α-Ketoglutarate Improves Meiotic Maturation of Porcine Oocytes and Promotes the Development of PA Embryos, Potentially by Reducing Oxidative Stress through the Nrf2 Pathway.

α-Ketoglutarate (α-KG) is a metabolite in the tricarboxylic acid cycle. It has a strong antioxidant function and can effectively prevent oxidative damage. Previous studies have shown that α-KG exists in porcine follicles, and its content gradually increases as the follicles grow and mature. However, the potential mechanism of supplementation of α-KG on porcine oocytes during in vitro maturation (IVM) has not yet been reported. The purpose of this study was to explore the effect of α-KG on the early embryonic development of pigs and the mechanisms underlying these effects. We found that α-KG can enhance the development of early pig embryos. Adding 20 µM α-KG to the in vitro culture medium significantly increased the rate of blastocyst formation and the total cell number. Compared with to that of the control group, apoptosis in blastocysts of the supplement group was significantly reduced. α-KG reduced the production of reactive oxygen species and glutathione levels in cells. α-KG not only improved the activity of mitochondria but also inhibited the occurrence of apoptosis. After supplementation with α-KG, pig embryo pluripotency-related genes (OCT4, NANOG, and SOX2) and antiapoptotic genes (Bcl2) were upregulated. In terms of mechanism, α-KG activates the Nrf2/ARE signaling pathway to regulate the expression of antioxidant-related targets, thus combating oxidative stress during the in vitro culture of oocytes. Activated Nrf2 promotes the transcription of Bcl2 genes and inhibits cell apoptosis. These results indicate that α-KG supplements have a beneficial effect on IVM by regulating oxidative stress during the IVM of porcine oocytes and can be used as a potential antioxidant for IVM of porcine oocytes.

Sodium arsenate induced genotoxicity, morphometric and morphological changes in mice embryo and protective role of Moringa oleifera extracts.

The present study was carried out to find the comparative ameliorative role of Moringa oleifera leaf and flower extracts against sodium arsenate induced genotoxic, morphometric and morphological changes in mice embryo. Seven to eight week old pregnant females (N=44) with body weight of 20-25g at gestation day zero were divided randomly in groups (A, B, C, D, E, F, G, H, I, J and K). Group A was of control while all others were experimental groups and administered with selected doses of sodium arsenate as toxicant (6mg/kg B.W and 12mg/kg/B.W) and Moringa oleifera leaf and flower extracts as antidote (150mg/kg and 300mg/kg B.W). Significant (p<0.05) amelioration at dose 300mg/kg of Moringa oleifera leaf extract was observed against sodium arsenate induced morphological abnormalities like micromelia, excencephally, cryptothalmia, anopthalmia, laproschisis and morphometric changes like fetus weight, head circumference, crown rump and snout length were observed. Significant protection of DNA was showed in Moringa oleifera leaf extract treated groups (27.50±2.51) as compared to sodium arsenate (66.25±2.75). So concluded that sodium arsenate induced teratogenicity can be decreased using Moringa extract especially of Moringa oleifera leaf extract as it contains bioactive compounds like phenolics.

Spatiotemporal evaluation of the mouse embryonic redox environment and histiotrophic nutrition following treatment with valproic acid and 1,2-dithiole-3-thione during early organogenesis.

Redox regulation during metazoan development ensures that coordinated metabolic reprogramming and developmental signaling are orchestrated with high fidelity in the hypoxic embryonic environment. Valproic acid (VPA), an anti-seizure medication, is known to increase markers of oxidation and also increase the risk of neural tube defects (NTDs) when taken during pregnancy. It is unknown, however, whether oxidation plays a direct role in failed neural tube closure (NTC). Spatial and temporal fluctuations in total glutathione (GSH) and total cysteine (Cys) redox steady states were seen during a 24 h period of CD-1 mouse organogenesis in untreated conceptuses and following exposure to VPA and the Nrf2 antioxidant pathway inducer, 1,2-dithiole-3-thione (D3T). Glutathione, glutathione disulfide (GSSG), and Cys, cystine (CySS) concentrations, measured in conceptal tissues (embryo/visceral yolk sac) and fluids (yolk sac fluid/amniotic fluid) showed that VPA did not cause extensive and prolonged oxidation during the period of NTC, but instead produced transient periods of oxidation, as assessed by GSH:GSSG redox potentials, which revealed oxidation in all four conceptal compartments at 4, 10, and 14 h, corresponding to the period of heartbeat activation and NTC. Other changes were tissue and time specific. VPA treatment also reduced total FITC-Ab clearance from the medium over 3 h, indicating potential disruption of nutritive amino acid supply. Overall, these results indicated that VPA's ability to affect cellular redox status may be limited to tissue-specific windows of sensitivity during the period of NTC. The safety evaluation of drugs used during pregnancy should consider time and tissue specific redox factors.

Toxicity of Methanolic Extracts of Seeds of Moringa stenopetala, Moringaceae in Rat Embryos and Fetuses.

Moringa stenopetala is a medicinal plant that has been used in Ethiopian traditional medicine as a remedy for the treatment of hypertension, diabetes, and stomach pain. The study is aimed at assessing the toxicity of the methanol extracts of the seeds of Moringa stenopetala on the developing embryo and fetuses of rats. The seeds of Moringa were extracted by maceration using 80% methanol. The extract (250-1000 mg/kg) was orally administered to pregnant Swiss albino rats from days 6 to12 of gestation. Embryos and fetuses were recovered by laparotomy on gestational day 12 and day 20, respectively, and were assessed for developmental anomalies. On day 20, significant prenatal growth retardation such as reduced litter weight and crown-rump length were observed in near term fetuses of 1000 mg/kg treated rats. Litter weight in 1000 mg/kg and pair-fed control groups was 2.41 g ± 0.108 and 3.08 g ± 0.093, respectively. Delay in the development of an otic, optic, and olfactory system, as well as a reduction in a number of branchial bars, occurred on day 12 embryos of 1000 mg/kg treated rats. The rate of fetal resorption in 1000 mg/kg and pair-fed control groups was 1.6 ± 0.55 and 0.42 ± 0.52, respectively. There was also a high incidence of fetal death in the 1000 mg/kg treated group but it was not statistically significant. The offspring's of Moringa-treated rats did not show gross external malformations at all doses. These findings suggest that the methanol seed extract of Moringa stenopetala is not safe to rat embryos and fetuses. Its toxic effects were evidenced by a significant delay in embryonic and fetal development and an increase in fetal resorptions and fetal death.

The effect of lutein and Urtica dioica extract on in vitro production of embryo and oxidative status in polycystic ovary syndrome in a model of mice.

BACKGROUND: Polycystic ovary syndrome (PCOS) is one of the most prevalent endocrinopathies in women during the reproductive age. Herbal medicines are used increasingly alone or in supplement with chemical medicines for the treatment of different diseases and dysfunctions. This study was aimed to evaluate the effects of lutein and nettle (Urtica dioica) extract on the biochemical parameters and the reproductive function in the PCOS model of mice. METHODS: Following the induction of PCOS by dehydroepiandrosterone (DHEA), the mice (n = 98) were randomly assigned into seven groups, each consisting of fourteen mice; the groups were included control group (received solvent), PCOS group (received 6 mg/100 g B.W/day IP, DHEA for 21 days), PCOS+ Nettle extract (200 and 400 mg/kg), PCOS+ Lutein (125 and 250 mg/kg), and PCOS+ NL (200 mg/kg nettle extract and 125 mg/kg lutein). The nettle extract and lutein were administrated using gavage for 30 consecutive days after PCOS induction. Malondialdehyde (MDA), total antioxidant capacity (TAC), and estrogen were measured in serum, ovary, and uterus samples by the ELISA method. The total number of oocytes, oocyte quality, fertilization rate, 2-cell blastocyst, and arrested embryos (type I, type II, and type III) were also investigated. RESULTS: A combination treatment of the nettle and lutein produced the lowest concentration of MDA in comparison to other groups which affected by the PCOS. The lowest level of TAC was observed in the PCOS group without treatment. The number of oocytes, oocyte quality, fertilization rate, and 2-cell blastocyst were significantly higher in the control group, but the lowest values were observed in the PCOS group without any treatment. CONCLUSIONS: The most favorable findings include improving antioxidant capacity, oocyte and embryo quality were observed in the PCOS+ 125 L group.

Knockout mouse models are predictive of malformations or embryo-fetal death in drug safety evaluations.

Traditionally, understanding potential developmental toxicity from pharmaceutical exposures has been based on the results of ICH guideline studies in two species. However, support is growing for the use of weight of evidence approaches when communicating the risk of developmental toxicity, where the intended pharmacologic mode of action affects fundamental pathways in developmental biology or phenotypic data from genetically modified animals may increasingly be included in the overall assessment. Since some concern surrounds the use of data from knockout (KO) mice to accurately predict the risk for pharmaceutical modulation of a target, a deeper understanding of the relevance and predictivity of adverse developmental effects in KO mice for pharmacological target modulation is needed. To this end, we compared the results of embryo-fetal development (EFD) studies for 86 drugs approved by the FDA from 2017 to 2019 that also had KO mouse data available in the public domain. These comparisons demonstrate that data from KO mouse models are overall highly predictive of malformations or embryo-fetal lethality (MEFL) from EFD studies, but less so of a negative outcome in EFD studies. This information supports the use of embryo-fetal toxicity data in KO models as part of weight of evidence approaches in the communication of developmental toxicity risk of pharmaceutical compounds.

Stage-specific response in early mouse embryos exposed to prednisolone in vitro.

Corticosteroids are increasingly being used during the peri-implantation period to treat women with repeated IVF failure and recurrent miscarriage. However, the direct effects of prednisolone (PRDL), one of the commonly used corticosteroids on early embryo development is not understood. To mimic the possible clinical scenario and to understand the embryonic response to direct PRDL exposure, this pilot study was conducted in a mouse model. Cleavage stage embryos exposed to 3 and 30 µM PRDL in vitro were assessed for peri-implantation developmental potential, genetic integrity, inner cell mass (ICM) proliferation and pluripotency markers in the proliferated ICM cells. Exposure to 30 µM PRDL delayed the embryonic progression beyond compaction (P < 0.05) in comparison to vehicle control and, had reduced total cell number (P < 0.001) than all other groups. In addition, 30 µM PRDL exposure resulted in poor hatching potential (P < 0.05) and increased apoptosis in blastocysts (P < 0.05) compared to 3 µM PRDL. On the other hand, completely formed ICM outgrowths were significantly higher (P < 0.05) in 3 µM PRDL compared to control. However, no significant differences were observed in the expression of pluripotency genes. In conclusion, the trend observed in embryos exposed to PRDL in vitro provides important information concerning the use of this drug when treating patients at the peri-implantation phase of IVF cycles. However, the clinical value of this observation on human embryo development needs further research.

Induction of Oxidative Stress and Mitochondrial Dysfunction by Juglone Affects the Development of Bovine Oocytes.

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus-oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT-qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8-16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5-50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.

Ascorbic acid during the suckling period is required for proper DNA demethylation in the liver.

Ascorbic acid (AA, vitamin C) serves as a cofactor for ten-eleven translocation (TET) enzymes and induces DNA demethylation in vitro. However, its role in DNA demethylation in vivo remains unclear. We previously reported that DNA demethylation in the mouse liver was enhanced during the suckling period. Therefore, we hypothesized that DNA demethylation is enhanced in an AA-dependent manner during the suckling period. To examine our hypothesis, we employed wild-type (WT) mice, which synthesize AA, and senescence marker protein-30/gluconolactonase (SMP30/GNL) knockout (KO) mice, which cannot synthesize AA, and analyzed the DNA methylation status in the livers of offspring in both the suckling period and adulthood. SMP30/GNL KO offspring showed DNA hypermethylation in the liver possibly due to low plasma and hepatic AA levels during the suckling period despite the administration of rescue-dose AA to dams. Furthermore, DNA hypermethylation of the fibroblast growth factor 21 gene (Fgf21), a PPARα target gene, persisted into adulthood. In contrast, a high-dose AA administration to SMP30/GNL KO dams during the lactation period restored DNA demethylation in the livers of offspring. Even though a slight increase was observed in plasma AA levels with the administration of rescue-dose AA to WT dams during the gestation and lactation periods, DNA demethylation in the livers of offspring was minimally enhanced. The present results demonstrate that AA intake during the suckling period is required for proper DNA demethylation in the liver.

Low-Concentration Essential Amino Acids in PZM-3 Improve the Developmental Competence of Porcine Embryos Produced by Handmade Cloning.

Essential amino acids (EAA) of inappropriate concentration have been reported to compromise the development of embryo. This study aimed to investigate the effect of EAA on the developmental competence of porcine embryos produced by either handmade cloning (HMC) or parthenogenetic activation (PA). In experiment 1, we examined the in vitro developmental competence of PA embryos after culture in PZM-3 containing different concentrations (v/v) of EAA (0%, 1%, and 2%). The results indicated that reducing the concentration of EAA from 2% to 1% significantly improved the blastocyst formation (36% vs. 54%), while 0% would compromise the blastocyst formation rate (54% vs. 38%). In experiment 2, we further investigated the effect of EAA concentration (1% and 2%) on the in vitro developmental competence and gene expression of HMC embryos. Blastocyst rate significantly increased by reducing concentration of EAA (41% vs. 53%) and those genes upregulated were enriched in oxidative phosphorylation, PPAR signaling pathway, and metabolism-related pathways. In experiment 3, the in vivo developmental competence of HMC embryos cultured in the medium supplemented with 1% EAA was examined. Embryos derived from both non-gene-modified fetal fibroblasts (FFs) and gene-modified fetal fibroblasts (GMFFs) were transferred to recipients. The pregnancy rates were 83% and 78% separately. Out of the pregnancies, 5 (FFs) and 6 (GMFFs) were successfully developed to term. Our study indicates that supplementing EAA to embryo culture medium at a concentration of 1% can improve the in vitro developmental competence of porcine HMC embryos and the blastocyst obtained can successfully develop to term, which could be beneficial for the production of gene-modified piglets.

Daidzein supplementation enhances embryo survival by improving hormones, antioxidant capacity, and metabolic profiles of amniotic fluid in sows.

Daidzein (DAI) is a kind of natural isoflavonic phytoestrogen with estrogenic activity. However, little is known about its influence on early fetal growth in mammalian animals. The current study aimed to explore the characteristics of amniotic fluid exposure to dietary DAI using 1H NMR-based metabolomics and biochemical analysis. Here, we found that DAI supplementation at a dose of 200 mg kg-1 significantly enhanced the number of viable embryos at the early gestation stage (P < 0.05). DAI significantly elevated the concentrations of estrogen (E) and insulin-like growth factor-I (IGF-I) in the amniotic fluid (P < 0.05). Moreover, DAI tended to increase the concentration of progesterone, but decrease the concentration of tumor necrosis factor α (TNF-α) in the amniotic fluid (0.05 < P < 0.10). Interestingly, the activity of glutathione peroxidase (GSH-Px) was higher in the DAI group than in the CON group (P < 0.05). An 1H NMR-based metabolomics analysis identified and quantified more than 30 compounds in the amniotic fluid, and some critical metabolites such as arginine, creatine, and citrate were found to be significantly elevated upon DAI supplementation (P < 0.05). Importantly, the metabolic pathways involved in arginine and proline metabolisms were found to be significantly affected by DAI. Collectively, dietary DAI may improve embryo survival by improving hormones, antioxidant capacity, and metabolic profiles in the maternal amniotic fluid.

Effect of Rhodiola sachalinensis Aqueous Extract on In Vitro Maturation of Porcine Oocytes and Subsequent In Vitro Embryonic Development.

Oxidative stress can impede maturation of the nucleus and cytoplasm of oocytes during in vitro maturation (IVM). Rhodiola sachalinensis, an herb commonly used in traditional Chinese medicine, conveys antioxidative effects to cryopreserved bovine sperm. Therefore, the aims of this study were to evaluate the effects of different concentrations of R. sachalinensis aqueous extract (RSAE) on IVM and subsequent in vitro embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT). The results showed that RSAE supplementation (6 and 60 mg/L) significantly increased intracellular glutathione levels, but had no effect on maturation rates or reactive oxygen species. After in vitro culture, greater blastocyst formation was observed in PA embryos (6 mg/L RSAE), as well as in IVF and SCNT embryos (60 mg/L) matured in RSAE-supplemented IVM media. In conclusion, although there was no significant improvement in the maturation rate, RSAE supplementation conveyed an antioxidative effect during IVM, and improved subsequent embryonic development in vitro. Further studies are needed to explore gene expression pattern in oocytes and embryos treated with RSAE.

Human Pharmacokinetics and Safety of Subcutaneous Collagenase Clostridium Histolyticum in Women.

BACKGROUND: Clostridium collagenase histolyticum (CCH) is being evaluated in women as a cellulite treatment. OBJECTIVE: To report preclinical safety and human pharmacokinetics (PK) and safety data for CCH. METHODS: Across 3 PK studies, 41 women received 12 subcutaneous injections per thigh/buttock in 1 session (up to 3.36 mg/dose). Blood samples were taken at baseline; at 5, 10, 20, and 30 minutes postdose; and at 1, 2, 4, 8, 12, 24, 48, 168, and 504 hours postdose. In a preclinical study, rats received 0, 0.029, 0.13, or 0.29 mg/dose of CCH intravenously (IV) every other day (QOD) for 16 days (total, 8 doses) and were evaluated for histopathologic changes. RESULTS: In human PK studies, no quantifiable plasma concentrations of AUX-I or AUX-II were observed postdose (n= 39 evaluable). Adverse events were injection site–related (bruising [97.6%], pain [87.8%], and edema/swelling [46.3%]). Antidrug antibodies were seen in most women at 504 hours postdose. In rats, plasma concentrations of AUX-I and AUX-II (CCH components) were measurable for 30 minutes and 1-2 hours, respectively, after IV administration. At ≥43× proposed human therapeutic dose on a mg/kg basis, rats experienced elevated liver enzyme levels, increased liver weights, and histologic changes that were mostly reversed during a 14-day recovery period. CONCLUSIONS: In human studies, no quantifiable circulating CCH levels were observed after a single subcutaneous dose of CCH up to 3.36 mg. Preclinical data indicated that repeat IV dosing (QOD; 8 doses) at ≥43× proposed human dose on a mg/kg basis for CCH was generally well tolerated.J Drugs Dermatol. 2020;19(9):852-856. doi:10.36849/JDD.2020.5048THIS ARTICLE HAD BEEN MADE AVAILABLE FREE OF CHARGE. PLEASE SCROLL DOWN TO ACCESS THE FULL TEXT OF THIS ARTICLE WITHOUT LOGGING IN. NO PURCHASE NECESSARY. PLEASE CONTACT THE PUBLISHER WITH ANY QUESTIONS.

Vitamin C protects early mouse embryos against juglone toxicity.

Juglone, a naphthoquinone isolated from many species of the Juglandaceae (walnut) family, has been used in traditional Chinese medicine for centuries for its various pharmacological effects. Our previous research found its toxic effects on oocytes maturation. But we still know a little about its toxic effects on embryo development. Here, we used mouse embryo as a model to explore the effects of juglone on early mammalian embryo development. Exposure to juglone significantly decreased the development rate in early mouse embryos in vitro. Moreover, juglone exposure led to developmental arrest by disturbing mitochondrial function, producing abnormal epigenetic modifications, inducing high levels of oxidative stress and DNA damage, and increasing the rate of embryonic cell apoptosis. However, vitamin C (VC) ameliorated the toxic effects of juglone to a certain extent. Overall, juglone has a toxic effect on early embryo development through the generation of ROS and apoptosis. But VC was able to protect against these juglone-induced defects. These results not only give a new perspective on juglone's pharmacological effects on early mammalian embryo development, but also provide ideas for the better application of this agent in traditional Chinese medicine.

A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression.

Mechanical forces are important in the regulation of physiological homeostasis and the development of disease. The application of mechanical forces to cultured cells is often performed using specialized systems that lack the flexibility and throughput of other biological techniques. In this study, we developed a high throughput platform for applying complex dynamic mechanical forces to cultured cells. We validated the system for its ability to accurately apply parallel mechanical stretch in a 96 well plate format in 576 well simultaneously. Using this system, we screened for optimized conditions to stimulate increases in Oct-4 and other transcription factor expression in mouse fibroblasts. Using high throughput mechanobiological screening assays, we identified small molecules that can synergistically enhance the increase in reprograming-related gene expression in mouse fibroblasts when combined with mechanical loading. Taken together, our findings demonstrate a new powerful tool for investigating the mechanobiological mechanisms of disease and performing drug screening in the presence of applied mechanical load.

Supplementation of insulin-transferrin-sodium selenite in culture medium improves the hypothermic storage of bovine embryos produced in vitro.

Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations. After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P < 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P < 0.05) total cell number per blastocyst and lesser apoptotic cells number. Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P < 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P < 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P < 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P < 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage.

Media optimization to promote rat embryonic development to the blastocyst stage in vitro.

Efficient model production in rats that incorporates newly developed genetic editing and embryo transfer tools, such as CRISPR/Cas9 technology and non-surgical embryo transfer, requires availability of an optimal embryo culture system. However, current technologies for in vitro manipulation of rat gametes, including embryo culture techniques, are less advanced compared to those in mice. In this study, we (1) identified a culture medium that was able to support optimal rat embryonic development by comparing two rat culture media: mR1ECM (modified rat 1-cell embryo culture medium) and KSOM-R (modified potassium simplex optimized medium for rats), and (2) evaluated the effect of glutamine dipeptides: alanyl-l-glutamine and glycyl-l-glutamine, on rat embryonic development. We also investigated the possibility of simplifying the KSOM-R culture procedure by increasing the volume of culture medium, reducing the need for daily medium changes. The results showed that rat embryos cultured in KSOM-R developed faster than those cultured in mR1ECM. Both alanyl-l-glutamine and glycyl-l-glutamine showed detrimental effects on rat embryonic development when supplemented in KSOM-R at the same concentration as glutamine. By increasing the volume of KSOM-R, rat zygotes were able to develop without daily medium refreshment at a similar rate and developmental competence as those in smaller volumes with daily medium changes. These results represent important improvements to rat embryo culture methods and will assist in more efficient production of rat models.

Mitochondria-targeted therapy rescues development and quality of embryos derived from oocytes matured under oxidative stress conditions: a bovine in vitro model.

STUDY QUESTION: Can we use a mitochondrial-targeted antioxidant (Mitoquinone) during in vitro embryo culture to rescue developmental competence of oocytes matured under lipotoxic conditions, exhibiting mitochondrial dysfunction and oxidative stress? SUMMARY ANSWER: Supplementation of embryo culture media with Mitoquinone reduced oxidative stress and prevented mitochondrial uncoupling in embryos derived from metabolically compromised oocytes in vitro, leading to higher blastocyst rates and lower blastomeric apoptosis. WHAT IS KNOWN ALREADY: Maternal metabolic disorders, such as obesity and type-II diabetes are associated with hyperlipidemia and elevated free fatty acid (FFA) concentrations in the ovarian follicular fluid (FF). Oocyte maturation under these lipotoxic conditions results in increased oxidative stress levels, mitochondrial dysfunction, reduced developmental competence and disappointing IVF results. STUDY DESIGN, SIZE, DURATION: A well-described bovine oocyte IVM model was used, where a pathophysiologically relevant elevated FF concentrations of palmitic acid (PA; 150 µM or 300 µM) were added to induce oxidative stress. After fertilization (Day 0, D0), zygotes were in vitro cultured (IVC, from D1 to D8) in standard fatty acid-free media in the presence or absence of Mitoquinone or its carrier triphenyl-phosphonium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryo cleavage and fragmentation (D2) and blastocyst rates (D8) were recorded. Mitochondrial activity and oxidative stress in cleaved embryos at D2 were determined using specific fluorogenic probes and confocal microscopy. D8 blastocysts were used to (i) examine the expression of marker genes related to mitochondrial unfolded protein responses (UPRmt; HSPD1 and HSPE1), mitochondrial biogenesis (TFAM), endoplasmic reticulum (ER) UPR (ATF4, ATF6 and BiP) and oxidative stress (CAT, GPX1 and SOD2) using real time RT-PCR; (ii) determine cell differentiation and apoptosis using CDX-2 and cleaved caspase-3 immunostaining; and (iii) measure mtDNA copy numbers. This was tested in a series of experiments with at least three independent replicates for each, using a total of 2525 oocytes. Differences were considered significant if a P value was <0.05 after Bonferroni correction. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to PA during IVM followed by culture under control conditions resulted in a significant increase in oxidative stress in embryos at D2. This was associated with a significant reduction in mitochondrial inner membrane potential (uncoupling) compared with solvent control (P < 0.05). The magnitude of these effects was PA-concentration dependent. Consequently, development to the blastocysts stage was significantly hampered. Surviving blastocysts exhibited high apoptotic cell indices and upregulated mRNA expression indicating persistent oxidative stress, mitochondrial and ER UPRs. In contrast, supplementation of PA-derived zygotes with Mitoquinone during IVC (i) prevented mitochondrial uncoupling and alleviated oxidative stress at D2; and (ii) rescued blastocyst quality; normalized oxidative stress and UPR related genes and apoptotic cell indices (P > 0.01 compared with solvent control). Mitoquinone also improved blastocyst rate in PA-exposed groups, an effect that was dependent on PA concentration. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is a fundamental study performed using a bovine in vitro model using PA-induced lipotoxicity during oocyte maturation. PA is the most predominant FFA in the FF that is known to induce lipotoxicity; however, in vivo maturation in patients suffering from maternal metabolic disorders involve more factors that cannot be represented in one model. Nevertheless, focusing on the carryover oxidative stress as a known key factor affecting developmental competence, and considering the novel beneficial rescuing effects of Mitoquinone shown here, we believe this model is of high biological relevance. WIDER IMPLICATIONS OF THE FINDINGS: Human oocytes collected for IVF treatments from patients with maternal metabolic disorders are vulnerable to lipotoxicity and oxidative stress during in vivo maturation. The results shown here suggest that mitochondrial targeted therapy, such as using Mitoquinone, during IVC may rescue the developmental competence and quality of these compromised oocytes. After further clinical trials, this may be a valuable approach to increase IVF success rates for infertile patients experiencing metabolic disorders. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by a BOF/KP grant number 34399, from the University of Antwerp, Belgium. W.F.A.M. was supported by a postdoctoral fellowship from the Research Foundation-Flanders (FWO), grant number 12I1417N, Antwerp, Belgium. The Leica SP 8 confocal microscope used in this study was funded by the Hercules Foundation of the Flemish Government (Hercules grant AUHA.15.12). All authors have no financial or non-financial competing interests to declare.

Biomarkers of metal toxicity in embryos in the general population.

BACKGROUND: With the development of industrialization, public exposure to toxic metals could occur everywhere, eventually affecting individuals' reproductive systems and even embryos and leading to early pregnancy loss. The aim of the study was to determine the profile of toxic metal levels in pregnant women in the general population and to identify biomarkers for metal toxicity in embryos. METHODS: A case-control study with pregnant women was conducted at Peking Union Medical College Hospital in 2016-2018. Women who experienced spontaneous abortion within 12 weeks of gestation comprised the case group, and women with pregnancies showing fetal cardiac activity who requested an induced abortion almost simultaneously were included in the control group. Blood and urine specimen were tested for concentrations of cadmium, chromium, selenium, arsenic, and mercury. RESULTS: A total of 195 patients were enrolled, with 95 in the case group and 100 in the control group. Significant differences in gravidity, parity, history of miscarriage, mean blood cadmium levels, and mean urine chromium levels were present between the two groups (P1 = 0.013, P2 = 0.000, P3 = 0.000, P4 = 0.002, P5 = 0.046); the odds ratios in the spontaneous abortion with blood cadmium >0.4 µg/L, urine chromium >2 µg/L, gravity <3, parity <2, and history of miscarriage >1 compared with the induced abortion group were 1.26 (1.09, 1.85), 1.56 (1.23, 2.53), 1.39 (1.17, 1.98), 1.72 (1.21, 4.62), and 1.18 (1.06, 1.65), with P-values of 0.003, 0.031, 0.003, 0.247, and 0.001, respectively. CONCLUSION: Blood cadmium and urine chromium levels are two possible biomarkers of toxic metal embryotoxicity in the general population, which means that in the general population, blood cadmium >0.4 µg/L or urine chromium >2 µg/L might indicate an increased risk of spontaneous abortion.

Evaluation of in vitro embryotoxicity tests for Chinese herbal medicines.

Chinese herbal medicines (CHMs) have been widely used during pregnancy, but feto-embryo safety tests are lacking. Here we evaluated in vitro embryotoxicity tests (IVTs) as alternative methods in assessing developmental toxicity of CHMs. Ten CHMs were selected and classified as strongly, weakly and non-embryotoxic. Three well validated IVTs and prediction models (PMs), including embryonic stem cell test (EST), micromass (MM) and whole embryo culture (WEC), were compared. All strongly embryotoxic CHMs were predicted by MM and WEC PM2. While all weakly embryotoxic CHMs were predicted by MM and WEC PM1. All non-embryotoxic CHMs were classified by EST, MM, but over-classified as weakly embryotoxic by WEC PM1. Overall predictivity, precision and accuracy of WEC determined by PM2 were better than EST and MM tests. Compared with validated chemicals, performance of IVTs for CHMs was comparable. So IVTs are adequate to identify and exclude embryotoxic potential of CHMs in this training set.