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N Songsasen, T K Woodruff and D E Wildt

The present study examined the influences of the physical and hormonal microenvironment on in vitro growth and steroidogenesis of dog follicles. Follicles were enzymatically isolated and individually encapsulated in 0.5% (w/v; n=17) or 1.5% (n=10) alginate and cultured with 0.5 IU/ml equine chorionic gonadotropin for 192 h. In a separate experiment, follicles were encapsulated in 0.5% alginate and cultured with 0 (n=22), 1 (n=23), 10 (n=20) or 100 (n=21) μg/ml FSH for 240 h. Follicle diameter and steroid production were assessed every 48 h in both studies. Follicles encapsulated in the 0.5% alginate grew faster (P<0.05) than those cultured in the 1.5% concentration. Oestradiol (E2) and progesterone (P4) increased consistently (P<0.05) over time, and follicles in the 1.5% alginate produced more (P<0.05) P4 than those in the 0.5% solution. Follicles cultured in the highest FSH concentration (100 μg/ml) increased 100% in size after 240 h compared with 50 to 70% in lower dosages. E2 concentration remained unchanged over time (P>0.05) across FSH dosages. However, P4 increased (P<0.05) as culture progressed and with increasing FSH concentration. Results demonstrate that dog follicles cultured in alginate retain structural integrity, grow in size and are hormonally active. Lower alginate and increasing FSH concentrations promote in vitro follicle growth. However, the absence of an E2 rise in follicles cultured in FSH alone suggests the need for LH supplementation to support theca cell differentiation and granulosa cell function.

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Shuo Xiao, Francesca E Duncan, Lu Bai, Catherine T Nguyen, Lonnie D Shea and Teresa K Woodruff

Encapsulated in vitro follicle growth (eIVFG) has great potential to provide an additional fertility preservation option for young women and girls with cancer or other reproductive health threatening diseases. Currently, follicles are cultured for a defined period of time and analyzed as a cohort. However, follicle growth is not synchronous, and culturing follicles for insufficient or excessive times can result in compromised gamete quality. Our objective is to determine whether the selection of follicles based on size, rather than absolute culture time, better predict follicle maturity and oocyte quality. Multilayer secondary mouse follicles were isolated and encapsulated in 0.25% alginate. Follicles were cultured individually either for defined time periods or up to specific follicle diameter ranges, at which point several reproductive endpoints were analyzed. The metaphase II (MII) percentage after oocyte maturation on day 6 was the highest (85%) when follicles were cultured for specific days. However, if follicles were cultured to a terminal diameter of 300–350 μm irrespective of absolute time in culture, 93% of the oocytes reached MII. More than 90% of MII oocytes matured from follicles with diameters of 300–350 μm showed normal spindle morphology and chromosome alignment, 85% of oocytes showed two pronuclei after IVF, 81% developed into the two-cell embryo stage and 38% developed to the blastocyst stage, all significantly higher than the percentages in the other follicle size groups. Our study demonstrates that size-specific follicle selection can be used as a non-invasive marker to identify high-quality oocytes and improve reproductive outcomes during eIVFG.

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J E Hornick, F E Duncan, L D Shea and T K Woodruff

In vitro follicle growth in alginate hydrogels is a unique and versatile method for studying ovarian and follicle biology that may also have implications for fertility preservation. Current culture systems support the development of isolated mouse follicles from the secondary stage onward. However, it has been a challenge to grow smaller follicles in vitro due to the dissociation of the oocyte from companion somatic cells. Recent work has demonstrated that coculturing primary follicles with mouse embryonic fibroblasts or ovarian stromal cells supports follicle survival and growth. In this study, we demonstrate that follicles themselves can exert a beneficial coculture effect. When primary follicles were cultured in groups of five or ten (multiple follicle culture), there was increased growth and survival. The multiple follicle culture approach maintained follicle integrity and resulted in the formation of antral stage follicles containing meiotically competent gametes. The growth and survival of primary follicles were highly number dependent, with the most significant enhancement observed when the largest number of follicles was grown together. Our data suggest that the follicle unit is necessary to produce the secreted factors responsible for the supportive effects of multiple follicle culture, as neither denuded oocytes, oocyte-secreted factors, nor granulosa cells alone were sufficient to support early follicle growth in vitro. Therefore, there may be signaling from both the oocyte and the follicle that enhances growth but requires both components in a feedback mechanism. This work is consistent with current in vivo models for follicle growth and thus advances the movement to recapitulate the ovarian environment in vitro.