Alongside their contribution to research, human embryonic stem cells (hESC) may also prove valuable for cell-based therapies. Traditionally, these cells have been grown in adhesion culture either with or without feeder cells, allowing for their continuous growth as undifferentiated cells. However, to be applicable in therapy and industry they must be produced in a scalable and controlled process. Here we present for the first time a suspension culture system for undifferentiated hESC and induced pluripotent stem cells (iPSC), based on medium supplemented with the IL6RIL6 chimera (interleukin-6 receptor fused to interleukin-6), and basic fibroblast growth factor. Four hESC lines cultured in this system maintained all ESC features after 20 passages, including stable karyotype and pluripotency. Similar results were obtained when hESC were replaced with iPSC from two different cell lines. We demonstrate that the IL6RIL6 chimera supports the self-renewal and expansion of undifferentiated hESC and iPSC in suspension, and thus present another efficient system for large-scale propagation of undifferentiated pluripotent cells for clinical and translational applications.

Numerous aquatic invertebrates remain dormant for decades in a hydrated state as encysted embryos. In search for functional pathways associated with this form of dormancy, we used label-free quantitative proteomics to compare the proteomes of hydrated encysted dormant embryos (resting eggs; RE) with nondormant embryos (amictic eggs; AM) of the rotifer A total of 2631 proteins were identified in rotifer eggs. About 62% proteins showed higher abundance in AM relative to RE (Fold Change>3; = 0.05). Proteins belonging to numerous putative functional pathways showed dramatic changes during dormancy. Most striking were changes in the mitochondria indicating an impeded metabolism. A comparison between the abundance of proteins and their corresponding transcript levels, revealed higher concordance for RE than for AM. Surprisingly, numerous highly abundant dormancy related proteins show corresponding high mRNA levels in metabolically inactive RE. As these mRNAs and proteins degrade at the time of exit from dormancy they may serve as a source of nucleotides and amino acids during the exit from dormancy. Because proteome analyses point to a similarity in functional pathways of hydrated RE and desiccated life forms, REs were dried. Similar hatching and reproductive rates were found for wet and dried REs, suggesting analogous pathways for long-term survival in wet or dry forms. Analysis by KEGG pathways revealed a few general strategies for dormancy, proposing an explanation for the low transcriptional similarity among dormancies across species, despite the resemblance in physiological phenotypes.

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Objective: To study whether a powerful, in-house, embryo-selection model can be developed for a specific in vitro fertilization (IVF) laboratory where embryos were already selected for transfer using general models. Design: In total, 12,944 fertilized oocytes were incubated in an EmbryoScope (Vitrolife, G€ oteborg, Sweden) at our laboratory. Embryos were selected for transfer or freezing using general models. There were 1,879 embryos with known implantation data (KID), of which 425 had positive KIDs. For the outcome, we set 3 endpoints for KID's definition: gestational sac, clinical pregnancy, and live birth. Results of a comparison between KID-positive and -negative embryos for cell division timings were analyzed separately for intracytoplasmic sperm injection (ICSI) and IVF embryos in patients aged 18-41 years. Setting: IVF center. Patients: The study included 1,075 women undergoing IVF or ICSI treatment between June 2013 and February 2019. Intervention(s): None. Main Outcome Measure(s): The KID-positive and -negative embryos were analyzed for statistical differences in cell division timing and cell cycle intervals. We used the EmbryoScope Stats software (Unisense FertiliTech, Aarhus, Denmark) for model development. The statistically different timing parameters were tested for their contribution to scoring in the model. The algorithms were tested for area under the receiver operating characteristic curve (AUC) in the KID embryos for developing day-2, -3, and -5 embryo-selection models. The validation of these algorithms was performed using calibration/validation procedures. Results: Because significant differences in morphokinetics were found between the KID-positive and KID-negative embryos in our laboratory, it was possible to use our specific KID data to develop an in-house model. The algorithms were developed for embryo selection on days 2, 3, and 5 in the ICSI embryos. In most cases, AUC was >0.65, which indicated that these models were valid in our laboratory. In addition, these AUC values were obtained from all gestational sac, clinical pregnancy, and live birth KID embryo databases tested. An increase in the predictability of the models was observed from days 2-3 to day 5 models. The AUC test results ranged between 0.657 and 0.673 for day 2 and day 3, respectively, and 0.803 for the day 5 model. Conclusion:A model based on laboratory-specific morphokinetics was found to be complementary to general models and an important additive tool for improving single embryo selection. Developing an in-house laboratory-specific model requires many stages of sorting and characterization. Many insights were drawn about the model developing process. These may facilitate and improve the process in other laboratories. (Fertil Steril Sci Ò 2021;2:176-97. Ó2021 by American Society for Reproductive Medicine.

We report a live birth from a heavily fragmented embryo which continued cleavage to a fully expanded blastocyst. A 32-year-old patient underwent 2 IVF cycles without achieving pregnancy. In the first cycle, 2 embryos with fragmentation were transferred; in the second, all embryos were fragmented and no embryo transfer was performed. In a third cycle, 12 oocytes were retrieved and 11 of them were fertilized. On day 2, all 11 embryos started to unwind to fragments. By careful annotation, using the time-lapse EmbryoScope, we observed that one embryo continued division as expected, discarding all fragments aside. On day 5, this embryo showed promising annotation according to our lab model. The embryo was transferred into the uterus and resulted in the birth of a healthy baby at term. To our knowledge, this is the first case report assisted by EmbryoScope where a healthy baby was delivered from a fragmented embryo.