Cumulus oocyte complexes from small antral follicles during the early follicular phase of spontaneous cycles in rhesus monkeys can expand and yield oocytes capable of maturation in vitro

PELUFFO, MC; STOUFFER, RL; HENNEBOLD, JD; ZELINSKI, MB

Chicago, Illinois

Conferencia; Third Annual Oncofertility Consortium Meeting; 2009

Oncofertility Consortium

Introduction:  Cryopreservation of the ovarian cortex is one experimental option for restoring fertility in cancer survivors. This strategy relies on the development of primordial/preantral follicles post-thaw in vivo or in vitro to yield mature oocytes for subsequent in vitro fertilization of intracytoplasmic sperm injection. However, there are many small antral follicles (SAF) in the ovarian medulla that could be a potential source of oocytes for in vitro maturation (IVM) in tissue not used for cryopreservation. The aim of this study was to determine the minimal diameter of an antral follicle from spontaneous menstrual cycles in macaques that could yield a cumulus-oocyte complex (COC) capable of expansion and oocyte maturation in vitro. Materials & Methods:  Ovaries were removed from adult rhesus monkeys (n=6) during the early follicular phase (days 3-4) of spontaneous cycles.  SAF were dissected from the ovarian medulla after collagenase treatment.  The COCs from healthy SAF (devoid of dark oocytes or granulosa cells) were extracted (n=87).  SAF were divided into five groups according to their diameter; Group 1: < 0.5 mm; Group 2: 0.5-0.99 mm; Group 3: 1.0-1.49 mm; Group 4: 1.5-1.99 mm; and Group 5: 2.0-2.5 mm. Individual COCs from each group were cultured for 48 h in TALP (Tyrode’s, albumin, lactate, pyruvate) alone, TALP + follicle stimulating hormone (FSH) + luteinizing hormone (LH) (n=46 COCs), SAGE (SAGE®, CooperSurgical, Inc.) alone, or SAGE + FSH + LH (n=41 COCs).  Images of COCs were acquired at collection (0 h), 24 and 48 h post-treatment to assess cumulus cell expansion. At 48 h, oocyte maturation and diameter were measured after treatment of COCs with hyaluronidase. Results: Of the total COCs collected, the majority distributed into Group 3 (69%), with fewer in Groups 1 (2%), 2 (14%), 4 (9%) and 5 (6%).  In all groups, cumulus-oocyte expansion was seen 24 and/or 48 h post-treatment. There were no differences in the percentage of oocytes that resumed maturation to metaphase I (MI) or continued maturation to metaphase II (MII) at 48 h in vitro between media alone and media + gonadotropins, so data were combined within media. Oocyte maturation was not observed in Group 1 in either media. In TALP, the percentages of MI oocytes observed in Groups 2, 3 and 4 were 20% in each and were similar to those seen in SAGE (14% and 20% in Groups 2 and 3, respectively).  A greater proportion of oocytes achieved MII in both media.  The percentage of MII oocytes obtained were not different (p >0.05) when cultured in either TALP or SAGE in Groups 2 (40 vs. 29%), 3 (31 vs. 16%) and 4 (40 vs. 33%).  The diameter of MII oocytes was similar regardless of SAF diameter, and did not differ between TALP (108 ± 2 ìm, mean ±SEM) and SAGE (107 ± 6 µm). Conclusions: These data indicate, for the first time in primates, that cumulus-enclosed oocytes derived from healthy SAF at least 0.5 mm in diameter obtained during the early follicular phase can meiotically mature in vitro.  The competence of these oocytes to undergo fertilization after IVM with subsequent embryonic development is under investigation.  Thus, the cohort of SAF present in the ovaries of spontaneous menstrual cycles prior to selection of the dominant follicle can provide COCs as an additional source of oocytes for IVM and fertility preservation.  Whether COCs obtained from SAF outside of the early follicular phase are also suitable for IVM remains to be determined. Supported by Oncofertility Consortium NIH 1 UL1 RR024926 (R01-HD058294, PL1-EB008542), NCRR-RR00163 and U54 HD55744.