Human pluripotent stem cell-derived cells endogenously expressing follicle-stimulating hormone receptors: modeling the function of an inactivating receptor mutation.

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.

Assessing the intra- and inter-reader reliability of dynamic ultrasound images in power Doppler ultrasonography.

OBJECTIVE: To assess the intra-reader and inter-reader reliabilities of interpreting ultrasonography by several experts using video clips. METHOD: 99 video clips of healthy and rheumatic joints were recorded and delivered to 17 physician sonographers in two rounds. The intra-reader and inter-reader reliabilities of interpreting the ultrasound results were calculated using a dichotomous system (normal/abnormal) and a graded semiquantitative scoring system. RESULTS: The video reading method worked well. 70% of the readers could classify at least 70% of the cases correctly as normal or abnormal. The distribution of readers answering correctly was wide. The most difficult joints to assess were the elbow, wrist, metacarpophalangeal (MCP) and knee joints. The intra-reader and inter-reader agreements on interpreting dynamic ultrasound images as normal or abnormal, as well as detecting and scoring a Doppler signal were moderate to good (kappa = 0.52-0.82). CONCLUSIONS: Dynamic image assessment (video clips) can be used as an alternative method in ultrasonography reliability studies. The intra-reader and inter-reader reliabilities of ultrasonography in dynamic image reading are acceptable, but more definitions and training are needed to improve sonographic reproducibility.