To explore the clinical significance of seven diabetes-related serum microRNAs (miR-9, miR-29a, miR-30d, miR34a, miR-124a, miR146a and miR375) during the pathogenesis of type 2 diabetes (T2D), 56 subjects were recruited to this study: 18 cases of newly diagnosed T2D (n-T2D) patients, 19 cases of pre-diabetes individuals (impaired glucose tolerance [IGT] and/or impaired fasting glucose [IFG]) and 19 cases of T2D-susceptible individuals with normal glucose tolerance (s-NGT). Serum miRNAs were determined by real-time RT-PCR. Expression levels of single miRNAs and the expression signatures of miRNAs as a panel were analysed among the three groups. In n-T2D, all 7 miRNAs were significantly up-regulated compared with s-NGT and five were significantly up-regulated compared with pre-diabetes, while miRNA expression was not significantly different between s-NGT and pre-diabetes. By Canonical discriminant analysis, 70.6% of n-T2D subjects (12/17) were recognized by canonical discriminant function, while s-NGT and pre-diabetes subjects could not be discriminated from each other. Similar results were found in Hierarchical Clustering analysis based on the expression levels of all seven miRNAs. In different statistical analysis, miR-34a always showed the most significant differences. We conclude that the expression levels of seven diabetes-related miRNAs in serum were significantly elevated in n-T2D compared with pre-diabetes and/or s-NGT, and the latter two groups featured similar expression patterns of these miRNAs, suggesting that during the pathogenesis of T2D, the peripheral diabetes-related miRNAs have not changed significantly from s-NGT at pre-diabetic stage.

Oocytes grown in vitro are of low quality and yield few live births, thus limiting the ability to store or bank the ova of women wishing to preserve their fertility. We applied tissue engineering principles to the culture of immature mouse follicles by designing an alginate hydrogel matrix to maintain the oocyte's 3- dimensional (3D) architecture and cell-cell interactions in vitro. A 3D culture mimics the in vivo follicle environment, and hydrogel-encapsulated follicles develop mature oocytes within the capacity for fertilization similar to that of oocytes matured in vivo. Embryos derived from cultured oocytes fertilized in vitro and transferred to pseudopregnant female mice were viable, and both male and female offspring were fertile. Our results demonstrate that alginate hydrogel-based 3D in vitro culture of follicles permits normal growth and development of follicles and oocytes. This system creates new opportunities for discovery in follicle biology and establishes a core technology for human egg banks for preservation of fertility.

The mechanical properties and density of natural and synthetic extracellular matrices are known to affect cellular processes and regulate tissue formation. In this report, these factors were independently investigated for their role in ovarian follicle development. The matrix composition was controlled through decreasing the solids concentration or the molar mass of the encapsulating biomaterial, alginate. Decreasing matrix stiffness and solids concentration enhanced follicle growth, and coordinated differentiation of the follicle cell types, as evidenced by antral cavity formation, theca cell differentiation, oocyte maturation, and relative hormone production levels. While a stiff environment favored high progesterone and androgen secretion, decreasing alginate stiffness resulted in estrogen production which exceeded progesterone and androgen accumulation. These studies reveal, for the first time, a direct link between the biomechanical environment and follicle function, and suggest a novel non-hormonal mechanism regulating follicle development.

The availability of viable oocytes is the limiting factor in the development of new reproductive techniques. Many attempts have been made to grow immature oocytes in vitro during recent decades. Recently, a modified alginate-based three-dimensional culture system was designed to support the growth and maturation of multilayered secondary follicles. This system was able to produce oocytes that successfully completed meiosis, fertilization, and development to the blastocyst stage. Subsequent attempts to culture two-layered secondary follicles were unsuccessful under the original conditions. Herein, we investigated the effect of alginate consistency on two-layered follicle growth and oocyte developmental competence by encapsulating follicles into alginate scaffolds of various concentrations. Although there were no significant differences in survival rates, 0.25% and 0.5% alginate supported more rapid growth of follicles and antrum formation compared with 1.5% and 1.0% alginate after 8 days of culture. Alginate scaffold concentration also affected the proliferation and differentiation of somatic cells (theca and granulosa cells), measured in terms of morphological changes, steroid profiles (androstenedione, estradiol, and progesterone), and specific molecular markers (Fshr, Lhcgr, and Gja1). Theca cell proliferation and steroid production were hindered in follicles cultured in 1.5% alginate. In vitro fertilization and embryo culture revealed that oocytes obtained from 0.25% alginate retained the highest developmental competence. Overall, the present study showed that the alginate scaffold consistency affects folliculogenesis and oocyte development in vitro and that the alginate culture system can and should be tailored to maximally support follicle growth depending on the size and stage of the follicles selected for culture.

Our data support the notion that human follicle development can be achieved in vitro in a bio-engineered culture system. More studies are required to investigate whether the fully sized oocytes obtained from in vitro grown follicle are competent to resume meiosis and be fertilized.