Karolina Lundin

Background: Induced pluripotent stem cells (iPSCs) from patients have been little used to model polyendocrine metabolic ovarian syndrome (PMOS, formerly polycystic ovary syndrome, PCOS), and differences between unrelated donors can masquerade as disease effects. Earlier comparisons of three or four donors per group reported hundreds to thousands of differentially expressed genes. We asked whether a disease signature is detectable in a larger, genotyped panel, balanced for BMI category and BMI-adjusted, of full-phenotype patients. Methods: We studied 14 clonal, transgene-free iPSC lines reprogrammed by CRISPR activation from endometrial stromal fibroblasts of seven women with PMOS meeting all three Rotterdam criteria, and seven controls frequency-matched for BMI category (30 kg/m²). Bulk RNA-seq (16,489 expressed genes) was related to diagnosis, hormones, metabolic traits, age, and X-inactive specific transcript (XIST) status using gene-wise models, exact enumeration of all 3,432 group splits, and permutation-calibrated gene-set tests. Donors were genotyped within a cohort of 140 women and scored with an imputation-based genome-wide PMOS polygenic risk score (PRS). PMOS risk-locus genes were examined against a 202-line iPSC proteome. Results: PMOS donors had higher anti-Müllerian hormone (AMH), testosterone and free androgen index than controls. No gene was differentially expressed between PMOS and control lines (false discovery rate < 0.1), and the number of nominally associated genes was no greater than for random 7-versus-7 splits of the lines (p = 0.75). This was unchanged with an alternative read quantification and with count-based models. With seven donors per group, the design could detect 1.8-fold differences per gene, compared with roughly 3-to 6-fold for the three-or four-donor designs used previously. Neither diagnosis nor any hormonal or metabolic trait explained more expression variance than chance, and neither PMOS GWAS-locus genes nor Hallmark pathways were enriched. Two control lines showed near-complete loss of XIST, potentially indicative of X-chromosome inactivation (XCI) erosion, and a third was borderline. Endocrine PMOS-locus genes (e.g., FSHR, AMH) were not expressed, whereas DNA-repair and growth genes were. Across 129 genotyped women, the PRS was higher in PMOS (odds ratio 1.81 per SD; AUC 0.65), but PMOS and control iPSC donors overlapped fully in polygenic risk. Conclusions: In this panel of 14 undifferentiated iPSC lines from women with the full PMOS phenotype, no robust PMOS-associated transcriptomic differences were detected. Expression variation was dominated by line-to-line differences, consistent with donor background as their main source, although with one clone per donor a contribution of clonal variation cannot be excluded. Reduced XIST expression in some control lines is potentially indicative of, but does not establish, X-chromosome inactivation erosion. Disease-associated differences in differentiated, hormone-responsive cells cannot be excluded, and differences reported in smaller panels may partly reflect inter-donor variability. Future studies should use adequately sized panels, control for XIST erosion, select donors by polygenic risk, and examine hormone-responsive derivatives.

Polyendocrine metabolic ovarian syndrome (PMOS) is a prevalent endocrine disorder with complex reproductive, metabolic, and psychological features. A total of fourteen induced pluripotent stem cell (iPSC) lines were generated from endometrial stromal fibroblasts using a CRISPRa-based reprogramming method, comprising seven iPSC lines obtained from women with PMOS (all three Rotterdam criteria) and seven from body mass index (BMI)-matched non-PMOS controls. This well-characterized resource provides a unique platform for investigating cell-intrinsic PMOS mechanisms, supporting targeted pathogenesis studies and translational advances.

STUDY QUESTION: What is the effect of different oxygen (O ₂ ) levels on the transcriptomic profile of bovine embryos during the in vitro culture? SUMMARY ANSWER: Embryos grown in hypoxia (6% O ₂ ) from zygotes until the blastocyst stage had the highest blastocyst formation rate, whereas normoxia (20% O ₂ ) delayed transcriptomic reprogramming and embryonic genome activation, and induced changes in energy metabolism gene expression. WHAT IS KNOWN ALREADY: Mammalian preimplantation embryo development is a complex sequence of events where, within a week, the zygote is reprogrammed to totipotency and subsequently diverges to embryonic and extraembryonic cell lineages for post-implantation embryo development. This period of development is sensitive to oxygen levels that can affect various cellular processes. STUDY DESIGN, SIZE, DURATION: In this study, we used triplicate bovine embryos as a model for human embryogenesis to compare the influence of O ₂ levels on preimplantation embryonic development by culturing embryos either in normoxic (20% O ₂ ) or physiological hypoxic (6% O ₂ ) conditions, or sequential hypoxia until 16-cell stage and then switching to ultrahypoxic culture (2% O ₂ ). PARTICIPANTS/MATERIALS, SETTING, METHODS: As the readout for varied O ₂ effects, we performed RNA sequencing using 5’ targeted STRT-N method on single embryos. We compared zygotes, 4-, 8-, 16-cell and blastocyst stage embryos grown in either normoxic or hypoxic condition, adding ultrahypoxia for blastocyst stage embryos as the third condition. MAIN RESULTS AND THE ROLE OF CHANCE: We found that the initial cleavage rate was not affected by O ₂ levels but there was a clear difference in blastocyst formation rate. In hypoxia, 36% of embryos reached blastocyst stage while in normoxia the blastocyst formation rate was 13%. In final ultrahypoxia condition only 4.6% of embryos reached blastocyst stage. Transcriptomic profiles showed that normoxic conditions slowed down oocyte transcript degradation and embryonic genome activation. Key metabolic enzyme genes were also altered between hypoxic and normoxic conditions at the blastocyst stage. Both hypoxic and ultrahypoxic conditions induced energy production by upregulating genes involved in glycolysis and lipid metabolism typical to in vivo embryos. In contrast, normoxic conditions failed to upregulate glycolysis genes and only depended on primitive oxidative phosphorylation metabolism. We conclude that constant hypoxia culture of in vitro embryos provided the highest blastocyst formation rate and appropriate energy metabolism. Normoxia altered the energy metabolism and decreased the blastocyst formation rate. Even though ultrahypoxia at blastocyst stage resulted in a drop of blastocyst formation, the transcriptional profile of surviving embryos was normal. LARGE SCALE DATA: The raw data (BCL files) are available at Zenodo: XXXXX FASTQ files generated are available in the EMBL’s European Bioinformatics Institute (EMBL-EBI)-BioStudies with accession number X-XXXX. LIMITATIONS, REASONS FOR CAUTION: The limitation of this study is the use of bovine as an animal model instead of human embryos. Due to this, the direct translation of the results to human should be taken with caution. WIDER IMPLICATIONS OF THE FINDINGS: This study supports previous literature on hypoxic culture conditions being the most suitable for in vitro embryo culture. In addition, we provide new insights on why embryos grown in normoxia do not have the same success rate as embryos grown in hypoxia. We did not observe any benefits of lowering the oxygen levels to 2%, calling for caution of switching to this culture system. STUDY FUNDING/COMPETING INTEREST(S): This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 813707.; Work in the JK laboratory is supported by Jane and Aatos Erkko Foundation, Sigrid Jusélius Foundation, Liv och Hälsa (Finland), Swedish Brain Foundation and Swedish Research Council. The study was also supported by the Estonian Research Council (grant no. PRG1076) and the Horizon Europe NESTOR project (grant no. 101120075). What this means for patients?: During assisted reproduction treatment (ART) oocytes are fertilized in vitro by sperm to form an embryo. The embryos are then exposed to environmental effects during the 5 days of culture before implantation. Embryo culture takes place inside the incubator, where different oxygen levels can be used. In this study, we compared 3 different oxygen concentrations for embryo culture: atmospheric or normoxia (20%), physiologic or hypoxia (6%) and combined low and ultra-low concentration with initial culture at 6% oxygen until day 3 followed by 2% (ultrahypoxia) until day 5 of culture. We conducted this study because many ART clinics use different oxygen concentrations for embryo culture, and we aimed to provide insight on how these different concentrations impact embryo development by monitoring gene expression. As using human embryos for this type of experiments is not commonly approved, we used bovine embryos whose early development resembles that of humans. We found that culturing embryos for 5 days at constant low oxygen concentration (hypoxia) gives the highest number of embryos that reach the blastocyst stage (day 5) and have expected gene expression that provides the physiological path of embryo development. Culturing embryos at atmospheric oxygen concentration showed that only 13% of fertilised eggs reached the blastocyst stage at day 5, while culturing at the final ultra-low oxygen concentration had the lowest percentage of matured embryos, and we did not observe any benefits that would justify the use of such a culture system. We suggest that in vitro culturing of embryos at constant 5-6% oxygen gives the best results.

Follicle-stimulating hormone (FSH) is crucial in the development and regulation of reproductive functions. The actions of human FSH and its receptor (FSHR) and mutations therein have mainly been studied using in vivo models, primary cells, cancer cells and cell lines ectopically expressing the FSHR. To allow studies of endogenous FSHR function in vitro, we differentiated FSHR-expressing cells from human pluripotent stem cells. FSH stimulation of the wild-type (WT), but not the inactivating Finnish founder mutant (A189V) receptor, activated the canonical cyclic adenosine monophosphate (cAMP)-dependent signaling pathway and downstream mediators. To investigate protein–protein interaction partners of FSHR at resting state and upon FSH stimulation, we expressed FSHR in HEK293 cells followed by affinity purification mass spectrometry analyses. We found 19 specific high-confidence interacting proteins for WT FSHR and 14 for A189V FSHR, several of which have been linked to infertility. Interestingly, while only WT FSHR interacted with FSH, insulin-like growth factor 1 receptor (IGF1R), for example, interacted with both WT and A189V FSHR upon FSH stimulation. In conclusion, our protocol allows detailed studies of FSH action and disease modeling in human cells endogenously expressing FSHR.