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The developmental competence of an oocyte is a measure of its intrinsic quality, this is characterised by the oocyte's ability to undergo maturation, to get fertilised, to cleave and develop post-fertilisation to the blastocyst stage and to implant and result in a viable pregnancy. Sirard and Blondin (1996) showed that the intrinsic quality of the oocyte is the major factor determining the proportion of oocytes that develop to the blastocyst stage. During folliculogenesis, as the oocytes grow and their surrounding somatic cells differentiate, they acquire their developmental competence (Eppig et al., 1994). Studies have shown that different ultrastructural and molecular changes taking place during the development of the oocyte are connected to its developmental competence (Assey et al., 1994; Hyttel et al., 1997). Another study confirmed that a higher percentage of in vivo matured oocytes develop to the blastocyst stage when compared to those matured in vitro (Greve et al., 1987; Riszo et al., 2002; Sutton et al., 2003). This is probably due to their removal from the follicular environment and therefore not exposed to the adequate factors within the ovary that are necessary for acquiring developmental competence (Wells et al., 2009). Factors that affect the oocyte's ultimate developmental competence include the following :(1) the size of its follicle of origin, with oocytes derived from larger follicles being more competent than those derived from smaller follicles (Pavlok et al., 1992; Lonergan et al., 1994; Dubey et al., 1995), (2) the state of health of the follicle, where dominance and atresia of follicles are linked with developmental competence (Blondin and Sirard, 1995; Hagemann, 1999), (3) ovarian stimulation, where the hormones improve oocyte developmental competence (Sirard et al., 2006) and (4) the importance of the interaction between the oocyte and its cumulus cells (CCs) in the oocyte's competence (Krisher, 2004).
A relationship between follicular size, oocyte maturation and pregnancy rates in women following in vitro fertilisation has been reported in different studies (Dubey et al., 1995; Bergh et al., 1998). In the clinical setting, the size of the follicle and the concentration of serum estradiol are used to detect oocyte maturity during ovarian stimulation (McArdle et al.,1983; Mikkelsen et al., 2000). Final maturation of oocytes with human chorionic gonadotropin (hCG) is done when the leading follicle is 16-18mm in size (Wittmaack et al., 1994) or when most of the follicles are 18-20mm in diameter (Ectors et al., 1997). Studies have shown that oocytes from follicles having a mean diameter â‰¥ 12(? Mope) have high fertilisation and cleavage rates (Wittmaack et al., 1994). There is also positive relationship between oocytes at metaphase II and the size of the follicle (Ectors et al., 1997; Tsuji et al., 1985, Scott et al., 1989). However, no mutual agreement has been reached concerning the maturity and developmental competence of oocytes derived from small follicles (Bergh et al., 1998; Dubey et al., 1995).
During folliculogenesis, the bidirectional relationship between the oocyte and the granulosa cells is important for a healthy oocyte and development of the CCs (Buccione et al., 1990a, b). The oocyte relies on its cumulus cells to supply nutrients and signals that regulate the maturation of its nucleus and cytoplasm, this is absolutely essential in acquiring its competence (Chian and Sirard, 1995; Ka et al., 1997). Recent studies carried out by Fatehi et al. (2002) and Zhang et al. (1995) also showed that early oocyte denudation during in vitro maturation or fertilisation impairs the competence of the oocyte.
As the antrum forms, the oocyte and its secreted growth factors maintain the differentiation of the granulosa cells and divide them into two groups based on their proximity to the oocyte and the difference in their gene expression, the cumulus granulosa cells being the ones next to the oocyte and the mural granulosa cells are proximal to the follicle wall and separated from the oocytes by the antral fluid (Epigg et al., 1997; Li et al., 2000). The mechanism by which this occurs is yet to be fully understood. According to Matzuk (2000), bone morphogenetic protein 15 (BMP15) and growth-differentiation factor 9 (GDF9) are often referred to as oocyte-secreted factors (OSFs) because evidence have shown that they are oocyte specific and are required for follicular development beyond the primary stages in human and sheep (GDF9 & BMP15) and mice (GDF alone) (Dong et al., 1996; Galloway et al., 2000; Yan et al., 2001; Montgomery et al., 2001; Hanrahan et al., 2004; Dixit et al., 2006). Abnormality in GDF9 expression in human is associated with polycystic ovarian failure (Teixeira et al., 2002) while mutation in both Gdf9 and Bmp15 were linked with premature ovarian failure and dizygotic twins (Di Pasquale., 2004; Dixit et al., 2006; Montgomery et al., 2006).Their recombinant forms were also found to regulate the activities of granulosa cells when applied in vitro (Elvin et al., 1999; Hayashi et al., 1999; Joyce et al., 2000; Otsuka et al., 2001b). BMP15 and GDF9 importance have also been demonstrated in the early phase of folliculogenesis with animals that became infertile or had impaired development of their follicles after been immunised against or been deficient of BMP15 and GDF9 (Dong et al., 1996; Galloway et al., 2000; Juengel et al., 2002; Hanrahan et al., 2004). Also, BMP15 and GDF9 have been identified in promoting cells, preventing their death and luteinisation by controlling steroidogenesis and synthesis of inhibin and curtailing the expression of luteinising hormone receptor (Hussein et al., 2005; Eppig, 2001).
BMP15 and GDF9 are members of the transforming growth factor beta (TGF-Î²) superfamily (Chang et al. 2002). BMP15 utilises the SMAD1/5/8 messengers and the TGFÎ²/activin pathway using SMAD 2 and 3 with BMPRIB (ALK6) and BMPRII receptors while GDF9 elicits its signals by interacting with TGF-bRI (activin-like kinase 5, ALK5) and bone morphogenetic protein receptor II (BMPRII) on the cell surface of the target and activates SMAD 2/3 molecules (Kaivo-oja et al., 2006). Evidence revealed that BMP15 affects the maturation and eventual quality of oocytes by stimulating and promoting granulosa cell growth and differentiation from the primary to the FSH dependent stage (Otsuka et al. 2000; Chang et al. 2002; Shimasaki et al. 2004). Although variation in the role of BMP15 was noticed in different species in the development of follicles in early preantral stage, studies still suggested that BMP15 is required for follicular development to the ovulatory stage (Juengel et al., 2002). Also, BMP15 level obtained in the follicular fluid of eggs that got fertilised and cleaved was higher when compared with the eggs that did not (Yan et al., 2007). Yan et al. (2007) also showed a positive relationship between BMP15 level in follicular fluid and estradiol. GDF9 have also been confirmed to be involved in the progression of primary follicle in the human ovaries (Hreinsson et al.2002) and in rodents in vitro (Hayashi et al.1999). Juengel et al. (2002) and Hanrahan et al. (2004) further established that the follicles did not develop beyond primary stage when mutations or immunisation against GDF9 was done. Lastly, Oocytes via OSFs have been shown to stimulate gene expression necessary for oocyte maturation and subsequent embryo development (Pangas et al., 2004), participate in cumulus expansion (Buccione et al., 1990b), maintain the cumulus cells in a non-luteinised state to promote growth and limit steroidogenesis (Li et al., 2000) and prevent CC apoptosis by ensuring a the desired gradient of anti-apoptotic factors in the cumulus oocyte complex (Hussein et al., 2005).
Based on the above evidence, we hereby hypothesised that the size of the follicle of origin and ability of the oocyte to secrete the OSFs determine the developmental competence of the oocyte. The aim of this study was to study the effect of follicular size and oocyte-secreted factors on the ability of human oocytes to be fertilised and develop in vitro.
Materials and Methods
30 patients undergoing fertility treatment at our clinic participated in this study. The follicles used were â‰¤17mm and â‰¥18mm in size. A total of 180 follicles were sampled, 6 from each patient. This study was carried out with the approval of Human Fertilization Embryology Authority (study license 26-035). The patients gave informed consent to donate follicular fluid (FF) and oocytes from six follicles each. Unless otherwise specified, all chemicals and reagents were bought from Sigma-Aldrich (Poole, Dorset, UK).
Ovarian Stimulation, sampling of follicular fluid and oocyte collection
This was done according to ............(Mendoza et al. , 1999?). Buserelin, gonadotropin-releasing hormone agonist (GnRHa, Suprecur; Hoechst, Frankfurt Germany), was commenced in all the patients in the mid-luteal phase of the previous cycle. After complete pituitary down regulation, the patients were stimulated with recombinant follicle stimulating hormone (r-FSH; Gonal-F; Serono Laboratories, Aubonne, Switzerland). Human chorionic gonadotropin (10000 IU, Profasi, Serono Laboratories) was administered when there was at least one follicle >18 mm in mean diameter. Oocyte retrieval was performed 36-38 hours after hCG administration. At the time of oocyte retrieval, under ultrasound guide, the follicles were measured and based on their sizes, each patient had three small follicles (â‰¤17 mm) and three large follicles (â‰¥18 mm) aspirated per vaginal. Aspiration of each follicle was done separately, the oocytes were immediately removed and the FF was put on ice until the end of the oocyte pickup. The FF of each follicle was transferred into a 15ml Falcon tube and centrifuged at 3000g for 10 minutes in the laboratory. The supernatant was transferred into a fresh falcon bottle, frozen immediately and stored at -70Â° for subsequent analysis.
Assessment of oocytes for maturity and IVF
After separation of the oocytes from their individual follicles, they were scored for maturity using the first polar body. The immature oocytes with germinal vesicles were noted. In vitro fertilization was done using donor sperm of proven fertility and normal karyotype (Cryos International Bank Aarhus, Denmark) 4 hours after oocyte collection according to their individual follicular size. Fertilization was checked for 18 hours post insemination, the oocytes with 2 pronuclei (PN) were normal and cultured. The oocytes with 1PN or 3PN were abnormal while oocytes without PN were taken to be unfertilized (Mendoza et al., 1999). On day 5, the embryos were checked for blastocyst development and the blastocysts were scored using Dokras et al. (1993).
Differential labeling of inner cell mass (ICM) and trophectoderm (TE) of blastocysts.
The counting of ICM and TE was carried out with polynucleotide- specific fluorochromes, a modification of Handyside and Hunter (1984) technique. In brief, the blastocysts were washed thrice in phosphate-buffered saline (PBS) and fixed in paraformaldehyde (PFA). The blastocysts were then placed in 200Âµl drop of acid Tyrode's solution containing 4mg/ml polyvinylpyrrolidone (PVP) to remove their zona pellucidae. The zona-free blastocysts were washed once in 4mg/ml BSA in HTF-HEPES (HTF-HEPES-BSA) and twice in 8mg/ml PVP in HBSS (HBSS-PVP). The blastocysts were then incubated in 10mM trinitrobenzine sulphonic acid (TNBS) in HBSS-PVP on ice in a dark room for 20 minutes at pH of 8.5. The blastocysts were washed thrice in HTF-HEPES-BSA and incubated again in 0.2mg/ml anti-dinitrophenol-BSA antibody at 37Â°C for 30 minutes. They were washed again as above and incubated in 1:10 guinea-pig complement serum: HTF-HEPES-BSA at 37Â°C for 30 minutes. Following another wash, the blastocysts were incubated at 37Â°C for 30 minutes in 10Âµl/ml propidium iodide to stain TE and then 0.05M bisbenzimide, to stain both ICM and TE. The blastocyst were washed, fixed in fixing solution for a minute then washed again. Each blastocyst was then mounted on a glass slide in a drop of 80% glycerol in PBS and covered firmly with a cover slip. The blastocysts were examined under the ultraviolet microscope (Leica TCS SP2 A0CS). ICM nuclei were stained blue with bisbenzimide while TE nuclei were stained red.