Quality of embryos on day 7 after medium refreshment on day 6: a prospective trial

STUDY QUESTION Are embryos that fail to meet biopsy or freezing criteria on day 6 (D6) more likely to meet these criteria on day 7 (D7) if cultured in fresh medium from D6 to D7? SUMMARY ANSWER Refreshment of medium on D6 did not increase the proportion of usable embryos on D7, with an adverse effect for women ≥40 years old. WHAT IS KNOWN ALREADY Embryo development in continuous single-step medium, from fertilization to the blastocyst stage, is equivalent to that using a sequential media protocol. However, there remains a theoretical benefit of refreshing the culture environment by transitioning slowly developing D6 embryos to a fresh medium droplet of the same composition, with a renewed source of nutrients and a milieu free of metabolic toxins. STUDY DESIGN, SIZE, DURATION This was a prospective trial of culture media exposure in which embryos were randomized on D6 to remain in the same culture medium from D3 to D7 (continuous, n = 620) or be moved to fresh medium (fresh, n = 603) on D6, with re-evaluation on D7. Data were collected from IVF cycles, with or without ICSI, between 29 March 2019 and 17 February 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS Embryos from 298 women, aged 18–44 years, from cycles with or without preimplantation genetic testing (PGT) that did not meet criteria for biopsy and/or freeze on D6 were included in the study. Embryos were only included if there was a minimum of two embryos meeting the inclusion criteria in any cohort. Only the first cycle undertaken by each woman in the study period from which embryos were randomized was included. MAIN RESULTS AND THE ROLE OF CHANCE A total of 1254 embryos were randomized from 312 cycles (209 non-PGT and 103 PGT) including 200 women undergoing IVF without PGT and 98 women who underwent PGT. The proportion of usable blastocysts on D7 did not differ between groups: 10.1% (61/603) in fresh versus 9.7% (60/620) in continuous medium (relative risk (RR) 1.05, 95% CI 0.74–1.47)). Embryos from women ≥40 years old had a significantly decreased likelihood of achieving a usable blastocyst on D7 after culture in fresh versus continuous medium: 3.5% versus 12.2%; RR 0.29, 95% CI 0.08–0.98. In total, 9.9% of embryos otherwise discarded on D6 met the criteria for biopsy and/or freeze on D7. LIMITATIONS, REASONS FOR CAUTION Future work investigating implantation, clinical pregnancy and miscarriage rates with D7 embryos is still needed. WIDER IMPLICATIONS OF THE FINDINGS Refreshment of medium on D6 did not increase the proportion of usable embryos on D7 overall. Younger women were more likely to develop D7 embryos after refreshment of medium on D6, while an adverse effect was seen in women ≥40 years old. However, by extending the culture of embryos to D7, additional blastocysts become available for clinical use. STUDY FUNDING/COMPETING INTEREST(S) Funding was provided through the Department of Obstetrics and Gynecology at Brigham and Women’s Hospital. I.G.I. works with Teladoc Health. A.L. has no disclosures. E.S.G. works as a consultant for Teladoc Health, and a writer and editor for UpToDate and BioMed Central. C.R. is a board member of the American Society for Reproductive Medicine and works with UpToDate. TRIAL REGISTRATION NUMBER N/A.

Plantlet regeneration via nucellar embryogenesis in Citrus jambhiri

To produce true-to-type and rapid multiplication micropropagation technique was utilized to the nucellar tissue with ovular halves of C. jambhiri. Nucellar tissues were cultured in a modified MS medium supplemented with different concentrations of 2ip viz. 0.25,0.50 mg l-1 alone and in combination of 0.50 mg l-1 NAA. Initiation of cell division and differentiation of proembryogenic tissue became apparent in first 80 days.These proembryos developed into embryos through subculturing in fresh medium. Low concentration of 0.25 2ip was found more suitable for the development of embryo producing large number of fully developed embryo in comparision to the embryo produced in the high concentration of 2ip (0.50 mg l-1) and in combination of 0.25 2ip and 0.50 mg l-1 NAA. Normally developed embryos in 0.25 2ip showed best germination in the fresh medium supplemented with 0.25 mg l-1 IAA, 100 ME mg l-1 and 5mg mg l-1 amino acids within 30 days as compared to other treatments. These germinated embryos were utilized for producing disease free saplings after hardening and nurturing in laboratory conditions. The disease free saplings thus produced can be used to establish new Citrus orchards within short time.

Identification of novel genes that promote persister formation by repressing transcription and cell division in Pseudomonas aeruginosa

ObjectivesBacterial persisters are a small subpopulation of cells that are highly tolerant of antibiotics and contribute to chronic and recalcitrant infections. Global gene expression in Pseudomonas aeruginosa persister cells and genes contributing to persister formation remain largely unknown. The objective of this study was to examine the gene expression profiles of the persister cells and those that regained growth in fresh medium, as well as to identify novel genes related to persister formation.MethodsP. aeruginosa persister cells and those that regrew in fresh medium were collected and subjected to RNA sequencing analysis. Genes up-regulated in the persister cells were overexpressed to evaluate their roles in persister formation. The functions of the persister-contributing genes were assessed with pulse–chase assay, affinity chromatography, fluorescence and electron microscopy, as well as a light-scattering assay.ResultsAn operon containing PA2282–PA2287 was up-regulated in the persister cells and down-regulated in the regrowing cells. PA2285 and PA2287 play key roles in persister formation. PA2285 and PA2287 were found to bind to RpoC and FtsZ, which are involved in transcription and cell division, respectively. Pulse–chase assays demonstrated inhibitory effects of PA2285 and PA2287 on the overall transcription. Meanwhile, light-scattering and microscopy assays demonstrated that PA2285 and PA2287 interfere with cell division by inhibiting FtsZ aggregation. PA2285 and PA2287 are conserved in pseudomonads and their homologous genes in Pseudomonas putida contribute to persister formation.ConclusionsPA2285 and PA2287 are novel bifunctional proteins that contribute to persister formation in P. aeruginosa.

Accelerated H2 Evolution during Microbial Electrosynthesis with Sporomusa ovata

Microbial electrosynthesis (MES) is a process where bacteria acquire electrons from a cathode to convert CO2 into multicarbon compounds or methane. In MES with Sporomusa ovata as the microbial catalyst, cathode potential has often been used as a benchmark to determine whether electron uptake is hydrogen-dependent. In this study, H2 was detected by a microsensor in proximity to the cathode. With a sterile fresh medium, H2 was produced at a potential of −700 mV versus Ag/AgCl, whereas H2 was detected at −500 mV versus Ag/AgCl with cell-free spent medium from a S. ovata culture. Furthermore, H2 evolution rates were increased with potentials lower than −500 mV in the presence of cell-free spent medium in the cathode chamber. Nickel and cobalt were detected at the cathode surface after exposure to the spent medium, suggesting a possible participation of these catalytic metals in the observed faster hydrogen evolution. The results presented here show that S. ovata-induced alterations of the cathodic electrolytes of a MES reactor reduced the electrical energy required for hydrogen evolution. These observations also indicated that, even at higher cathode potentials, at least a part of the electrons coming from the electrode are transferred to S. ovata via H2 during MES.

In vitro arrow cane (Gynerium sagitatum Aubl.) multiplication in double phase medium

Arrow cane (Gynerium sagitatum Aubl.) is a Poaceae species used as fiber source to make traditional and valuable handmade craftsmanship by indigenous communities in Northern Colombia. Since no commercial crops are established fiber needs are taken from natural plant populations affecting ecosystem. A micropropagation protocol to clonally multiply large quantities of arrow cane plant material for planting commercial crops has been developed; however, micropropagated plants are costly compared to naturally extracted plant material. To reduce micropropagated plants costs, in the present research a double phase medium formulation along with continuous shoot culture with no periodic transfers to fresh medium was compared to semisolid medium system with subculture every four weeks with respect to multiplication rate and costs of micropropagated plants. The results showed that continuous culture of explants with double phase medium and no periodic transfers resulted in higher multiplication rates and larger shoots compared to shoots cultured using the conventionalsemisolid medium system and transfer to fresh medium every four weeks. Plants from both, semisolid and double phase culture system, fully adapted and recovered when transferred to ex vitro conditions. The cost analysis showed that double phase cultured shoots are ≥20% less expensive.

Hydrogenotrophic Methanogenesis and Autotrophic Growth ofMethanosarcina thermophila

Although Methanosarcinales are versatile concerning their methanogenic substrates, the ability ofMethanosarcina thermophilato use carbon dioxide (CO2) for catabolic and anabolic metabolism was not proven until now. Here, we show thatM. thermophilaused CO2to perform hydrogenotrophic methanogenesis in the presence as well as in the absence of methanol. During incubation with hydrogen, the methanogen utilized the substrates methanol and CO2consecutively, resulting in a biphasic methane production. Growth exclusively from CO2occurred slowly but reproducibly with concomitant production of biomass, verified by DNA quantification. Besides verification through multiple transfers into fresh medium, the identity of the culture was confirmed by 16s RNA sequencing, and the incorporation of carbon atoms from13CO2into13CH4molecules was measured to validate the obtained data. New insights into the physiology ofM. thermophilacan serve as reference for genomic analyses to link genes with metabolic features in uncultured organisms.

66 CUMULUS-OOCYTE-COMPLEX SECRETIONS FROM THE FIRST 8 HOURS OF IN VITRO MATURATION AFFECT OOCYTE DEVELOPMENTAL COMPETENCE

Mammalian oocytes are surrounded by cumulus cells, forming a structure known as the cumulus-oocyte complex (COC). Cumulus cells play important protective functions during oocyte maturation, for example, protecting the oocyte against reactive oxygen species. However, it is not yet fully understood how the cumulus complex modulates the developmental competence of the enclosed oocyte. It was investigated whether direct contact between an oocyte and its cumulus cells is essential throughout the maturation process. To this end, bovine oocytes aspirated from ovarian follicles were matured in vitro. Eight hours after the onset of maturation the cumulus cells were removed, and the oocytes either placed back in the original medium or cultured further in fresh maturation medium. In all experiments, COCs/oocytes were matured for 23 h in M199 supplemented with 0.05 IU FSH and penicillin/streptomycin. All experiments were performed in triplicate, with 35 to 45 COCs per group. Student’s t-test was used for a paired comparison. Denudation after 8 h and return to the same maturation medium had no effect on the cleavage rate (93%) compared with culture without denudation (90.7%). Only if the oocytes were transferred to fresh medium did the cleavage rate decrease slightly (75.4%; P = 0.038). By contrast, blastocyst formation was reduced nearly four times if COCs were denuded before being returned to the medium, compared with controls (14% v. 50.8%; P < 0.001). If the oocytes were transferred to fresh medium after denudation, very few blastocysts resulted (0.9%; P < 0.001). In a second study, oocytes denuded immediately after removal from the follicle were matured in the absence or presence of cumulus cells in a Corning® Transwell® system. Culturing denuded oocytes in the presence of cumulus cells resulted in similar cleavage rates (83.5%) to control conditions (84.8%). However, blastocyst formation was markedly lower (4.3%) than for controls (29.6%; P = 0.003). We conclude that COCs secrete substances during the first 8 h of maturation that are beneficial for oocyte acquisition of developmental competence. Moreover, intimate contact between the cumulus cells and oocyte is essential. This work was supported by EU FP7 EpiHealthNet, PITN-GA-2012–317146.

Cryo-injury in algae and the implications this has to the conservation of micro-algae

As with all products and processes exploiting biological materials algal biotechnology has an absolute requirement for stable, function-fulfilling master stockcultures. Conventionally microalgae and cyanobacteria are maintained by serial transfer with inocula (2.5-25% v/v) being transferred to fresh medium and the culture being held under controlled environmental conditions. This method is satisfactory for many algal taxa, but by its nature cannot provide an absolute guarantee of phenotypic or genotypic stability. Cryopreservation at ultra-low temperatures (> -130oC) is the only methodology that can provide this level of security to master stock-cultures; however, many algae are recalcitrant to cryopreservation with low or no survival. This review explores the reasons of this cryo-recalcitrance on the application of conventional colligative, two-step cryopreservation protocols and points towards the options available to enhance postcryopreservation viability.