Size-specific follicle selection improves mouse oocyte reproductive outcomes

Abstract

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.

FIG. 1

(A) Mouse follicle development in vitro in alginate-based hydrogels. a–d. Follicles developed from (a) multilayer secondary follicle to (b) early antral, and (c) antral stages during eIVFG. (d) The oocyte extruded the first polar body after oocyte maturation in vitro. Corresponding histology of follicles in different developmental stages during eIVFG: (e) pre-antral stage, (f) early antral stage, (g) antral stage, and (h) after oocyte maturation. (B) Follicle and oocyte growth during eIVFG from day 0 to day 10. Follicle distribution on different culture days (C) from preantral to antral stage with the presence of antral cativity and (D) cumulus-oocyte complex formation with cumulus cells surrounding the oocyte. Error bars: standard deviation; * Follicle antrum; scale bar: 100 μm. N=20–40 follicles for three replicates.

FIG. 2

Representative images of follicles and the chromatin configuration within their enclosed oocytes during eIVFG (A). (a–c) follicle containing an oocyte with non-surrounded nucleus (NSN); (d–f) follicle containing an oocyte with an intermediate stage (I) chromatin configuration; (g–i) follicle containing an oocyte with surrounded nucleus (SN). (B) Chromatin configuration distribution from day 2 to day 10 during eIVFG. (C) Oocyte diameters associated with different oocyte chromatin configurations during eIVFG. (D) Follicle diameters associated with different oocyte chromatin configurations during eIVFG. Scale bars: 100 μm for a, d, and g; 50 μm for b, e, and h; 10 μm for c, f, and i. Error bars: standard deviation. *P < 0.05, compared to diameter of follicles with oocyte in I stage in C, and compared to the diameter of follicles with oocyte in SN stage in C and D. N=40–80 follicles for three replicates.

FIG. 3

MII percentages after oocyte maturation for eIVFG cultured follicles. (A) MII percentages for follicles cultured for specific periods of time from day 2 to day 10. (B) MII percentages for follicles cultured to specific diameters. (C) Follicle diameter associated with different oocyte maturation outcomes. MII: metaphase II. GV: germinal vesicle. GVBD: germinal vesicle break down. D: degenerated oocyte. *P < 0.05, compared to the other groups in (A) and (B), and compared to the diameter of follicles with oocyte in MII stage after oocyte maturation in (C). N=100–160 follicles for five replicates.

FIG. 4

Spindle morphology and chromosome alignment of MII oocytes after oocyte maturation. (A–C) Representative images of meiotic spindles of MII oocytes with well-organized microtubule fibers (green) and tightly aligned chromosomes (blue). (D–F) Representative images of MII oocytes with abnormal spindle morphology and/or chromosomal alignment. (G) Incidence of spindle abnormality and chromosome misalignment in MII oocytes from follicles cultured to different sizes. MII: metaphase II. *P < 0.05. N=20–40 follicles for three replicates.

FIG. 5

Follicle size is associated with IVF and preimplantation embryo development outcomes. Representative images of MII oocytes after IVF (A) showing 2 pronuclei (2-PN) (B). Fertilized oocyte developed to 2-cell (C), 4-cell (D), morula (E), and blastocyst stages (F). (G) Developmental outcomes for oocytes from follicles cultured up to specific follicle sizes or from in vivo control superovulated oocytes. a, b and c: P < 0.05 compared to the percentage of corresponding embryo development stage with follicle size group of 300–350 μm and the group of superovulated oocytes. d: P < 0.05 between follicle size group of 300–350 μm and superovulated oocytes. Scale bar: 50 μm. N=30–60 for three replicates.