In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium

Abstract

In-vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side-effects of gonadotrophin stimulation for in-vitro fertilization (IVF). The pregnancy rates from oocytes matured in vitro are much lower than those of in-vivo stimulation cycles indicating that optimization of IVM remains a challenge. Therefore, we investigated the effect of supplementation of the medium with gonadotrophins, oestradiol and epidermal growth factor (EGF) and the effect of retaining or removing the cumulus cells on nuclear and cytoplasmic maturation of immature oocytes. Human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation for intracytoplasmic sperm injection (ICSI) were cultured in a complex defined medium either supplemented with gonadotrophins, oestradiol and physiological concentrations of EGF (2 ng/ml) or gonadotrophins and oestradiol alone. The cumulus cells were either removed or kept intact. In GV stage oocytes cultured without cumulus (group I) significantly more oocytes reached the metaphase II (MII) stage at 30 h in media supplemented with EGF (64.3 versus 33.9%, P < 0.003). For oocytes cultured with intact cumulus (group II), more oocytes reached MII at 30 h than in group I, but there was no difference in medium with or without EGF supplementation (81.8 and 79.8% respectively). Cytoplasmic maturation of MII oocytes was judged from their capability to activate and fertilize after ICSI. In group I, the rates of activation and normal fertilization were similar. However, in group II, significantly more oocytes underwent normal fertilization in the EGF-supplemented than the unsupplemented group (71.7 versus 45.6%, P < 0.05). The cleavage rates of the fertilized oocytes were similar in the sibling oocyte subgroups cultured with or without EGF supplementation, but the overall cleavage rates were higher in cumulus-intact compared to cumulus-denuded oocytes (88.9 versus 47.8%, P < 0.001). Thus, supplementation of the maturation medium with EGF and maintenance of the cumulus during culture improve the nuclear and cytoplasmic maturation of human oocytes in vitro.

Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders

“…However, only four oocytes were at the MII stage, the others were still at the MI stage. The matured oocytes underwent the IVF procedure, whereas the immature oocytes were cultured first in medium that contained follicle stimulating hormone (FSH) and human chorionic gonadotrophin (HCG) [15]. On the second day, 14 oocytes had matured in vitro and were fertilized by intracytoplasmic sperm injection (ICSI).…”

Differences in the transcriptional profiles of human cumulus cells isolated from MI and MII oocytes of patients with polycystic ovary syndrome

“…The cross talk between an oocyte and the surrounding cumulus cells (CCs) is critical for achieving oocyte competence, early embryonic development and CC expansion (Salustri et al 1989, Cha & Chian 1998, Goud et al 1998. Previous researches have proved that different gene expressions of CCs could indicate oocyte competence or predict the efficiency of embryo development and pregnancy outcome (McKenzie et al 2004, Zhang et al 2005, Feuerstein et al 2007, Assou et al 2008, van Montfoort et al 2008, Kenigsberg et al 2009, Adriaenssens et al 2010, Assou et al 2010.…”

Effect of bovine cumulus–oocyte complexes‐conditioned medium on in‐vitro maturation of canine oocytes

“…In contrast with most mammalian species, in which the oocyte completes its first meiotic division in the preovulatory follicle and matured oocytes are ovulated and ready for fertilization within the oviduct, the domestic bitch ovulates immature oocytes at the germinal vesicle stage and the oocytes undergo a 48-72-h period of postovulatory maturation in the upper regions of the oviduct, and during oocyte maturation in vivo oocytes are exposed to an ever-changing environment of gonadotropins, steroids, growth factors, and many other factors, any or all of which may interact to regulate maturational changes that occur in the oocyte and its surrounding cumulus cells during the preovulatory period [1][2][3][4][5][6][7]. Clearly, these factors are assumed to be beneficial and involved in nuclear and/or cytoplasmic maturation of oocytes during IVM for several mammalian species, for example porcine [8,9], human [10], mouse [11], equine [12], and bovine [13]. The peculiarities of reproductive physiology of the dog complicate the definition of a culture system for IVM, which is currently characterized by poor and highly variable results.…”

“…It has been reported that epidermal growth factor (EGF) stimulates the IVM of oocytes in mice [39], cattle [40], humans [41] and pigs [42]. Activin, transforming growth factor beta (TGF-b), and basic fibroblast growth factors (bFGF) have been shown to stimulate the mitotic activity of bovine granulosa cells and/or the growth of granulosa cells or follicles in culture [43,44].…”