ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses.

STUDY QUESTION: Do different human ART culture protocols prepare embryos differently for post-implantation development? SUMMARY ANSWER: The type of ART culture protocol results in distinct cellular and molecular phenotypes in vitro at the blastocyst stage as well as subsequently during in vivo development. WHAT IS KNOWN ALREADY: It has been reported that ART culture medium affects human development as measured by gestation rates and birthweights. However, due to individual variation across ART patients, it is not possible as yet to pinpoint a cause-effect relationship between choice of culture medium and developmental outcome. STUDY DESIGN, SIZE, DURATION: In a prospective study, 13 human ART culture protocols were compared two at a time against in vivo and in vitro controls. Superovulated mouse oocytes were fertilized in vivo using outbred and inbred mating schemes. Zygotes were cultured in medium or in the oviduct and scored for developmental parameters 96 h later. Blastocysts were either analyzed or transferred into fosters to measure implantation rates and fetal development. In total, 5735 fertilized mouse oocytes, 1732 blastocysts, 605 fetuses and 178 newborns were examined during the course of the study (December 2010-December 2011). PARTICIPANTS/MATERIALS, SETTING, METHODS: Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and recipients for embryo transfer, respectively. In vivo fertilized B6C3F1 oocytes were allowed to cleave in 13 human ART culture protocols compared with mouse oviduct and optimized mouse medium (KSOM(aa)). Cell lineage composition of resultant blastocysts was analyzed by immunostaining and confocal microscopy (trophectoderm, Cdx2; primitive ectoderm, Nanog; primitive endoderm, Sox17), global gene expression by microarray analysis, and rates of development to midgestation and to term. MAIN RESULTS AND THE ROLE OF CHANCE: Mouse zygotes show profound variation in blastocyst (49.9-91.9%) and fetal (15.7-62.0%) development rates across the 13 ART culture protocols tested (R(2)= 0.337). Two opposite protocols, human tubal fluid/multiblast (high fetal rate) and ISM1/ISM2 (low fetal rate), were analyzed in depth using outbred and inbred fertilization schemes. Resultant blastocysts show imbalances of cell lineage composition; culture medium-specific deviation of gene expression (38 genes, ≥ 4-fold) compared with the in vivo pattern; and produce different litter sizes (P ≤ 0.0076) after transfer into fosters. Confounding effects of subfertility, life style and genetic heterogeneity are reduced to a minimum in the mouse model compared with ART patients. LIMITATIONS, REASONS FOR CAUTION: This is an animal model study. Mouse embryo responses to human ART media are not transferable 1-to-1 to human development due to structural and physiologic differences between oocytes of the two species. WIDER IMPLICATIONS OF THE FINDINGS: Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the market has been optimized for human embryo development. The mouse embryo assay (MEA), which requires ART media to support at least 80% blastocyst formation, is in need of reform and should be extended to include post-implantation development.

Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success.

Establishment of totipotency after somatic cell nuclear transfer (NT) requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H(2b)-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI). Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.

Somatic cell nuclear reprogramming of mouse oocytes endures beyond reproductive decline.

The mammalian oocyte has the unique feature of supporting fertilization and normal development, while capable of reprogramming nuclei of somatic cells toward pluripotency, and occasionally even totipotency. While oocyte quality is known to decay with somatic aging, it is not a given that different biological functions decay concurrently. In this study, we tested whether oocyte's reprogramming ability decreases with aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT), giving rise to higher blastocysts rates compared to young donors ooplasts. Despite the differences in transcriptome between climacteric and young ooplasts, gene expression profiles of SCNT blastocysts were very similar. Importantly, embryonic stem cell lines with capacity to differentiate into tissues from all germ layers were derived from SCNT blastocysts obtained from climacteric ooplasts. Although apoptosis-related genes were down-regulated in climacteric ooplasts, and reprogramming by transcription factors (direct-induced pluripotency) benefits from the inhibition of p53-mediated apoptosis, reprogramming capacity of young ooplasts was not improved by blocking p53. However, more outgrowths were derived from SCNT blastocysts developed in the presence of a p53 inhibitor, indicating a beneficial effect on trophectoderm function. Results strongly suggest that oocyte-induced reprogramming outcome is determined by the availability and balance of intrinsic pro- and anti-reprogramming factors tightly regulated and even improved throughout aging, leading to the proposal that oocytes can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction.