Human oocyte maturation in vitro is improved by co-culture with cumulus cells from mature oocytes

Abstract

The conventional method of human oocyte maturation in vitro in the presence of gonadotrophins continues to be a relatively low-success procedure in the assisted conception programme owing to suboptimal maturation conditions in the absence of an ovarian ‘niche’ and poor understanding of this procedure at the molecular level in oocytes. In this study, the gene expression profiles of human oocytes were analysed according to their manner of maturation: in vivo (in the ovaries) or in vitro (matured either by the conventional method or by a new approach – co-cultured with cumulus cells of mature oocytes from the same patient). Our results show that the in-vitro maturation procedure strongly affects the gene expression profile of human oocytes, including several genes involved in transcriptional regulation, embryogenesis, epigenetics, development, and the cell cycle. The in-vitro maturation of oocytes co-cultured with cumulus cells from mature oocytes provides an ovarian ‘niche’ to some degree, which improves oocyte maturation rates and their gene expression profile to the extent that they are more comparable to oocytes that naturally mature in the ovarian follicle.

Introduction

The conventional human oocyte in-vitro maturation (IVM) procedure is a relatively low-success procedure owing to suboptimal maturation conditions in the absence of an ovarian ‘niche’ and the poor understanding of this process at the molecular level in oocytes. Some investigators have reported on structural and morphological differences in human oocytes after IVM compared with standard IVF (Coticchio et al, 2016, Walls et al, 2016). For in-vitro matured oocytes, worse IVF clinical results, i.e., embryonic development, pregnancy and live birth, are achieved compared with oocytes that naturally mature in the ovarian niche and that are supported by surrounding cumulus cells, especially granulosa cells, during their growth and maturation in the follicle (Roesner et al, 2012, Walls et al, 2015). There is no generally accepted procedure for oocyte maturation in vitro, and the techniques used for IVM differ substantially across infertility clinics, resulting in extremely variable clinical outcomes (Dahan et al., 2016). Therefore, the procedure for oocyte maturation in vitro continues to be an experimental procedure (American Society for Reproductive Medicine (ASRM), 2013). This procedure, however, represents an interesting model for studying the molecular mechanisms involved in the oocyte maturation process, and the IVM procedure should be optimized for the future. It is of great interest because it could be beneficial for some infertile women who have polycystic ovaries (Siristatidis et al., 2015), for women who have poor ovarian response to hormonal stimulation (Lee et al., 2016) and for oncological patients who cryopreserve their oocytes before oncotherapy to preserve their fertility (Revelli et al., 2012).

The ovarian follicle is the major functional unit of the ovary and female reproductive system. Bidirectional crosstalk between the oocyte and surrounding cumulus cells is essential for oocyte growth, for enabling nutrients and other small molecules to transfer between them, together ensuring that the oocyte properly acquires the molecular machinery required for subsequent early embryo development (Albertini et al, 2001, Gilchrist, 2011, Gilchrist et al, 2008, Zuccotti et al, 1998). A highly coordinated interplay between the oocyte and surrounding cumulus cells (granulosa cells) in the follicle depends on functional gap junctions (Granot and Dekel, 2002), which are mainly composed of connexin 37 (Cx37) (Furger et al, 1996, Gershon et al, 2008, Tsai et al, 2003) and directly mediate the cell–cell communication by allowing the passage of small molecules such as ions, metabolites, nutrients, and small signalling molecules between the neighbouring cells (Caspar et al, 1977, Makowski et al, 1977). Studies in knockout mice clearly demonstrated that the ablation of Cx37 leads to an interruption of intercellular coupling between oocytes and cumulus cells, disruption of follicle development, incompetent oocytes and ovulatory dysfunction (Simon et al., 1997).

Experimental data on cumulus cell transcriptome revealed that bidirectional communication via gap junctions between the human oocyte and cumulus cells is essential for the maturation and production of a competent oocyte (Huang, Wells, 2010, Ouandaogo et al, 2011, Ouandaogo et al, 2012). Among important substances exchanged between the oocyte and cumulus cells (granulosa cells) are bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which play key roles in promoting follicle growth beyond the primary stage (Chang et al, 2002, Knight, Glister, 2006); the absence of these two substances causes infertility, as shown in the animal models (Dong et al, 1996, Galloway et al, 2000). Cumulus cell–oocyte gap junctions have also been reported to regulate the oocyte reduced glutathione synthesis and accumulation, which plays an important role as a reducing agent and important antioxidant in oocyte maturation, fertilization, and embryonic development (Curnow et al, 2008, de Matos et al, 1997, de Matos et al, 2002, Li et al, 2016, Nagai, 2001, Sutovsky, Schatten, 1997, Takeo et al, 2015); oocytes are quite susceptible to oxidative stress during the maturation process, and their most important antioxidant glutathione is mostly supplied by surrounding cumulus cells via gap junctions.

The human oocytes are usually matured in vitro without cumulus cells and ovarian ‘niche’. The human oocyte maturation process involves several known components, but there are still many unknown components that may be lacking in the present maturation media owing to the absence of cumulus cells, which may have negative consequences at the genetic level. Several differences in the global mRNA transcript profile have been reported between mature (metaphase II) and immature (GV) oocytes in both mice and humans (Assou et al, 2006, Cui et al, 2007, Gasca et al, 2007, Wang et al, 2004, Yoon et al, 2006, Zhang et al, 2007). Published data show that immature human oocytes express a higher number of genes than mature oocytes (Assou et al, 2006, Wells, Patrizio, 2008). A set of genes whose expression gradually increased during human oocyte maturation was identified, including phosphatase CDC25A, PCNA and SOCS7 (Assou et al., 2006). Fewer data are available on the molecular status of human oocytes during the IVM process. Existing data show that more than 2000 genes were differentially expressed in human oocytes matured in vitro compared with those matured in vivo (Jones et al., 2008); this group included several genes involved in transcriptional regulation. Reduced HDAC1 expression and insufficient histone deacetylation are associated with metaphase defects in human oocytes matured in vitro (Huang et al., 2012). Moreover, it has been found that inefficient SIRT3 expression induced decreased mitochondrial DNA copy number and biogenesis, and therefore impaired the developmental competence of human oocytes matured in vitro (Zhao et al., 2016).

The aims of this study were to elucidate how the IVM procedure affects the molecular status of oocytes compared with oocytes that matured in vivo and to elucidate the effects of oocyte co-culture with cumulus cells, which provides some degree of an ovarian niche during the maturation procedure. The gene expression profile of human oocytes was analysed according to whether they matured in vivo or in vitro (either by the conventional or in co-culture with cumulus cell). We evaluated the effects of the two IVM procedures on the gene expression profiles of human oocytes compared with oocytes that matured in vivo.