sanford Fertility and Reproductive Medicine, Gameto, and Buffalo IVF, and travel support from the Upper Egypt Assisted Reproduction Society. C.C.K., S.P., M.M., A.G., B.P., K.S.P., G.R., and A.D.N. are listed on a patent covering the use of OSCs for IVM: U.S. Provisional Patent Application No. 63/492,210. Additionally, C.C.K. and K.W. are listed on three patents covering the use of OSCs for IVM: U.S. Patent Application No. 17/846,725, U.S Patent Application No. 17/846,845, and International Patent Application No.: PCT/US2023/026012. C.C.K., M.P.S., and P.C. additionally are listed on three patents for the transcription factor-directed production of granulosa-like cells from stem cells: International Patent Application No.: PCT/US2023/065140, U.S. Provisional Application No. 63/326,640, and U.S. Provisional Application No. 63/444,108. The remaining authors have no conflicts of interest to declare.

Progesterone and Androstenedione Are Important Follicular Fluid Factors Regulating Porcine Oocyte Maturation Quality.

Oocytes matured in vitro are useful for assisted human and farm animal reproduction. However, the quality of in vitro matured oocytes is usually lower than that of in vivo matured oocytes, possibly due to the absence of some important signal regulators in vitro. In this study, untargeted metabolomics was used to detect the changes in the metabolites in the follicular fluid (FF) during in vivo pig oocyte maturation and in the culture medium during in vitro maturation. Our results showed that the total metabolite changing profile of the in vivo FF was different from that of the in vitro maturation medium, but the levels of 23 differentially expressed metabolites (DEMs) changed by following the same trend during both in vivo and in vitro pig oocyte maturation. These 23 metabolites may be important regulators of porcine oocyte maturation. We found that progesterone and androstenedione, two factors in the ovarian steroidogenesis pathway enriched from the DEMs, were upregulated in the FF during in vivo pig oocyte maturation. The levels of these two factors were 31 and 20 fold, respectively, and they were higher in the FF than in the culture medium at the oocyte mature stage. The supplementation of progesterone and androstenedione during in vitro maturation significantly improved the pig oocyte maturation rate and subsequent embryo developmental competence. Our finding suggests that a metabolic abnormality during in vitro pig oocyte maturation affects the quality of the matured oocytes. This study identified some important metabolites that regulate oocyte maturation and their developmental potential, which will be helpful to improve assisted animal and human reproduction.

Alpha-lipoic acid supplementation reverses the declining quality of oocytes exposed to cyclophosphamide.

Cyclophosphamide (CY) is a chemotherapeutic drug that is commonly used to treat malignancies of the ovary, breast, and hematology, as well as autoimmune disorders. As a cofactor of mitochondrial multienzyme complexes, alpha lipoic acid (ALA) is well known for its antioxidant characteristics, which operate directly on the scavenging of reactive oxygen species (ROS) and indirectly on the intracellular recycling of other antioxidants. However, the underlying mechanisms through which CY exerts its toxic effects on meiosis and oocyte quality, as well as a viable approach for protecting oocyte quality and preserving fertility, remain unknown. In present study, immunostaining and fluorescence intensity quantification were applied to assess the effects of CY and ALA supplementation on the key processes during the oocyte meiotic maturation. Our results show that supplementing oocytes with ALA, a well-known antioxidant and free radical scavenger, can reverse CY-induced oocyte meiotic maturation failure. Specifically, we found that CY exposure caused oocyte meiotic failure by disrupting meiotic organelle dynamics and arrangement, as well as a prominently impaired cytoskeleton assembly. In addition, CY caused an abnormal distribution of mitochondrion and cortical granules, two indicators of oocyte cytoplasmic maturation. More importantly, we show that ALA supplementation effectively reverses CY-induced meiotic failure and oocyte quality decline by suppressing oxidative stress-induced DNA damage and apoptosis in oocytes. Collectively, our data reveal that ALA supplementation is a feasible approach to protect oocytes from CY-exposed deterioration, providing a better understanding of the mechanisms involved in chemotherapy-induced meiotic failure.

Salidroside Supplementation Affects In Vitro Maturation and Preimplantation Embryonic Development by Promoting Meiotic Resumption.

Salidroside (Sal) possesses several pharmacological activities, such as antiaging, and anti-inflammatory, antioxidant, anticancer activities, and proliferation-promoting activities, but the effects of Sal on oocytes have rarely been reported. In the present study, we evaluated the beneficial effects of Sal, which is mainly found in the roots of Rhodiola. Porcine cumulus oocyte complexes were cultured in IVM medium supplemented (with 250 µmol/L) with Sal or not supplemented with Sal. The maturation rate in the Sal group increased from 88.34 ± 4.32% to 94.12 ± 2.29%, and the blastocyst rate in the Sal group increased from 30.35 ± 3.20% to 52.14 ± 7.32% compared with that in the control group. The experimental groups showed significant improvements in the cumulus expansion area. Sal reduced oocyte levels of reactive oxygen species (ROS) and enhanced intracellular GSH levels. Sal supplementation enhanced the mitochondrial membrane potential (MMP), ATP level, and mtDNA copy number, which shows that Sal enhances the cytoplasmic maturation of oocytes. Oocytes in the Sal group exhibited slowed apoptosis and reduced DNA breakage. Cell cycle signals and oocyte meiosis play important roles in oocyte maturation. The mRNA expressions of the MAPK pathway and MAPK phosphorylation increased significantly in the Sal group. The mRNA expression of the oocyte meiosis gene also increased significantly. These results show that Sal enhances the nuclear maturation of oocytes. Moreover, Sal increased the number of blastocyst cells, the proliferation of blastocysts, and the expressions of pluripotency genes. Sal down-regulated apoptosis-related genes and the apoptotic cell rate of blastocysts. In summary, our results demonstrate that Sal is helpful to improving the quality of porcine oocytes in vitro, and their subsequent embryonic development.

Melatonin enhances the developmental potential of immature oocytes from older reproductive-aged women by improving mitochondrial function.

Aims: To evaluate whether melatonin (MT) supplementation during in vitro maturation (IVM) of human oocytes can reverse the age-related decline in oocyte quality. Main methods: We enrolled 172 patients aged ≥35 years (older reproductive-aged women) and 83 patients aged <35 years (young women) who underwent in vitro fertilization between 2019 and 2022. We conducted IVM with and without 10 µM MT in immature oocytes of different ages. Oocyte fertilization and embryo development were observed using a stereomicroscope. We assessed the immunofluorescence intensity of mitochondrial function, measured the copy number of mitochondrial DNA (mtDNA), and examined the spindle and chromosome composition in in vitro mature stage II (IVM-MII) oocytes using immunofluorescence and second-generation sequencing. Key findings: MT supplementation significantly improved the redox level in the IVM medium and IVM-MII oocytes in older reproductive-aged women. It also significantly increased the proportion of circular mtDNA and the adenosine triphosphate content in IVM-MII oocytes. In addition, the IVM-MII oocytes obtained with MT supplementation showed a significant improvement in the normal composition of the spindle and chromosomes. Thus, the aged immature oocytes also showed significantly improved maturation and blastocyst formation rates owing to the role of MT. Significance: Supplementation with 10 µM MT in the IVM medium reverses the age-related decline in oocyte quality. Our findings provide a viable solution for enhancing fertility in older reproductive-aged women.

Effect of variety and malting conditions on proteolytic activity, free amino nitrogen, and soluble protein contents of two maize varieties (Atp-Y and Coca-sr): amylolytic activity and physico-chemical and functional properties of optimal sample.

Introduction: The utilization of sprouted meals in beer production and enhancing the physicochemical properties of supplementary foods is widespread in Africa. This work aimed to determine the influence of soaking, germination, maturation and variety conditions on the physicochemical properties, proteolytic activity, free amino nitrogen (FAN) and soluble protein contents of Coca-sr and Atp-Y maize varieties. Methods: To achieve this, the central composite design (CCD) was used for the optimization of five parameters, namely soaking time (18-42 h), plant salt concentration (0.5-1.2%), soaking temperature (25-41°C), sprouting time (80-195 h) and ripening time (17.50-42 h), and following dependent variables were investigated: proteolytic activity, FAN content and soluble protein. Optimal samples flours obtained were then subsequently subjected to physicochemical and functional analysis. Results: The analysis of results showed that the linear, interactive and quadratic effects of the factors significantly (p<0.05) affected the proteolytic activity, FAN and soluble protein contents of both varieties. The direction of each factor's variation and its effects were not similar in the two varieties. The optimal malting conditions were 7.31 h soaking with 1.678% vegetable salt at a temperature of 34.65°C followed by sprouting for 245.59 h and maturation for 0.765 h for the Atp-Y variety. For the Coca-sr variety, it requires 1.608 h of soaking with 1.678% vegetable salt at a temperature of 51.93°C followed by 273.94 h and 58.73 h for sprouting and ripening time respectively. The meals of Coca-sr produces using these optimal conditions showed a significantly (p<0.05) higher proteolytic activity, FAN and soluble protein content. The amylolytic activity was more pronounced in the Atp-Y variety, as was the content of essential amino acids. The above optimal conditions reduced the content of anti-nutrients (phytates, saponins, oxalates, condensed and hydrolysable tannins), improved the availability of minerals (Ca and Mg), reduced the pH, mass density, water retention capacity and swelling rate. Conclusion: As a result, the optimal flours of these two maize varieties could be applied in the formulation of supplementary foods, bakery products and beer by industrialists.

Pre-culture with transferrin-Fe3+ before in vitro maturation improves the developmental competence of porcine oocytes matured in vitro.

Purpose: Since the developmental competence of oocytes cultured after in vitro maturation (IVM) is low, it is necessary to improve the IVM method for efficient offspring production. In this study, we revealed that transferrin (TF)-Fe3+ was accumulated in follicular fluid with increasing the follicular diameter, and that TF receptor (TFR1) was localized in granulosa cells of pig. Thus, we hypothesized that TF-Fe3+ would be a factor in the induction of developmental competence of porcine oocytes. Methods: To mimic the follicular development environment, cumulus-oocyte complexes (COCs) were cultured in pre-IVM medium (low dose of FSH) without or with Holo-TF (monoferric or diferric TF) or Apo-TF (non-iron bond TF). After pre-IVM without or with Holo-TF, COCs were cultured in IVM medium (high dose of FSH and EGF) without or with Holo-TF. Results: Cultivation with Holo-TF increased the expression of follicular development maker (Cyp19a1 and Ccnd2), E2 production, and proliferative activity of cumulus cells, whereas cultivation with Apo-TF did not show these positive effects. The treatment with Holo-TF during pre-IVM, but not during IVM, dramatically induced oocyte maturation with increasing the blastocyst rate. Conclusion: We succeeded in showing for the first time that the cultivation with Holo-TF in pre-IVM can produce embryos in pig with high efficiency.

Effects of lactate, super-GDF9, and low oxygen tension during bi-phasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte complex†.

In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.