Reprogramming of differentiated nuclei into a totipotent embryonic state following somatic cell nuclear transfer (SCNT) is not efficient. Previous studies in the hybrid B6D2F1 mouse strain revealed that a transient treatment of the SCNT embryos with the histone deacetylase inhibitor (HDACi) trichostatin A (TSA) significantly enhance the potential of the cloned embryos to develop in vitro and to term. Here, we compare two different SCNT protocols with TSA and explore, for the first time, the effect of another HDACi, valproic acid (VPA), on the in vitro development, blastocyst quality, and full-term development of mouse B6CBAF1 cloned embryos. Rates of blastocyst development in SCNT embryos treated with either 5 nM TSA during and after activation (31.8%) or with 100 nM TSA or 2 mM VPA before and during activation (34.5 and 38.3%, respectively) were clearly superior to those of nontreated SCNT embryos (22.9-25.1%). These increased in vitro development rates of the HDACi-treated embryos were correlated with an increased level of histone H3 lysine 14 acetylation and an improved blastocyst quality, as judged by the increased number of total and ICM cells in comparison to the nontreated embryos (30-35% increase). Treatment of SCNT embryos with TSA or VPA also allowed the obtention of viable cloned mice, whereas none could be produced from untreated SCNT embryos. In conclusion, we have demonstrated for the first time that VPA can improve the in vitro and full-term development of B6CBAF1 SCNT embryos, at a similar level as TSA. Our findings may open new opportunities to improve cloning efficiencies in other mouse strains or species.
Our findings support the idea that in a TLI with a controlled air purification system, human embryos can be successfully cultured continuously from day 0 onward in single medium with no need to renew it on day-3. This strategy does not affect embryo morphokinetics or development to term and offers more stable culture conditions for embryos as well as practical advantages and reduced costs for the IVF laboratory.
STUDY QUESTION
Which lab-related factors impact the culture system’s capacity to maintain a stable osmolality during human embryo culture?
SUMMARY ANSWER
Incubator humidity, the volume of mineral oil, the type of culture media and the design of time-lapse dishes have been identified as important parameters that can cause an impact on media evaporation and consequently osmolality during culture.
WHAT IS KNOWN ALREADY
Culture medium is a critical component in human embryo culture. Minimizing its evaporation during culture is an adequate strategy to stabilize osmolality and, as a result, improving culture conditions and clinical outcomes.
STUDY DESIGN, SIZE, DURATION
The studied variables included media composition and supplementation; volume of mineral oil; incubator humidification; and the type of dish and incubator used. Additionally, six time-lapse dish models were compared in their ability to prevent evaporation.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Dishes were incubated in parallel to analyze osmolality during culture between groups: synthetic oviductal medium enriched with potassium versus human tubal fluid medium; protein versus no protein supplementation; dry versus humid atmosphere; high versus low volume of mineral oil. Additionally, media evaporation was compared between six models of time-lapse dishes with distinct designs, cultured in a joint incubator. Two of them were retested in their corresponding incubator to analyze the dish-incubator fit. Daily osmolality measurements were compared between groups. Linear regression was performed to analyze evaporation rates.
MAIN RESULTS AND THE ROLE OF CHANCE
Protein supplementation did not significantly affect evaporation. Contrarily, humidity levels inside the incubators, the volume of mineral oil and the type of culture media, played an important role in osmolality stabilization. The design of time-lapse dishes and their recommended preparation protocol heavily influenced their evaporation rates, which were further altered by each incubator’s characteristics. Media with initially high osmolalities had a bigger risk of reaching hypertonic levels during culture.
LIMITATIONS, REASONS FOR CAUTION
While numerous, the studied variables are limited and therefore other factors could play a role in osmolality dynamics, as well. Incontrollable atmospheric factors could also result in some variation in the observed results between different centers and laboratories.
WIDER IMPLICATIONS OF THE FINDINGS
Published literature has extensively described how hypertonic media may impair embryo development and negatively affect clinical outcomes; therefore, maintaining a stable osmolality during culture should be considered essential. This work is of interest both for embryologists when analyzing their culture system and methodologies, as well as manufacturers in charge of designing IVF consumables.
STUDY FUNDING/COMPETING INTEREST(S)
This study was privately funded.
TRIAL REGISTRATION NUMBER
N/A.
The developmental potential of early embryos is mainly dictated by the quality of the oocyte. Here, we explore the utility of the maternal spindle transfer (MST) technique as a reproductive approach to enhance oocyte developmental competence. Our proof-of-concept experiments show that replacement of the entire cytoplasm of oocytes from a sensitive mouse strain overcomes massive embryo developmental arrest characteristic of non-manipulated oocytes. Genetic analysis confirmed minimal carryover of mtDNA following MST. Resulting mice showed low heteroplasmy levels in multiple organs at adult age, normal histology and fertility. Mice were followed for five generations (F5), revealing that heteroplasmy was reduced in F2 mice and was undetectable in the subsequent generations. This pre-clinical model demonstrates the high efficiency and potential of the MST technique, not only to prevent the transmission of mtDNA mutations, but also as a new potential treatment for patients with certain forms of infertility refractory to current clinical strategies.
Chemically assisted enucleation has been successfully applied to porcine and bovine oocytes to prepare recipient cytoplasts for nuclear transfer procedures. In this study, the antimitotic drugs demecolcine, nocodazole, and vinblastine were first assessed for their ability to induce the formation of cortical membrane protrusions in mouse, goat, and human oocytes. While only 2% of the treated human oocytes were able to form a protrusion, high rates of protrusion formation were obtained both in mouse (84%) and goat oocytes (92%), once the treatment was optimized for each species. None of the antimitotics applied was superior to the others in terms of protrusion formation, but mouse oocytes treated with vinblastine were unable to restore normal spindle morphology after drug removal and their in vitro development after parthenogenetic activation was severely compromised, rendering this antimitotic useless for chemically assisted enucleation approaches. Aspiration of the protrusions in mouse oocytes treated with demecolcine or nocodazole yielded 90% of successfully enucleated oocytes and allowed the extraction of a smaller amount of cytoplasm than with mechanical enucleation, but both enucleation methods resulted in the depletion of spindle-associated gamma-tubulin from the prepared cytoplasts. Treatment of mouse oocytes with demecolcine or nocodazole had no effect on their in vitro development after parthenogenetic activation, or on their ability to repolymerize a new spindle after the removal of the drug or the reconstruction of the treated cytoplasts with a somatic nucleus. Therefore, demecolcine- and nocodazole-assisted enucleation appears as an efficient alternative to mechanical enucleation, which can simplify nuclear transfer procedures.
primary objective was met, with a cumulative spontaneous abortion rate of 7.4% with upper bound of the 95% confidence interval (CI; 4.4%, 11.5%) below the predefined threshold of 15.0% set based upon the observed 10.0% rate (CI 7.6%, 12.8%) in the pivotal trial (Stadtmauer 2013). Clinical pregnancy rates were 43.2% at 10 weeks post OR.CONCLUSIONS: This trial established a safety bridge between PVR produced via enhanced manufacturing processes and the legacy PVR based upon a rate of spontaneous abortion comparable to that observed in the pivotal phase 3 trial. Weekly administration of the PVR was well-tolerated with good pregnancy outcomes associated with its use in conjunction with HP-hMG stimulation. Based upon demonstrated safety and efficacy coupled with more convenient dosing than existing therapeutics, PVR offers an important option for luteal phase supplementation.
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