WO2012061898A1

WO2012061898A1 - Method of assessing oocytes

Info

Publication number: WO2012061898A1
Application number: PCT/AU2011/001461
Authority: WO
Inventors: Kathryn Gebhardt; Darryl L. Russell
Original assignee: Adelaide Research And Innovation Pty Ltd
Priority date: 2010-11-12
Filing date: 2011-11-11
Publication date: 2012-05-18

Abstract

The present invention relates to methods for assessing the developmental competence of an oocyte. In particular, the methods comprise measuring expression of a gene in the one or more cumulus cells, wherein the gene is selected from one or more of Growth Arrest-Specific transcript 5 (GAS5), Versican (VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1), Phosphofructokinase Platelet (PFKP), Pentraxin 3 (PTX3), Pepsinogen 3 (PGA3), Pepsinogen 4 (PGA4), and Pepsinogen 5 (PGA5), wherein the expression of the gene is correlated with the developmental competence of the oocyte. The methods may be used as the basis for selecting oocytes for inclusion in an assisted reproduction method to ensure the greatest chance of achieving a successful pregnancy and subsequent live birth.

Description

METHOD OF ASSESSING OOCYTES

PRIORITY CLAIM

This international patent application claims priority to Australian provisional patent application 2010905055 filed on 12 November 2010, the content of which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods of assessing the developmental competence of an oocyte. In particular, the methods comprise measuring expression of a gene in one or more cumulus cells of a cumulus oocyte complex associated with an oocyte, wherein the gene is selected from one or more of Growth Arrest-Specific transcript 5 {GAS5), Versican { VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1), Phosphofructokinase Platelet {PFKP), Pentraxin 3 (PTX3), Pepsinogen 3 {PGA3), Pepsinogen 4 {PGA4), and Pepsinogen 5 {PGA5).

BACKGROUND OF THE INVENTION

A significant proportion of children in western countries are now born using assisted reproduction technologies, including the use of in vitro fertilization (IVF). IVF generally takes the form of stimulating the female to ovulate, contacting collected oocytes with sperm in vitro and introducing the resultant embryo into the uterus.

Gamete quality is a major factor in infertility and success in assisted reproductive technology. In each female reproductive cycle, a large cohort of oocytes develop in the ovary, but these do not all have equal developmental potential. In normal fertile cycles only one or two of these oocytes are ovulated and gain the capacity to be fertilised and form an embryo. Hence natural biological processes select the gamete with reproductive potential. In assisted reproductive technologies including IVF and ICSI a large oocyte cohort is collected and fertilised in vitro. However, the main approach currently available to predict successful implantation is embryo morphological assessment. This is only a crude tool that is effective at eliminating embryos with poor competence, but often does not provide enough information to select the embryo with best probability of implanting and progressing to a successful live birth. Despite considerable research and technical advances in the IVF field, the rate of successful pregnancy following IVF treatment is still quite low and is in the order of 15 to 25% per cycle. The poor success rate for IVF treatment is due in large part to an extraordinarily high rate of early embryonic loss due to impaired development and/or implantation failure.

Poor ooctye quality may lead to failed implantation, miscarriage, or one or more pregnancy complications including, for example, preeclampsia, pre-term birth, gestational hypertension, gestational diabetes, unexplained stillbirth, premature rupture of membranes and intrauterine growth restriction.

Accordingly, there is a need to provide improved methods of assessing the developmental competence of an oocyte.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method of assessing developmental competence of an oocyte, the method comprising:

measuring expression of a gene in the one or more cumulus cells, wherein the gene is selected from one or more of Growth Arrest-Specific transcript 5 (GAS5),

Versican { VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1),

Phosphofructokinase Platelet {PFKP), Pentraxin 3 (PTX3), Pepsinogen 3 {PGA3),

Pepsinogen 4 {PGA4), and Pepsinogen 5 {PGA5),

wherein the expression of the gene is correlated with the developmental competence of the oocyte.

In a second aspect, the present invention provides a method of assessing developmental competence of an oocyte, the method comprising:

obtaining a cumulus oocyte complex which includes the oocyte and one or more cumulus cells associated with the oocyte; and

Versican { VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1), Phosphofructokinase Platelet {PFKP), and Pentraxin 3 (PTX3), Pepsinogen 3 {PGA3),

Pepsinogen 4 {PGA4), and Pepsinogen 5 {PGA5),

wherein the expression of the gene is correlated with the developmental competence of the oocyte. In one embodiment of the aforementioned aspects of the invention the gene is GAS5. In some embodiments, the GAS5 gene is a human GAS5 gene. In some embodiments, measuring expression of the GAS5 gene comprises measuring an expression level of a GAS5 mRNA. In one embodiment, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the GAS5 mRNA is positively correlated with pregnancy success. In some embodiments, the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the GAS5 mRNA is positively correlated with probability of a live birth. In some embodiments, measuring expression of the GAS5 gene comprises measuring an expression level of one or more small nucleolar RNAs (snoRNAs) derived from the GAS5 gene. In one embodiment, the one or more snoRNAs comprise a sequence as set forth in any one of GenBank ID NOs: NR_002764 (SNORD47), NR_003944 (SNORD78), NR_002750 (SNORD44), NR_003942 (SNORD76), NR_003940 (SNORD 80), NR_003943 (SNORD77). In some embodiments, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the one or more snoRNAs is negatively correlated with pregnancy success. In some embodiments of the aforementioned aspects of the invention the gene is VCAN. In one embodiment, the VCAN gene is a human VCAN gene. In some embodiments, measuring expression of the VCAN gene comprises measuring an expression level of a VCAN mRNA. In one embodiment, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the VCAN mRNA is positively correlated with pregnancy success. In some embodiments, the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the VCAN mRNA is positively correlated with probability of a live birth. In some embodiments, the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the VCAN mRNA is positively correlated with birth weight.

In some embodiments of the aforementioned aspects of the invention the gene is PTGS2. In one embodiment, the PTGS2 gene is a human PTGS2 gene. In some embodiments, measuring expression of the PTGS2 gene comprises measuring an expression level of a PTGS2 mRNA. In one embodiment, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the PTGS2 mRNA is positively correlated with pregnancy success. In some embodiments, the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the PTGS2 mRNA is positively correlated with probability of a live birth.

In some embodiments of the aforementioned aspects of the invention the gene is GREM1. In one embodiment, the GREM1 gene is a human GREM1 gene. In some embodiments, measuring expression of the GREM1 gene comprises measuring an expression level of a GREM1 mRNA. In one embodiment, the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the GREM1 mRNA is positively correlated with birth weight.

In some embodiments of the aforementioned aspects of the invention the gene is PFKP. In one embodiment, the PFKP gene is a human PFKP gene. In some embodiments, measuring expression of the PFKP gene comprises measuring an expression level of a PFKP mRNA. In one embodiment, the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the PFKP mRNA is positively correlated with birth weight.

In some embodiments of the aforementioned aspects of the invention the gene is PTX3. In one embodiment, the PTX3 gene is a human PTX3 gene. In some embodiments, measuring expression of the PTX3 gene comprises measuring an expression level of a PTX3 mRNA. In one embodiment, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the PTX3 mRNA is positively correlated with pregnancy success. In some embodiments, the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the PTX3 mRNA is positively correlated with probability of a live birth. In some embodiments of the aforementioned aspects of the invention the gene is selected from one or more of PGA3, PGA4 and PGA5. In one embodiment, the PGA3, PGA4 and PGA5 genes are human PGA3, PGA4 and PGA5 genes. In some embodiments, measuring expression of one or more of the PGA3, PGA4 and PGA5 genes comprises measuring an expression level of a PGA3, PGA4 and/or PGA5 mRNA. In one embodiment, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of one or more of the PGA3, PGA4 and PGA5 mRNAs is positively correlated with pregnancy success.

In some embodiments of the aforementioned aspects of the invention the one or more cumulus cells are isolated from the cumulus oocyte complex prior to measuring expression of the gene in the one or more cumulus cells. In one embodiment, the cumulus oocyte complex is cultured prior to isolation of the one or more cumulus cells.

In some embodiments of the aforementioned aspects of the invention the cumulus oocyte complex is cultured prior to measuring expression of the gene in the one or more cumulus cells.

In some embodiments of the aforementioned aspects of the invention the method further comprises selecting an oocyte for fertilisation or transfer on the basis of the assessed developmental competence of the oocyte.

In some embodiments of the aforementioned aspects of the invention the method comprises part of an assisted reproductive technology. In one embodiment, the assisted reproductive technology comprises in vitro fertilisation or comprises intracytoplasmic sperm injection.

In some embodiments of the aforementioned aspects of the invention the assessed developmental competence of the oocyte is positively correlated with the developmental competence of an embryo derived from the oocyte. In a third aspect, the present invention provides a method of assisted reproduction, the method comprising:

(a) selecting an oocyte for fertilisation or transfer on the basis of the assessed developmental competence of the oocyte as determined by a method according to a first or second aspect of the invention;

(b) fertilising the selected oocyte to form a zygote;

(c) culturing the zygote to form an embryo; and

(d) implanting the embryo into a subject. In one embodiment the subject is a non-human animal. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a graph of the relative copy number of the GAS5 gene from cumulus cells associated with oocytes used in single embryo transfer assisted reproduction against pregnancy success (as determined by the presence of a fetal heartbeat). Relative copy number data is normalised to GAPDH. p= 0.002.

Figure 2A shows a graph of the relative copy numbers of snoRNAs from the GAS5 region in cumulus cells associated with oocytes that were used in assisted reproduction resulting in pregnancy or no pregnancy. Relative copy number data is normalised to GAPDH.

Figure 2B shows the GAS5 gene structure and arrangement of snoRNAs encoded within the human GAS5 gene. The exons coding GAS5 mRNA and intronic snoRNA regions (not drawn to scale) are denoted by black and gray boxes, respectively. The human GAS5 gene contains twelve exons and encodes ten snoRNAs. The snoRNA names are indicated above the gene structure. The snoRNAs are indicated by the same Roman numerals. Image obtained from Shao et al. 2009 BMC Genomics 10: 86.

Figure 3 shows a graph of the relative copy number of the VCAN gene from cumulus cells associated with oocytes used in single embryo transfer assisted reproduction against pregnancy success (as determined by the presence of a fetal heartbeat) and a subsequent live birth. Relative copy number data is normalised to GAPDH. p < 0.02.

Figure 4 shows a graph of the relative copy number of the PTGS2 gene from cumulus cells associated with oocytes used in single embryo transfer assisted reproduction against pregnancy success (as determined by the presence of a fetal heartbeat) and a subsequent live birth. Relative copy number data is normalised to GAPDH. p = 0.02.

Figure 5 shows a graph of the relative copy number of the PTX3 gene from cumulus cells associated with oocytes used in single embryo transfer assisted reproduction against pregnancy success (as determined by the presence of a fetal heartbeat) and a subsequent live birth. Relative copy number data is normalised to GAPDH. p = 0.06.

Figure 6 shows a dot plot of the relative copy number of the Versican gene from cumulus cells associated with oocytes used in single embryo transfer assisted reproduction against neonatal birth weight of babies derived from the oocytes. Relative copy number data is normalised to GAPDH. p = 0.08.

Figure 7 shows a dot plot of the relative copy number of the GREM1 gene from cumulus cells associated with oocytes used in single embryo transfer assisted reproduction against neonatal birth weight of babies derived from the oocytes. Relative copy number data is normalised to GAPDH. p = 0.06.

Figure 8 shows a dot plot of the relative copy number of the PFKP gene in cumulus cells associated with oocytes used in single embryo transfer assisted reproduction against neonatal birth weight of babies derived from the oocytes. Relative copy number data is normalised to GAPDH. p < 0.05.

Figure 9 shows a graph of the relative copy number of the GAS5 gene in cumulus cells associated with oocytes used in single embryo transfer assisted reproduction (in subjects with Male Factor Only infertility - study 2) against pregnancy success (as determined by the presence of a fetal heartbeat). Relative copy number data is normalised to GAPDH. p= 0.037. Figure 10 shows a graph of the relative copy number of the VCAN gene in cumulus cells associated with oocytes used in single embryo transfer assisted reproduction (in subjects with Male Factor Only infertility - study 2) against pregnancy success (as determined by the presence of a fetal heartbeat). Relative copy number data is normalised to GAPDH.

Figure 1 1 shows a graph of the relative copy number of the PTX3 gene in cumulus cells associated with oocytes used in single embryo transfer assisted reproduction (in subjects with Male Factor Only infertility - study 2) against pregnancy success (as determined by the presence of a fetal heartbeat). Relative copy number data is normalised to GAPDH. p = 0.041.

Figure 12 shows a graph of the relative copy number of the PTGS2 gene in cumulus cells associated with oocytes used in single embryo transfer assisted reproduction (in subjects from study 2) against pregnancy success (as determined by the presence of a fetal heartbeat). Relative copy number data is normalised to GAPDH. p = 0.023. Figure 13 shows a graph of the relative copy number of the PTX3 gene in cumulus cells associated with oocytes used in single embryo transfer assisted reproduction (in subjects from study 2) against pregnancy success (as determined by the presence of a fetal heartbeat). Relative copy number data is normalised to GAPDH. p = 0.07.

DESCRIPTION OF THE INVENTION

A typical cycle of in vitro fertilisation will yield multiple viable embryos, but it is medically desirable in the interests of the health of the mother and her offspring to transfer only one embryo to the mother's uterus. As described above, IVF clinics presently rely on morphology (the appearance through a microscope) to select embryos for transfer. However, embryos with similar morphological appearance have very divergent competence. In accordance with the present invention, a genetic test has been developed that discriminates oocytes with a greater potential to implant and progress to a healthy live birth. The test quantitatively measures the amount of key gene expression in cumulus cells that surround and nurture the oocyte during its development. The cumulus cells are typically removed from the oocyte and discarded prior to fertilisation in vitro.

Thus, in a first aspect, the present invention provides a method of assessing developmental competence of an oocyte, the method comprising:

measuring expression of a gene in one or more cumulus cells associated with an oocyte, wherein the gene is selected from one or more of Growth Arrest-Specific transcript 5 {GAS5), Versican { VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1), Phosphofructokinase Platelet {PFKP), Pentraxin 3 (PTX3), Pepsinogen 3 {PGA3), Pepsinogen 4 {PGA4), and Pepsinogen 5 {PGA5),

measuring expression of a gene in the one or more cumulus cells, wherein the gene is selected from one or more of Growth Arrest-Specific transcript 5 (GAS5), Versican { VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1), Phosphofructokinase Platelet {PFKP), and Pentraxin 3 (PTX3), Pepsinogen 3 {PGA3), Pepsinogen 4 {PGA4), and Pepsinogen 5 {PGA5), wherein the expression of the gene is correlated with the developmental competence of the oocyte.

As the expression level of one or more of the genes correlates with the developmental competence of the oocyte, the developmental competence may be readily and sensitively ascertained by using the method. Furthermore, the method measures the expression of the one or more genes in cumulus cells, which are normally discarded. Accordingly, the oocyte associated with the cumulus cells is not exposed to potentially harmful processing in carrying out the method.

Oocytes are female reproductive cells, which may be part of a follicle and/or part of a cumulus oocyte complex. Typically, the collection of oocytes includes the hormonal stimulation of follicles of a female subject and removal of the cumulus oocyte complex (COC) from the follicles using transvaginal ultrasound and a needle.

The term "one or more cumulus cells associated with the oocyte", and the like, as used throughout the specification is to be understood to mean one or more cumulus cells as a part of a cumulus oocyte complex, or one or more cumulus cells that were at one time part of a specific cumulus oocyte complex but which have been physically removed or dissociated from the cumulus oocyte complex. In this regard, for an oocyte undergoing in vitro fertilisation, the COC is generally trimmed of the outer layers of cumulus cells, while for an oocyte undergoing intracytoplasmic sperm injection, both the outer and inner cumulus cell layers are trimmed. As described above, the present method assesses the developmental competence of an oocyte. The term "developmental competence" in relation to oocytes is intended to mean the capacity of an oocyte to achieve any one or more of the following indicators: complete nuclear maturation (i.e. resumption and completion of meiosis from arrest, such as metaphase II arrest); fertilisation; progression through early embryogenesis; implantation; placentation; initiation of pregnancy; normal fetal development; absence of pregnancy complications; full term birth; and normal birth weight. For example, an oocyte with a high developmental competence is more likely to achieve one or more of these indicators than an oocyte with a low developmental competence. The term "pregnancy success" as used herein, relates to any one or more of fertilisation, implantation, placentation or a positive pregnancy test (chemical test or detection of a fetal heart beat). If a gene is positively correlated with pregnancy success, an oocyte associated with a cumulus cell with high level expression of the gene is more likely to result in pregnancy success than an oocyte associated with a cumulus cell with a lower level of expression of the gene. If a gene is negatively correlated with pregnancy success, an oocyte associated with a cumulus cell with low level expression of the gene is more likely to result in pregnancy success than an oocyte associated with a cumulus cell with a higher level of expression of the gene.

As pregnancy success relates to one or more early stages in pregnancy, it may or may not correlate with live birth rates or birth weight. If a gene is positively correlated with the probability of a live birth, an oocyte associated with a cumulus cell with high level expression of the gene is more likely to result in a live birth than an oocyte associated with a cumulus cell with a lower level of expression of the gene. If a gene is negatively correlated with the probability of a live birth, an oocyte associated with a cumulus cell with low level expression of the gene is more likely to result in a live birth than an oocyte associated with a cumulus cell with a higher level of expression of the gene.

If a gene is positively correlated with birth weight, an oocyte associated with a cumulus cell with high level expression of the gene is more likely to result in a higher birth weight than an oocyte associated with a cumulus cell with a lower level of expression of the gene. If a gene is negatively correlated with birth weight, an oocyte associated with a cumulus cell with low level expression of the gene is more likely to result in lower birth weight than an oocyte associated with a cumulus cell with a higher level of expression of the gene. Birth weight is an important indicator with respect to the health of the baby and long term health outcomes for the individual. For example, low birth weight for gestational age correlates with increased risk of cardiovascular disease including hypertension, type 2 diabetes mellitus, obesity and other diseases. Birth weight should be indexed against the gestational age of the baby. For example, a baby with a gestational age of 40 weeks and a birth weight of 2500 g would be classified as small for its gestational age, while a baby with the same birth weight but with a gestational age of 35 weeks may be appropriate for its gestational age.

As set out above, the method involves measuring the expression of one or more genes selected from Growth Arrest-Specific transcript 5 (GAS5), Versican ( VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1), Phosphofructokinase Platelet {PFKP), Pentraxin 3 (PTX3), Pepsinogen 3 {PGA3), Pepsinogen 4 {PGA4), and Pepsinogen 5 (PGA5). The term "gene" as used throughout the specification is to be understood to mean a region of genomic nucleotide sequence (nuclear or mitochondrial) associated with a coding region and/or producing a transcript. The term includes regulatory regions (e.g. promoter regions), transcribed regions, exons, introns, untranslated regions and other functional and/or non-functional sequence regions associated with the gene.

Methods for measuring the expression of a gene are generally known in the art and may include, for example, detection and/or quantitation of mRNA or protein. Levels of mRNA may be measured by Northern blotting, RT-PCR, microarrays, or "tag based" technologies such as SAGE (serial analysis of gene expression). Microarrays and SAGE may be used to simultaneously quantitate the expression of multiple genes. Primers or probes may be designed based on nucleotide sequences of the genes or transcripts thereof. Methodology similar to that disclosed in Paik et al. (2004) NEJM 351 (27): 2817-2826 or Anderson et al. (2010) Journal of Molecular Diagnostics 12(5): 566-575) may be used to measure the expression of one or more genes of interest.

For protein encoding genes, gene expression may be measured by determining the amount of protein produced. Methods of protein quantification may include Western blotting, ELISA or other methods known in the art. Details of the genes according to embodiments of the present invention may be accessed from the GenBank database at the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). For example, the Gene ID number for human GAS5 is 60674, human VCAN is 1462, human PTGS2 is 5743, human GREM1 is 26585, human PFKP is 5214, human PTX3 is 5806, human PGA3 is 643834, human PGA4 is 643847, and human PGA5 is 5222. The contents of these GenBank records are incorporated herein by reference.

In some embodiments, measuring expression of one or more of the genes may comprise measuring the mRNA levels associated with one or more of the genes. For example, the GenBank ID for human GAS5 mRNA is NR_002578.2, human VCAN transcript variant 1 is NM_004385.4, human VCAN transcript variant 2 is NM_001 126336.2, human VCAN transcript variant 3 is NM_001 164097.1 , human VCAN transcript variant 4 is NM_001 164098.1 , human PTGS2 is NM_000963.2, human GREM1 transcript variant 1 is NM_013372.6, human GREM1 transcript variant 2 is NM_001 191323.1 , human GREM1 transcript variant 3 is NM_001 191322.1 , human PFKP transcript variant 1 is NM_002627.4, human PFKP transcript variant 2 is NM_001242339.1 , human PTX3 is NM_002852.3, human PGA3 is NM_001079807.1 , human PGA4 is NM_001079808.1 , and human PGA5 is NM_014224.2.

While human genes and mRNA sequences have been identified above, it should be appreciated that the present invention is not limited to methods of assessing developmental competence of human oocytes. The oocyte and associated cumulus cells may be from a female human, a female mammal including a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig), or an animal of veterinary significance. Details of the specific genes and mRNA sequences for different species may be readily accessed from the GenBank database (e.g. the Gene ID for Mus musculus GAS5 is 14455 and Rattus norvegicus GAS5 is 81714) or sequences may be identified by Blast searching. In some instances, primers, probes or antibodies that may be used to measure gene expression in one species may also be used for unrelated species.

In some embodiments, the gene may be GAS5. Reference herein to a GAS5 gene should be understood to refer to a growth arrest-specific 5 gene. Such growth arrest- specific genes are typically non-protein coding.

In some embodiments, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the GAS5 mRNA is positively correlated with pregnancy success. In some embodiments, the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the GAS5 mRNA is positively correlated with probability of a live birth.

In some embodiments, measuring expression of the GAS5 gene comprises measuring an expression level of one or more small nucleolar RNAs (snoRNAs) derived from the GAS5 gene. The snoRNAs may comprise a sequence as set forth in any one of the following GenBank IDs: NR_002764 (SNORD47), NR_003944 (SNORD78), NR_002750 (SNORD44), NR_003942 (SNORD76), NR_003940 (SNORD 80), NR_003943 (SNORD77). Small nucleolar RNAs are a class of small RNA molecules that primarily function to guide modifications of other RNAs, such as ribosomal RNAs, transfer RNAs and small nuclear RNAs. In some embodiments, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the one or more snoRNAs is negatively correlated with pregnancy success. Without wishing to be bound by theory, the differential correlation between GAS5 mRNA and GAS5 snoRNAs with respect to pregnancy success is consistent with an expected inverse relationship between GAS5 and its derivative snoRNA transcripts through RNAse P degradation of GAS5 pre-RNA releasing the snoRNAs.

In some embodiments, the gene may be VCAN including, for example, the human VCAN gene. Measuring expression of the VCAN gene may comprise measuring an expression level of a VCAN mRNA. Details of the human VCAN gene and mRNA are provided above.

In some embodiments, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the VCAN mRNA is positively correlated with pregnancy success. In some embodiments, the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the VCAN mRNA is positively correlated with probability of a live birth. In some embodiments, the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the VCAN mRNA is positively correlated with birth weight.

In some embodiments, the gene may be PTGS2 including, for example, a human PTGS2 gene. Measuring expression of the PTGS2 gene may comprise measuring an expression level of a PTGS2 mRNA. Details of the human PTGS2 gene and mRNA are provided above.

In some embodiments, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the PTGS2 mRNA is positively correlated with pregnancy success. In some embodiments, the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the PTGS2 mRNA is positively correlated with probability of a live birth.

In some embodiments, the gene may be GREM1 including, for example, a human GREM1 gene. Measuring expression of the GREM1 gene may comprise measuring an expression level of a GREM1 mRNA. Details of the human GREM1 gene and mRNA are provided above.

In some embodiments, the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the GREM1 mRNA is positively correlated with birth weight.

In some embodiments, the gene may be PFKP including, for example, a human PFKP gene. Measuring expression of the PFKP gene may comprise measuring an expression level of a PFKP mRNA. Details of the human PFKP gene and mRNA are provided above.

In some embodiments, the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the PFKP mRNA is positively correlated with birth weight.

In some embodiments, the gene may be PTX3 including, for example, a human PTX3 gene. Measuring expression of the PTX3 gene may comprise measuring an expression level of a PTX3 mRNA. Details of the human PTX3 gene and mRNA are provided above.

In some embodiments, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the PTX3 mRNA is positively correlated with pregnancy success. In some embodiments, the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the PTX3 mRNA is positively correlated with probability of a live birth.

In some embodiments, the gene may be one or more of PGA3, PGA4 and PGA5 including, for example, one or more of a human PGA3, human PGA4 or human PGA5 gene. Measuring expression of one or more of the PGA3, PGA4 and PGA5 genes may comprise measuring an expression level of one or more of a PGA3, PGA4 and PGA5 mRNA. Details of the human PGA3, PGA4 and PGA5 genes and mRNAs are provided above.

In some embodiments, the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of one or more of PGA3 mRNA, PGA4 mRNA and PGA5 mRNA is positively correlated with pregnancy success.

In some embodiments, the developmental competence of an oocyte may be determined by measuring the level of gene expression of more than one of the aforementioned genes.

As described above, cumulus cells may be removed from the COC as part of an assisted reproductive method. In some embodiments, one or more cumulus cells may be isolated from the cumulus oocyte complex prior to measuring expression of the gene in the one or more cumulus cells. Methods for isolating one or more cells from a COC are known in the art and may involve removal of the one or more cells using a needle. Measuring the expression of one or more of the genes may be performed using the cumulus cell mass as a whole or using a single cell suspension.

Upon collection of one or more cumulus oocyte complexes, the cumulus oocyte complexes may be maintained in culture medium until ready for use in the assisted reproductive technology. Methods are known in the art for maintaining cumulus oocyte complexes in culture medium. Accordingly, in some embodiments, the cumulus oocyte complex may be cultured prior to isolation of the one or more cumulus cells. In some embodiments, the cumulus oocyte complex may be cultured prior to measuring expression of the gene in the one or more cumulus cells.

As described above, the present invention may be used to determine the developmental competence of oocytes. Accordingly, in some embodiments, the method may further comprise selecting an oocyte for fertilisation or transfer on the basis of the assessed developmental competence of the oocyte. In this regard, an ooctye with high developmental competence may be selected for use in an assisted reproduction method.

It follows that the method of the present invention may therefore comprise part of an assisted reproduction method. The term "assisted reproduction" as used throughout the specification is to be understood to mean any technique involving the production of an embryo from an oocyte or other cell, such that the embryo is capable of implantation. As will be appreciated, generally the purpose of assisted reproduction technologies is to produce a viable pregnancy. For example, an assisted reproduction technology may include a technique using an oocyte in vitro, in vitro fertilization (IVF; aspiration of an oocyte, fertilization in the laboratory and transfer of the embryo into a recipient), gamete intrafallopian transfer (GIFT; placement of oocytes into the fallopian tube), zygote intrafallopian transfer (ZIFT; placement of fertilized oocytes into the fallopian tube), tubal embryo transfer (TET; the placement of cleaving embryos into the fallopian tube), peritoneal oocyte and sperm transfer (POST; the placement of oocytes and sperm into the pelvic cavity), intracytoplasmic sperm injection (ICSI), testicular sperm extraction (TESE), microsurgical epididymal sperm aspiration (MESA), nuclear transfer, expansion from a totipotent stem cell, and parthenogenic activation. These and other methods of assisted reproduction are known in the art.

In some embodiments, the assessed developmental competence of the oocyte may be positively correlated with the developmental competence of an embryo derived from the oocyte. In this regard, an embryo that is formed from the oocyte with high developmental competence may be selected for use in an assisted reproductive method. Accordingly, the present invention provides a method which enables for an increase in the rate of pregnancy success per assisted reproduction technology cycle.

A third aspect of the present invention provides a method of assisted reproduction, the method comprising:

(b) fertilising the selected oocyte to form a zygote;

(c) culturing the zygote to form an embryo; and

(d) implanting the embryo into a subject.

The subject may be a human or non-human. For example, the subject may be a female human, a female mammal including a primate, a livestock animal (eg. a horse, a cow, a sheep, a pig, a goat), a companion animal (eg. a dog, a cat), a laboratory test animal (eg. a mouse, a rat, a guinea pig), or an animal of veterinary significance.

The present invention is further described by the following non-limiting examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description. EXAMPLE 1

Gene Expression Analysis - Study 1

Materials and Methods

Patient Selection

Ethical approval for the use of human samples was obtained from the Women's and Children's Hospital Human Research Ethics Committee, Adelaide, Australia and the Repromed Scientific Advisory Committee, Adelaide, Australia. Written consent for the use of cumulus cells for research was obtained from thirty-eight patients undergoing routine IVF/ICSI with Single Embryo Transfer (SET) at Repromed, Dulwich, South Australia between May and October 2005. Patients with clinical indications of polycystic ovary syndrome (PCOS) were not included in this study. All patients had treatment with their own gametes and were not undergoing pre-implantation genetic diagnosis. Patient Stimulation

Patients were stimulated using a long down-regulation or antagonist protocol involving the administration of a Gonadotropin Releasing Hormone analog (Nafarelin (Synarel), Organon, Australia) with down regulation confirmed with blood estrogen levels below 0.2 nM/L. Patients were then administered recombinant Follicle Stimulating Hormone (Gonal-F, Serono, Sydney, Australia or Puregon, Organon, Sydney, Australia) for 9-12 days and monitored by ultrasound and serum hormone levels until the lead follicle was a size of 18mm. Patients were then given 5,000 IU human Chorionic Gonadotropin (Pregnyl, Organon, Sydney, Australia) and the oocyte collection scheduled for 36h later. Cumulus oocyte complexes (COCs) from follicles greater than 14mm were collected using transvaginal ultrasound and a 17 gauge needle.

Laboratory Procedures and Cumulus Sampling

All media and oil products were purchased from Vitrolife, Kungsbacka, Sweden, or produced in-house, unless otherwise indicated. All culture was performed at 37°C, 6% C0₂, 5% 0₂, 89% N₂, in a humidified atmosphere, with manipulations being performed at 37°C, 6% C0₂. COCs were isolated from follicular fluid and washed in glucose supplemented (2.5mM glucose) GFERT plus medium. COCs were stored in 25 μΙ of the same medium for 3 hours post-oocyte collection for both IVF and ICSI inseminations. Following the 3 hour period, COCs were trimmed of their outer layers of cumulus cells with a 30 gauge needle on a syringe and collected individually. All cumulus cell samples were collected prior to insemination and stored frozen in a minimal volume of culture media at -80°C until required for analysis. Each cumulus mass and its respective oocyte were numbered to track which cumulus mass came from which oocyte. Oocytes undergoing ICSI were then exposed to 75IU Hyaluronidase (Hyalase®, Aventis Pharma Pty Ltd, Lane Cove, Australia) in glucose supplemented GFERT and then ICSI performed in G1 .3 plus medium and cultured singly in ~\ 0[\L drops of the same medium under oil for 16-18h. Oocytes undergoing IVF had the inner layers of cumulus left intact and were co-incubated with 1 ,000 motile sperm in ~\ 0[\L drops of glucose supplemented GFERT plus under oil for 17-19h. All oocytes underwent individual culture in order to track resultant embryos with respect to fertilization, timed cleavage, embryo cell number and quality, as well as pregnancy following SET. Fertilization was assessed 16-19h post-insemination and all embryos with two pronuclei continued culture individually in ~\ 0[\L drops of G1 .3 plus medium under oil for the next 48h. All embryos were then washed thoroughly through G2.3 Plus medium and cultured for another 48h of culture at which point embryos were transferred to fresh G2.3 medium for the final 24h of culture.

RNA Isolation and Real Time RT-PCR

Cumulus cell RNA was extracted using a commercial RNA isolation kit (Ambion RNAqueous Micro or Cells-to CT, Austin, TX, USA) as per the manufacturer's instructions. Total RNA extracted using Ambion RNAqueous Micro was eluted in 20 μΙ of elution buffer and stored at -80°C. Total RNA was then treated with 1 U of DNase as per manufacturer's instructions (Ambion Inc., Austin, TX, USA). First-strand complementary DNA (cDNA) was synthesized from total RNA using random hexamer primers (Geneworks, Hindmarsh, SA, Australia) and Superscript III reverse transcriptase (Invitrogen Australia Pty. Ltd). Specific gene primers for real time RT- PCR were designed against published mRNA sequences (NCBI Pubmed database) using Primer Express software (PE Applied Biosystems, Foster City, CA) and synthesised by Sigma Genosys (Sigma-Aldrich Pty. Ltd., Castle Hill, NSW, Australia). Real time RT-PCR was performed in triplicate for each sample using a Corbett Rotor Gene 6000 thermocycler (Corbett Life Science, Qiagen, Doncaster, Victoria, Australia) or an Applied Biosystems StepOnePlus Real Time RT-PCR system (Applied Biosystems, Life Technologies, Foster City, CA, USA). In each reaction, 2 μΙ of cDNA (equivalent to 10ng of total RNA), 0.2 μΙ of forward and reverse primers and 10 μ I of SYBR Green (Applied Biosystems, Scoresby, VIC, Australia) master mix were added, with H₂0 added to make a final volume of 20 μΙ. All primers were used at an optimised concentration of 50 μΜ. PCR cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 amplification cycles of 95°C for 15 s and 60°C for 1 min. Controls included omission of the cDNA template or RT enzyme in otherwise complete reaction mixtures; each showed no evidence of product amplification or primer dimers. Following real time RT-PCR, analysis of the dissociation curves confirmed that a single product was amplified in all reactions. The most stable real time RT-PCR loading control was determined using the GeNorm housekeeper analysis software (Vandesompele, et al. 2002 Genome Biol. 3(7): 1-12).

Real time RT-PCR standard curves were produced for all genes of interest and internal controls using plasmid DNA. PCR products were generated and inserted into TOPO 2.1 or TOPO 4.0 vectors (Invitrogen, Carlsbad, CA, USA), then transformed into chemically competent E. coli host strain TOP10 cells (Invitrogen) as per the manufacturer's instructions. The confirmed positive recombinant plasmids were purified using the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, Wl, USA). The presence of gene inserts was confirmed by PCR and restriction digestion of recombinant plasmids. The mass of plasmids containing cloned target sequences were calculated based on the size of the TOPO vector and PCR insert based on the derivation of DNA mass formula available from Applied Biosystems (PE Applied Biosystems). For each gene, a standard curve was generated ranging from 30 copies to 3x10⁷ copies per reaction then refined to an appropriate standard curve of five logarithmic concentrations matching the concentration range found in human cumulus cells. Each gene standard curve was linear and showed similar efficiency. Copy number was determined for each gene from the standard curve then divided by the copy number for the internal control gene to give the relative copy number for each gene. Embryo Quality Assessments

Embryo quality assessments were based on multiple observations. Embryo transfers were performed on days 2/3 or days 4/5 for extended culture. The day of embryo transfer was a clinical decision determined prior to the commencement of the patient's treatment by their clinician, and was independent of the response to stimulation or the number of embryos resulting from treatment. Embryo selection for transfer was based on morphology assessment, and in all cases the single highest morphological quality embryo, as determined by the appropriate scoring system (cleavage or extended culture stage), was transferred. The cleavage stage morphology scoring system used was based on the number of cells, the degree of fragmentation and the presence of multinucleated cells. Good quality embryos were assessed to have 4-cells on day 2 or 7-9 cells on day 3 with limited fragmentation and absence of multinucleated blastomeres. Extended culture selections performed for day 4 embryo replacements were performed according to the scoring system described by Feil et al. (2008) Hum. Reprod. 23(7): 1505-10 with degree of compaction and early cavitation being assessed. Blastocyst development on day 5 was assessed as per the grading system described by Gardner and Schoolcraft (1999) Curr. Opin. Obstet. Gynecol. 1 1 (3): 307- 1 1 , describing degree of expansion and quality of the inner cell mass and trophectoderm. Independent of the day of transfer embryos were scored using a scale of 1-4, where grade 1 indicates best quality and grade 4 indicates poor quality. Embryos selected for transfer were incubated in EmbryoGlue (Vitrolife) for 0.5-4h before transfer and transferred into the uterus in a volume of approximately 10μΙ, under ultrasound guidance.

Pregnancy Outcomes

Clinical pregnancy was determined by the presence of a foetal heart beat 6 weeks following embryo transfer and was retrospectively analysed. Pregnancy outcomes were obtained from the obstetrician in charge of the patient care. Obstetricians were asked to provide delivery dates, birth weights, sex and whether there were any maternal or neonatal interventions or complications.

Statistical Analysis

Data are presented as dot plots or box and whisker plots where the horizontal line represents the median copy number and the box encompasses 50% of data points (first quartile to third quartile). Data points within the 10th and 90th percentiles are represented by the upper adjacent value and lower adjacent whiskers (vertical lines). Values lower than the first percentile and greater than the 99th percentile are indicated by closed dots. Data is normalised to GAPDH. Pregnancy outcomes were analysed using a Wilcoxon-Mann-Whitney test. Differences were considered significant at a p- value < 0.05.

Results

Patient Demographics

Patient demographic characteristics are shown in Table 1 . All data are presented as mean ± SEM. There was no significant difference in maternal age between treatment groups of patients who had a live birth compared with those which failed to establish pregnancy (32.9±1 .13 and 33.6±0.64 years, respectively). The number of previous cycles undergone by patients, the rates of ICSI and IVF, the number of oocytes collected and rate of fertilization in the different embryo transfer day groups were also not significantly different. Patients had an average of 12±1 .0 oocytes collected and 91 % of patients underwent ICSI as the insemination method. All patients had a single embryo transferred.

TABLE 1

Patient demographic characteristics for thirty eight patients recruited to investigate cumulus cell markers of oocyte quality and pregnancy success.

(Age data is presented as years ± SEM. All other data is presented as mean ± SEM) Cumulus Cell Gene Expression as a Biomarker of Pregnancy Success, Pregnancy Outcome and Birth Weight

A total of 38 trimmed cumulus masses from patients (pregnant n = 12 or non-pregnant n = 26) were subjected to gene expression analysis to test for correlations with successful pregnancy and/or birth weight. All patients had individual oocyte and embryo culture and underwent a single embryo transfer.

As shown in Figure 1 , cumulus cell gene expression analysis showed GAS5 expression was significantly positively correlated (p = 0.002) with pregnancy success. Analysis of different snoRNAs from the GAS5 region revealed that they are consistently lower in pregnant versus non-pregnant patient cumulus RNA (Figure 2A). The arrangement of the snoRNAs encoded within the human GAS5 gene are shown in Figure 2B. As indicated in Figure 2A, the expression of SNORD 47, SNORD 78, SNORD 44 and SNORD 76 negatively correlates with pregnancy success. Investigation of cumulus cell gene expression also revealed that VCAN (Figure 3) and PTGS2 (Figure 4) median relative gene copy numbers were significantly higher (p < 0.02) compared to cumulus cell gene expression from oocytes which failed to result in a successful pregnancy. Cumulus cell PTX3 median relative gene copy numbers showed a trend (p = 0.066) towards significance with a higher relative copy number in cumulus cells from oocytes which resulted in a successful pregnancy and live birth (Figure 5). In cumulus cells from oocytes which resulted in a live birth when compared to cumulus cells not associated with pregnancy the median relative gene copy number of VCAN (Figure 3) was found to be 1.9-fold higher (0.4553 vs. 0.239 respectively), PTGS2 (Figure 4) was 1.82-fold higher (0.326 vs. 0.1788 respectively) and PTX3 (Figure 5) was 1 .74-fold higher (3.1 15 vs. 1.787 respectively).

Cumulus cell gene expression analysis also demonstrated positive correlations between birth weight and VCAN (Figure 6), GREM1 (Figure 7) and PFKP (Figure 8) expression. Additional genes analysed included the matrix genes HAS2 and TNFAIP6 which showed no significant difference in the median relative copy numbers between pregnant and non-pregnant patients (data not shown). There was no significant difference in the expression of the signalling genes STS and AHR between the pregnant and non-pregnant patient cohorts (data not shown). Surprisingly cumulus cell median copy numbers of ALDOA, PFKP, LDHA and PKM2 genes which encode rate limiting enzymes of the glycolytic metabolic pathway were not significantly different between patients with a successful pregnancy and those which were not pregnant (data not shown). No significant correlation was found linking maternal age or Body Mass Index (BMI) with cumulus cell gene expression and live birth (data not shown).

EXAMPLE 2

Gene Expression Analysis - Study 2

A different sample set of patients was used in the study compared to study 1 above. The study 2 sample set included a total of 83 female patients undergoing routine IVF/ICSI with Single Embryo Transfer (SET) at Repromed, Dulwich, South Australia between February and September 201 1. Patients with clinical indications of polycystic ovary syndrome (PCOS) and diminished ovarian reserve (DOR) were included in this study. Furthermore, patients in which infertility diagnosis related solely to sperm quality ("Male Factor Only" cohort) were also included in this study. The majority of patients had treatment with their own gametes, with the remaining patients making use of donor sperm. All patients were not undergoing pre-implantation genetic diagnosis. All other methods used for study 2 are as described above with respect to study 1.

As shown in Figure 9, in the women selected for Male Factor Only infertility (35 patients), a significant (p = 0.037) 3-fold elevation in median GAS5 expression was found in cumulus cells from oocytes that established successful pregnancy compared to those that failed. Similarly, Figure 10 shows that in the same group of women, a 4- fold higher median level of VCAN expression was found in cumulus cells from oocytes that achieved pregnancy compared to those that failed. Finally, in the same group of women, a significant (p = 0.041 ) 4-fold elevation in median PTX3 expression was found in cumulus cells from oocytes that established successful pregnancy compared to those that failed (Figure 1 1 ).

When the patient group was considered as a whole, as shown in Figure 12 a significant (p = 0.023) 3-fold elevation in median PTGS2 expression was found in cumulus cells from oocytes that established successful pregnancy compared to those that failed. Similarly, as shown in Figure 13, a 3-fold elevation in median PTX3 expression was found in cumulus cells from oocytes that established successful pregnancy compared to those that failed (p = 0.07). EXAMPLE 3

Gene Expression Analysis - Study 3

Microarray analysis was also used to identify genes, the expression of which, are an indicator of the developmental competence of an oocyte. Cumulus cell RNA was obtained from at least three patients in study 1 using the methods described above was pooled into six pools of RNA representing three replicate samples from individual oocytes that either resulted in pregnancy success (n=3) or no pregnancy (n=3). Patients were allocated to pools based on the amount of RNA sample available. cDNA was synthesised from the RNA samples then labelled with Cy5. Labelled cDNA was hybridised overnight at 42°C with Affymetrix GeneChip 1.0 ST Array System for Human. The arrays were then washed by immersion in 0.2 x SSC, 0.01 % SDS for 2 x 5 mins then in 0.2 x SSC for 3 mins. Analysis was performed using Partek Genomics Suite as per the manufacturer's software instructions. For the data set background correction was performed by robust multichip averaging (RMA) followed by probe affinity adjustment (based on the probe GC content). Quantile normalisation was performed to make the arrays comparable, and a mean probeset summary was obtained by averaging the intensity values for the multiple probes representing a gene. Linear modelling statistics using a mixed model Analysis of Variance (ANOVA) were performed and an adjusted p-value obtained. As shown in Table 2, probes for the three Pepsinogen isozymogens {PGA3I PGA4I PGA5) all showed similar highly increased expression in the pregnancy success samples compared to the no pregnancy samples. TABLE 2

Differential expression of the pepsinogen metagene between human cumulus cells from pregnant vs. non-pregnant patients

Gene Gene Fold logFC t B (log t-test GenBank

Symbol Change odds) p-value Accession No.

Pepsinogen 3 PGA3 2.05 1.03 7.44 -3.65 0.0007 NM_001079807

Pepsinogen 4 PGA4 1.99 0.99 6.94 -3.67 0.001 NM_001079808

Pepsinogen 5 PGA5 1.88 0.91 6.36 -3.70 0.001 NM_014224

Results from microarray analysis of human cumulus cell samples from oocytes that proceeded to successful pregnancy vs those that failed to establish pregnancy.

Specifically, expression of PGA3, PGA4 and PGA5 was up to 2.05-fold higher in cumulus cells from patients with a successful pregnancy outcome. The three isozymogens of human Pepsinogen are known to be co-expressed transcripts from consecutive genes forming a metagene on Chromosome 1 1 q 13.

The reproducibility of this result supports the conclusion that expression of the Pepsinogen genes is positively associated with ART pregnancy success. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Also, it must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise. A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Claims

1 . A method of assessing developmental competence of an oocyte, the method comprising:

measuring expression of a gene in the one or more cumulus cells, wherein the gene is selected from one or more of Growth Arrest-Specific transcript 5 {GAS5), Versican ( VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1), Phosphofructokinase Platelet (PFKP), Pentraxin 3 (PTX3), Pepsinogen 3 (PGA3), Pepsinogen 4 (PGA4), and Pepsinogen 5 (PGA5),

2. A method of assessing developmental competence of an oocyte, the method comprising:

measuring expression of a gene in the one or more cumulus cells, wherein the gene is selected from one or more of Growth Arrest-Specific transcript 5 (GAS5), Versican { VCAN), Prostaglandin Synthase 2 (PTGS2), Gremlin 1 (GREM1), Phosphofructokinase Platelet (PFKP), and Pentraxin 3 (PTX3), Pepsinogen 3 (PGA3), Pepsinogen 4 (PGA4), and Pepsinogen 5 (PGA5),

3. The method of claim 1 or claim 2, wherein the gene is GAS5.

4. The method of any one of claims 1 to 3, wherein the GAS5 gene is a human GAS5 gene.

5. The method of any one of claims 1 to 4, wherein measuring expression of the GAS5 gene comprises measuring an expression level of a GAS5 mRNA.

6. The method of claim 5, wherein the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the GAS5 mRNA is positively correlated with pregnancy success.

7. The method of claim 5 or claim 6, wherein the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the GAS5 mRNA is positively correlated with probability of a live birth.

8. The method of any one of claims 1 to 4, wherein measuring expression of the GAS5 gene comprises measuring an expression level of one or more small nucleolar RNAs (snoRNAs) derived from the GAS5 gene.

9. The method of claim 8, wherein the one or more snoRNAs comprise a sequence as set forth in any one of GenBank ID NOs: NR_002764 (SNORD47), NR_003944 (SNORD78), NR_002750 (SNORD44), NR_003942 (SNORD76), NR_003940 (SNORD 80), NR_003943 (SNORD77).

10. The method of claim 8 or claim 9, wherein the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the one or more snoRNAs is negatively correlated with pregnancy success.

1 1 . The method of claim 1 or claim 2, wherein the gene is VCAN.

12. The method of claim 1 1 , wherein the VCAN gene is a human VCAN gene.

13. The method of claim 1 1 or claim 12, wherein measuring expression of the VCAN gene comprises measuring an expression level of a VCAN mRNA.

14. The method of claim 13, wherein the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the VCAN mRNA is positively correlated with pregnancy success.

15. The method of claim 13 or claim 14, wherein the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the VCAN mRNA is positively correlated with probability of a live birth.

16. The method of any one of claims 13 to 15, wherein the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the VCAN mRNA is positively correlated with birth weight.

17. The method of claim 1 or claim 2, wherein the gene is PTGS2.

18. The method of claim 17, wherein the PTGS2 gene is a human PTGS2 gene.

19. The method of claim 17 or claim 18, wherein measuring expression of the PTGS2 gene comprises measuring an expression level of a PTGS2 mRNA.

20. The method of claim 19, wherein the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the PTGS2 mRNA is positively correlated with pregnancy success.

21 . The method of claim 19 or claim 20, wherein the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the PTGS2 mRNA is positively correlated with probability of a live birth.

22. The method of claim 1 or claim 2, wherein the gene is GREM1.

23. The method of claim 22, wherein the GREM1 gene is a human GREM1 gene.

24. The method of claim 22 or claim 23, wherein measuring expression of the GREM1 gene comprises measuring an expression level of a GREM1 mRNA.

25. The method of claim 24, wherein the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the GREM1 mRNA is positively correlated with birth weight.

26. The method of claim 1 or claim 2, wherein the gene is PFKP.

27. The method of claim 26, wherein the PFKP gene is a human PFKP gene.

28. The method of claim 26 or claim 27, wherein measuring expression of the PFKP gene comprises measuring an expression level of a PFKP mRNA.

29. The method of claim 28, wherein the assessed developmental competence of the oocyte is birth weight following fertilisation of the oocyte and subsequent gestation, and the expression level of the PFKP mRNA is positively correlated with birth weight.

30. The method of claim 1 or claim 2, wherein the gene is PTX3.

31 . The method of claim 30, wherein the PTX3 gene is a human PTX3 gene.

32. The method of claim 30 or claim 31 , wherein measuring expression of the PTX3 gene comprises measuring an expression level of a PTX3 mRNA.

33. The method of claim 32, wherein the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of the PTX3 mRNA is positively correlated with pregnancy success.

34. The method of claim 32 or claim 33, wherein the assessed developmental competence of the oocyte is the probability of a live birth following fertilisation of the oocyte and subsequent gestation, and the expression level of the PTX3 mRNA is positively correlated with probability of a live birth.

35. The method of claim 1 or claim 2, wherein the gene is selected from one or more of PGA3, PGA4 and PGA5.

36. The method of claim 35, wherein the PGA3, PGA4 and PGA5 genes are human PGA3, PGA4 and PGA5 genes.

37. The method of claim 35 or claim 36, wherein measuring expression of one or more of the PGA3, PGA4 and PGA5 genes comprises measuring an expression level of a PGA3, PGA4 and/or PGA5 mRNA.

38. The method of claim 37, wherein the assessed developmental competence of the oocyte is pregnancy success following fertilisation of the oocyte, and the expression level of one or more of the PGA3, PGA4 and PGA5 mRNAs is positively correlated with pregnancy success.

39. The method of any one of claims 1 to 38, wherein the one or more cumulus cells are isolated from the cumulus oocyte complex prior to measuring expression of the gene in the one or more cumulus cells.

40. The method of claim 39, wherein the cumulus oocyte complex is cultured prior to isolation of the one or more cumulus cells.

41 . The method of any one of claims 1 to 40, wherein the cumulus oocyte complex is cultured prior to measuring expression of the gene in the one or more cumulus cells.

42. The method of any one of claims 1 to 41 , wherein the method further comprises selecting an oocyte for fertilisation or transfer on the basis of the assessed developmental competence of the oocyte.

43. The method of any one of claims 1 to 42, wherein the method comprises part of an assisted reproductive technology.

44. The method of claim 43, wherein the assisted reproductive technology comprises in vitro fertilisation.

45. The method of claim 43, wherein the assisted reproductive technology comprises intracytoplasmic sperm injection.

46. The method of any one of claims 1 to 45, wherein the assessed developmental competence of the oocyte is positively correlated with the developmental competence of an embryo derived from the oocyte.

A method of assisted reproduction, the method comprising:

(a) selecting an oocyte according to the method of claim 42;

(b) fertilising the selected oocyte to form a zygote;

(c) culturing the zygote to form an embryo; and

(d) implanting the embryo into a subject.

48. The method of claim 47, wherein the subject is a non-human animal.