WO2017064482A1

WO2017064482A1 - Methods and compositions

Info

Publication number: WO2017064482A1
Application number: PCT/GB2016/053160
Authority: WO
Inventors: Ali Akbar FOULADI-NASHTA; Geraldine Mary HARTSHORNE
Original assignee: The Royal Veterinary College
Priority date: 2015-10-14
Filing date: 2016-10-12
Publication date: 2017-04-20

Abstract

Low molecular weight hyaluronic acid (LMWHA) or very low molecular weight hyaluronic acid (VLMWHA) or PH20 for use in the culture of an embryo, zygote, blastocyst and/or an oocyte. A composition comprising low molecular weight hyaluronic acid (LMWHA) and/or very low molecular weight hyaluronic acid (VLMWHA) and/or PH20 may be used in the culture of an embryo, zygote, blastocyst and/or an oocyte. The LMWHA may be between 15kDa and 40kDa in size. The VLMWHA may be <10kDa, optionally the VLMWHA is a tetrasaccharide or a hexasaccharide, optionally the VLMWHA is 4-8 monosaccharides or less in size. A method for selecting an embryo or blastocyst generated through assisted fertilisation, optionally in vitro fertilisation or ICSI, as suitable for implantation, wherein the method comprises determination of the level of hyal-2 in the culture medium used to culture the embryo or blastocyst.

Description

Methods and Compositions

Assisted conception in humans is highly commercial and expanding rapidly. In the UK, approximately 1.8% of all newborns are conceived in vitro. The cost per completed IVF (in vitro fertilisation) or ICSI (intra-cytoplasmic sperm injection) cycle in UK is estimated at an average of £3375 (NICE, 2013) and the average 'take home baby rate' per cycle is 25.6% (in 2010, the latest complete national dataset). In addition to NHS expenditure, individuals often pay for their own treatment if they fall outside NHS funding criteria for IVF, for example due to age, previous attempts, body mass index, or having had a child already. Such privately funded cycles account for 60% of the total. Nearly 62,000 cycles of IVF treatment took place in the UK in 201 1 and the number has increased year on year since records began. Internationally, assisted conception is increasing rapidly, including in the developing world. In addition to human assisted conception, similar embryo transfer procedures are used in many animals for a range of purposes, for example to produce genetic modifications, breeding of high value animals by transfer into lower value gestational carriers and preservation of exotic species and rare breeds. The financial investment in such work is substantial and any increase in implantation rates would be valuable and welcome.

It is widely appreciated that the development of an IVF system and medium that closely mimics the natural fertilization process represents the ideal, but it is less clear how to achieve this. For example, culture of IVF-produced embryos in the oviduct improves development to the blastocyst stage (Lazzari et al., 2010) compared to those cultured using standard IVF techniques. This can potentially be attributed to a number of supporting factors present in the oviduct and absent from current embryo culture media.

Some factors which are found in the natural environment of the oocyte and developing embryo have been described. Among those factors is hyaluronic acid (HA), a ubiquitous component of the extracellular matrix of vertebrates. HA is present in the follicular, oviductal and uterine fluids and is known to play an important role in the regulation of many reproductive events like ovulation (Salustri et al., 1989), fertilization (Shimada et al., 2008) and embryogenesis (Camenisch et al., 2000). HA is a linear polysaccharide which belongs to the family of glycosaminoglycans (GAGs). It is composed of repeating alternating units of d-glucuronic acid (GlcUA) and N-acetyl-d- glucosamine (GlcNAc) forming up to 25 000 disaccharides with a molecular mass of up to 10 million Da (Laurent, 1998). HA is synthesized by hyaluronic acid-synthase (HAS) enzymes (Itano and Kimata, 2002) located at the inner cytoplasmic face of the plasma membrane which adds units of GlcUA and GlcNAc while extruding the HA chain through the membrane during its synthesis to the outside of the cell (Laurent, 1998). There are three different mammalian HASs which synthesize HA chains with different molecular stability average lengths (molecular sizes) and different biological functions (Dougherty and van de Rijn, 1993, Crater and van de Rijn, 1995, Itano and Kimata, 2002, Stern, 2005). HAS1 synthesises HA with a molecular weight of 0.2-2 million Daltons; HAS2 synthesises HA with a molecular weight of 2 to 10 million Daltons; and HAS3 synthesises HA with a molecular weight of 0.1 to 1 million Daltons.

Polymer size, location and concentration govern the function of HA (Stem et al., 2006). The functions of high-molecular-weight (HMVV) HA polymers (>1000-5000 disaccharides) are mainly attributed to the hydrodynamic properties of the molecule. HMW HA is a space- filling hydrating molecule that impedes cell differentiation and it is anti-angiogenic (Feinberg and Beebe, 1983) and immunosuppressive (McBride, 1979, Delmage et al., 1986). On the other hand, the functions of low-molecular-weight (LMW) HA are mainly mediated through interaction with HA receptors, like CD44 and receptor for HA-mediated motility (RHAMM). CD44 is a cell surface glycoprotein and is involved in cell signaling. RHAMM is a receptor for HA mediated motility. Other relevant receptors include HARE - which is a HA receptor for endocytosis and ICAM-1 (CD54). Moreover, the interaction between HA and CD44 has been shown to induce cell signalling (Ohno-Nakahara et al., 2004, Toole, 2001 ), involving multiple signalling pathways like Rac1 -mitogen-activated protein kinase (MAPK), PI3-AKT and NFkB (Toole, 2001 ), and in some cases, influences cell proliferation (Bourguignon et al., 1997).

LMW HA competes with the larger molecules for receptors and acts in an opposing manner; they are anti-apoptotic and function as a survival factor and stimulate cell proliferation (Xu et al., 2002). LMW HAs are also angiogenic (West et al., 1985) and immunostimulatory molecules (Alaniz et al., 201 1 ).

HA has a high rate of turnover at cellular and tissue levels, mainly by the enzymatic hydrolysis by hyaluronidases (HYALs) which include HYAL1 , HYAL-2, HYAL3, HYAL4, HYALP1 and PH-20. Predominantly, HYAL1 and HYAL-2 are considered the major HA- degrading enzymes in somatic tissue, whereas PH-20 is abundant in spermatozoa (Bastow et al., 2008). As formulated by Stern (Stern, 2004, Stern, 2003), a sequence of enzymatic reactions cleave the HMW HA progressively generating HA fragments of decreasing sizes.

HYAL-2 is a glycosylphosphatidylinositol-anchored enzyme attached to the external surface of the plasma membrane expressed in many tissues (Lepperdinger et al., 2001 ). HYAL-2 initially cleaves HMW HA into fragments -20 000 Da (50-60 saccharides) in size. CD44-HYAL-2 interaction facilitates the endocytosis of HA which undergoes further degradation by lysosomal HYAL1 into smaller HA fragments (4-8 saccharides in size) enabling cellular migration, proliferation and mitosis (Lepperdinger et al., 2001 ).

Hyal1 is expressed in all stage embryos. However, its level of expression reduces in morula and blastocyst stages (in contrast to Hyal-2 which starts to be expressed at these stages). HyaM is located in the lysosome, along with two other enzmes, β-Ν- acetylglucosaminidase and β-glucoronidase. HyaM in conjunction with these two enzymes produces 800Dalton HA tetrasacharides which move to the cytoplasm. β-Ν- acetylglucosaminidase and β-glucoronidase are also present in the cytoplasm and break the HA tetrasaccharides further to 200 Dalton monosaccharides before breaking it to single sugars (Stern, 2004). Hyal 1 inhibition does not affect blastocyst development, as demonstrated by the work of the inventors, disclosed herein. PH-20 is a GPI-anchored hyaluronidase (also known as SPAM-1 ) which depolymerises HA into very small tetrasaccharide and hexasaccharide products (Thompson et al., 2010, Hofinger et al., 2008, Kim et al., 2005). PH-20 is located on the sperm surface and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH-20 facilitates penetration of sperm through the cumulus ECM and zona pellucida of the ovum, and is also necessary for fertilization.

Several studies have confirmed that PH-20 is the only hyaluronidase in mammalian sperm, including the sperm of guinea pigs, rats, macaques and humans (Cherr et al., 2001 , Zheng et al., 2001 ). It is originally synthesized as a polypeptide with an apparent molecular weight of 64 kDa. During the course of sperm maturation, part of PH-20 is processed into two fragments that are linked through disulfide bridges, such as one at the N-terminal domain of 41-48 kDa and at the C-terminal domain of 27 kDa. PH-20 is unique among hyaluronidases, in that it shows enzyme activity at both acidic and neutral pH and that these activities appear to involve two different domains in the protein (Gmachl and Kreil, 1993, Cherr et al., 2001 ). PH-20 is only produced by sperm. During ovulation and fertilisation the dominant hormone affecting the reproductive system is progesterone. Progesterone production results in stimulation of HAS-3, which produces smaller HA molecules (<1000KDa) whilst upregulating Hyal-2 expression in the oviduct which breaks HA to 20 KDa. Therefore, it is considered that in vivo, embryos are exposed mainly to small sized HA sizes.

Recently, the inventors have shown that HA is made during all stages of embryogenesis, and that despite hyaluronidase-2 (Hyal-2) being expressed in the oviduct, embryos do not express Hyal-2 until the morula and blastocyst stages (Marei et al., 2013). Therefore, during the early stages of development of a naturally fertilized embryo, the embryo is exposed to Hyal-2 from the oviduct at a time when it cannot produce its own. Exposure to Hyal-2 at the early stages of an IVF embryo would not occur under standard conditions. In addition, the culture medium is renewed approximately every 48 hours in standard IVF procedures, resulting in any HA made by the embryo being washed away. The inventors found that Hyal-2 supplementation of embryo culture media significantly improved blastocyst formation (1.6-2x) and enhanced quality in cow/sheep embryos, whilst in vivo infusion of Hyal-2 into sheep oviducts resulted in 1.4x increased blastocyst formation (Size Specific Effects of Hyloronan (HA) on Sheep Blastocyst Formation and Quality In Vivo Waleed Fawzy Marei and Ali A. Fouladi-Nashta (2012). BIOLOGY OF REPRODUCTION 85 : 237; Critical role of hyaluronan system in pre-implantation embryo development and establishment of pregnancy 2013, Fouladi-Nashta and Marei, Int Journal of Fertility and Sterility 7: page 34; Critical role of hyaluronidase-2 during preimplantation embryo development 2013 Marie et al Molecular Human Reproduction 19: 590-599)) Further detailed understanding of the precise composition of the environment of the embryo, at various stages of development, can lead to improvements in IVF technique, resulting in an increased number of better quality embryos with potential benefits including higher success rate; reduced need for transfer of multiple embryos which often result in unwanted multiple pregnancy in and may risk mother's health; and a reduction in cost and emotional stress to those involved.

Brief summary of the invention

The inventors of the present invention have surprisingly found that low molecular weight HA (LMWHA), and in particular very low molecular weight HA (VLMWHA) and combinations of the two, has a particularly beneficial effect on the development of the embryos to blastocyst stage and embryo quality, an effect which could not have been predicted from the prior art.

Before the current invention, it was known that the developing blastocyst is exposed in vivo to Hyal-2 produced by the oviduct, and that addition of the Hyal-2 to IVF culture media enhanced the development of the blastocyst. However, there was no reason to suppose that the in vitro effect, or the native in vivo role of Hyal-2, was due to the production of LMWHA (approximately 25KDa as produced by Hyal-2) from HMWHA, the reduction in the amount of HMWHA by Hyal-2, or some other effect of Hyal-2.

For example, it is known that HMWHA at the uterus hinders implantation and it is possible that HMWHA at the blastocyst may have a similar role, suggesting that the role of Hyal-2 in the oviduct (and the effects of supplementation of Hyal-2 in culture medium) may be to degrade the HMWHA in order to, for example, aid implantation. (Critical role of hyaluronan system in pre-implantation embryo development and establishment of pregnancy 2013 Fouladi and Marei, Int Journal of Fertility and Sterility 7: page 34). However, the present inventors have shown that the addition of LMWHA to the embryo culture medium has significant benefits on blastocyst number and quality, suggesting that although the above may be one factor in the improvement of blastocyst quality and number when treated with Hyal-2, that is not the only factor, and the small sized HA fragments produced by Hyal-2 have their own role in the process..

Thus, rather than the in vitro role of Hyal-2 being the reduction in amount of HMWHA (though this still may be of importance), it is considered that the actual generation of the smaller HA fragments is key. It is also possible that, as the HA receptor in the cell membrane is co-localised with Hyal-2, that the complex formed between HA, CD-44 and Hyal-2 creates a signal which benefits embryo development. The ability to bypass the use of enzyme additivities in culture media, which are prone to degradation and loss of function, has clear advantages over the potentially expensive or time consuming production and purification of the Hyal-2 enzyme.

The inventors have further shown that the inclusion of VLMWHA (4-8 monosaccharaides in culture media has an even greater effect than that of the LMWHA. The only hyaluronidase capable of producing such a small polymer is PH20, which is only found in the acrosome of sperm. PH20 in the sperm was considered, before the present invention, to aid in penetration of the oocyte, with no further role in development associated with the enzyme and no role associated to sperm other than the one that fertilises the oocyte. Without wishing to be bound by any theory, the inventors propose that the additional sperm present in the oviduct (beyond the single sperm that fertilises the oocyte) may have a key role in the proper and timely development of the embryo, as addition of either PH20 or VLMWHA to embryo culture media has been shown by the inventors to have a significant effect on blastocyst development and quality. However, this effect appears to be dose restricted, with only low doses of PH20 having an effect. This may mimic the effect in vivo, where it is not expected that the developing blastocyst will be exposed to a high dose of PH20.

Additionally, the inventors have found that a combination of both VLMWHA and LMWHA has an even greater beneficial effect. This is more likely to mimic the environment of a naturally fertilised oocyte, wherein Hyal-2 produced by the oviduct acts on HA, producing LMWHA, whilst at the same time the sperm carrying PH20 will, for the lifetime of the sperm, release PH20 into the environment, exposing the developing blastocyst to VLMWHA. Thus it appears that the true natural environment of the early embryo will contain both LMWHA and VLMWHA and mimicking this in IVF culture conditions is considered to improve both number of fertilised oocytes developing to the blastocyst stage, and the blastocyst quality. Moreover, the addition of PH20 resulted in better hatching, an essential step in embryogenesis and implantation

Thus the present invention provides agents and combinations of agents, which include fragments of HA and hyaluronidase enzymes that can be used as additives or supplements or core ingredients of media used for assisted fertilisation/conception, such as IVF and ICSI and related procedures. The invention also provides methods of culture and methods of IVF and ICSI.

It will be appreciated that following the in vitro step of fertilisation of an oocyte or the step of intra-cytoplasmic sperm injection, for example, the developing zygote/blastocyst/embryo requires culture until such a time as it is considered appropriate for the embryo to be implanted. References herein to assisted fertilisation or assisted conception, for example to IVF or ICSI, refer (unless the context requires otherwise) to the process that incorporates the in vitro step of fertilisation of an oocyte or the step of intra- cytoplasmic sperm injection, for example, not just to the step of fertilisation or the step of intra-cytoplasmic sperm injection. Thus, references herein to assisted fertilisation or assisted conception, for example to IVF or ICSI, relate to the culture of the zygote/blastocyst/embryo which results from the actual fertilisation or intra-cytoplasmic sperm injection step. Accordingly, the term IVF, for example, encompasses not only the step of fertilisation of the oocyte, but also the culture of the developing zygote/blastocyst/embryo. The inventors have also found that hyal-2 produced by the developing embryo can be detected in the culture media, and that the level of hyal-2 produced positively correlates with the quality of the embryo and the likelihood of pregnancy resulting from embryo transfer. This thus represents an easy and importantly non-invasive method to aid in assessing the quality of embryos prior to implantation. Further, the inventors have also surprisingly found that embryos produced via IVF rather than ICSI may produce more hyal- 2, and may therefore also be more likely to result in pregnancy.

Detailed description of the invention During an assisted fertilisation/conception procedure, such as the IVF procedure, oocytes typically are stripped of the surrounding loose and expanded cumulus cells (rich in HMWHA) prior to incubation with sperm to facilitate fertilisation (day 0). Stripping of the cumulus cells is generally accomplished by the use of Hyal-2. One day after fertilisation (24h, day 1 ), the presumptive zygotes and early cleaved embryos are completely denuded from the remaining cumulus cells (further removal of hyaluronan) and transferred into embryo culture medium. The culture media is then renewed every 48h on day 3 and 5. Embryos develop to 4 or 8 cells within approximately 48 hours, and reach the morula stage (32+ cells) on day 4 and blastocyst stage 5-7 days post fertilisation (variable in species). The embryos are then screened for suitability for implantation, using techniques such as assessment of the morphology, and division pattern where time lapse microscopy is used.

There are therefore several stages in which the gametes or developing embryos are incubated in artificial media. The inventors of the present invention have found that addition of LMWHA and/or VLMWHA and/or PH20 to the culture medium results in high numbers of zygotes reaching the blastocyst stage, better quality of blastocysts, and better hatching rates. The invention provides LMWHA and/or VLMWHA and/or PH20 for use in the culture of all of an oocyte, a zygote, an embryo and/or a blastocyst. By this we mean that at all stages of the culture of an oocyte, a zygote, a blastocyst and/or an embryo, for example one that results from assisted conception such as IVF or ICSI, until implantation, LMWHA and/or VLMWHA and/or PH20 can be used to better simulate the environment of a natural conception. In a first aspect, therefore, the invention provides LMWHA and/or VLMWHA and/or PH20 for use in the culture of an oocyte, a zygote, an embryo, and/or a blastocyst. LMWHA and/or VLMWHA and/or PH20 are collectively herein termed agents of the invention. The invention also provides a composition comprising LMWHA and/or VLMWHA and/or PH20. The invention further provides a composition comprising LMWHA and/or VLMWHA and/or PH20 for use in the culture of an oocyte, a zygote, an embryo, and/or a blastocyst. Compositions comprising LMWHA and/or VLMWHA and/or PH20 are herein collectively termed compositions of the invention. For the avoidance of doubt, in this and other aspects of the invention, the invention typically concerns culture of a zygote, a pre-implantation embryo (from 4+cell stage to blastocyst) rather than the culture of an oocyte, as will be apparent herein.

Similarly, the invention provides a method of culturing an oocyte, a zygote, an embryo and/or a blastocyst comprising culture in the presence of LMWHA and/or VLMWHA and/or PH20, and also provides LMWHA and/or VLMWHA and/or PH20 for use in the manufacture of a medicament for the treatment of infertility, wherein the LMWHA and/or VLMWHA and/or PH20 is for use in a method of culturing an oocyte, a zygote, an embryo or a blastocyst.

In one embodiment the composition comprises LMWHA and not VLMWHA and PH20. In another embodiment the composition comprises VLMWHA and not LMWHA and PH20. In a further embodiment the composition comprises PH20 and not LMWHA and VLMWHA. In yet another embodiment the composition comprises both LMWHA and VLMWHA and not PH20. In another embodiment the composition comprises LMWHA and PH20 and not VLMWHA. In another embodiment the composition comprises VLMWHA and PH20 and not LWMHA. In a further embodiment the composition comprises all of VLMWHA and LMWHA and PH20.

In another or further embodiment, the composition comprises one or more additional agents, such as Hyal-2 and/or hyaluronic acid (for example hyaluronic acid that is suitable for use as a substrate for PH20 and/or Hyal-2). For example the composition may comprise hyaluronic acid of a size which does not adversely affect development, but which provides a suitable substrate for PH20 and Hyal-2. All of the compositions may comprise one or more additional agents, further agents, such as Hyal-2 and/or hyaluronic acid suitable for use as a substrate for PH20 and/or Hyal-2. By low molecular weight hyaluronic acid (LMWHA) we include the meaning of hyaluronic acid with a molecular weight of less than about 40 kDa but more than about 10KDa. For example LMWHA may be hyaluronic acid with a molecular weight of between 15kDa and 40kDa, for example between 20kDa and 30kDa, for example 25 kDa. Typically, LMWHA acid is about 20kDa or 25kDa in weight. LMWHA can be purchased from commercial supplies, for example from R&D Systems (catalog number GLR001 , which has a size of 20 kDa). Hyaluronic acid which is only several saccharides or disaccharides in length, for example similar to that produced by the action of PH20, is referred to herein as very low molecular weight hyaluronic acid (VLMWHA). This includes the hyaluronic acid oligosaccharides (tetrasaccharide or hexasaccharide) referred to elsewhere, and may be as little as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 saccharides in length. VLMWHA for example may be less than about 10KDa. In one embodiment VLMWHA is a tetrasaccharide and/or hexasaccharide. In one embodiment the VLMWHA is less than about 10kDa. VLMWHA can be purchased from commercial supplies, for example from R&D Systems (catalog number GLR003, which has a size of 4.8 kDa). Any of the hyaluronic acids disclosed herein, for example HMWHA, LMWHA and VLMWHA may be modified by ay modification known in the art, for example the HA may be cationic HA (which is commonly used in transfections) or N-acetylated HA (which modulates the pro-inflammatory cytokine production and has a therapeutic effect). It is considered that particular modifications may have a beneficial effect.

By PH20 we include the meaning of a PH20 enzyme from any species. The skilled person will appreciate that a PH20 enzyme is considered useful if it has the activity of a PH20 hyaluronidase, i.e. is capable of producing VLMWHA as defined above. It will be appreciated that the actual sequence of the polypeptide can deviate, sometimes significantly, whilst still retaining appreciable enzymatic activity. For example, although the human PH20 enzyme has the sequence of SEQ ID NO:1 , a polypeptide with a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%. 99% or 100% identity to this sequence is still considered to be a PH20 enzyme, providing it has hyaluronidase activity. Therefore, the term PH20 is to be taken to encompass all PH20 enzymes from all species, including those polypeptides that show at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%. 99% or 100% identity to the naturally occurring enzyme. Therefore, in one embodiment, the PH20 is defined as a protein or polypeptides with PH20-like activity. The skilled person will be readily able to determine whether a given protein has PH20-like activity, by, for example, assessing the ability of the protein to cleave HA into, for example, tetrasaccharide or hexasaccharides. In another embodiment, PH20 is defined as a protein capable of producing VLMWHA.

The PH20 enzyme of the invention may be homogeneously produced, i.e. produced by the native organism from which the PH20 is derived. Or in other embodiments the PH20 enzyme of the invention may be heterogeneously produced, i.e. produced by an organism which is not the native organism of the PH20 enzyme. In a further embodiment the PH20 enzyme may be synthetically produced.

It is considered that PH-20 from a different species to the oocyte, zygote, embryo and/or blastocyst is useful in the compositions and methods of the invention. Therefore, in one embodiment, the PH20 is from a species other than that of the oocyte, zygote, embryo and/or blastocyst. In a preferred embodiment, the PH20 is from a species that is the same as the oocyte, zygote, embryo and/or blastocyst. Accordingly by PH20 we include the meaning of any sperm hyaluronidase. For example, the hyaluronidase may be from any species. It is well within the skilled person's abilities to be able to identify PH20 enzymes.

The PH20 may be, for example, a recombinant bovine SPAM1 protein (Accession AAI10184). It has 91 % similarity to sheep and 78.7% similarity to human. Additional non- limiting examples of PH20 enzymes that are considered to be useful include: Mouse (Accession 20690 ) sperm adhesion molecule 1 ; Rat (Accession 117037 ) sperm adhesion molecule 1 (PH-20 hyaluronidase, zona pellucida binding); Zebrafish (Accession 791203 ) sperm adhesion molecule 1 ; chicken (Accession 417749 ) sperm adhesion molecule 1 (PH-20 hyaluronidase, zona pellucida binding); dog (Accession 47521 1 ) sperm adhesion molecule 1 (PH-20 hyaluronidase, zona pellucida binding); domestic cat (Accession 101095731 ) sperm adhesion molecule 1 (PH-20 hyaluronidase, zona pellucida binding); Horse (Accession 100051243 ) sperm adhesion molecule 1 (PH-20 hyaluronidase, zona pellucida binding); Domestic Rabbit (Accession 100009391 ) sperm adhesion molecule 1 (Accession PH-20 hyaluronidase, zona pellucida binding); domestic guinea pig (Accession 100135624 ) sperm adhesion molecule 1 ; and naked mole-rat (101700218 ) sperm adhesion molecule 1 (Accession PH-20 hyaluronidase, zona pellucida binding). For example, bovine PH-20 has been shown to work well with sheep embryos. This is likely due to mammalian species (including humans) having 60-91% similarity in PH-20 mRNA to bovine PH20. The invention therefore provides vectors and nucleic acids for the expression of any of the PH20 enzymes of the invention. The vectors and nucleic acids may comprise any promoter known in the art.

In one embodiment, the oocyte, zygote, embryo and/or blastocyst is exposed to only one of LMWHA or VLMWHA or PH20 throughout the entire culturing process. In another embodiment, the oocyte, zygote, embryo and/or blastocyst is exposed to LMWHA and/or VLMWHA and/or PH20 sequentially, i.e. not at the same time. For example, in a specific embodiment, during early development, for example during fertilisation, the oocyte, zygote, embryo or blastocyst are exposed to VLMWHA, as this is considered to be the size of HA produced by the action of the PH20 enzyme located in the sperm, and is thus considered to reflect the natural and optimal situation fertilisation in vivo. At a later stage in development, where the sperm would no longer be present following fertilisation in vivo, the zygotes and/or blastocysts may be exposed only to LMWHA. Alternatively, the oocyte/sperm/early zygote may be exposed to LMWHA and then be exposed only to VLMWHA later in development.

However, it is considered beneficial if the oocyte, zygote, embryo, and/or blastocyst are exposed to a combination of at least two of VLMWHA and/or LMWHA and/or PH20, in particular to a combination of LMWHA and VLMWHA, as this is considered to most accurately reflect natural conception and provide the optimal signalling required for development. During natural fertilisation, whilst the sperm surrounding the oocyte are considered to release PH20 degrading HA to VLMWHA, hyal-2 released by the oviduct during very early development is considered to degrade HA to LMWHA, exposing the oocyte/zygote to LMWHA in addition to VLMWHA.

Thus, the invention provides a composition comprising VLMWHA and LMWHA for use in any of the methods, or for inclusion in any of the culture media described herein. The composition may also further comprise PH20. The composition may comprise any of the agents of the invention. The oocyte, zygote, embryo and/or blastocyst may be exposed to any combination of the agents, at any time and for any duration. The composition may be constant, or may alter in concentration and/or composition over time.

The ratio of VLMWHA to LMWHA may be altered to mimic what is considered to be the ratio of VLMWHA and LMWHA found in nature. For example, during fertilisation, it may be considered that the oocyte is exposed to VLMWHA at a greater concentration than LMWHA. Therefore, during fertilisation and very early development, in one embodiment, the amount of VLMWHA is higher than the amount of LMWHA, i.e. the ratio of VLMWHA to LMWHA is greater than 1. Alternatively, the ratio of VLMWHA to LMWHA during fertilisation and very early development may be less than 1.

The ratio of the two sizes of HA may change over time. For example, wherein the ratio of VLMWHA to LMWHA is greater than 1 during fertilisation and/or very early development, through changes in culture media, or addition of further LMWHA, the ratio may be changed such that during later development the embryo is exposed to a ratio of VLMWHA to LMWHA of less than 1.

Any ratio or change in ratio of LMWHA and VLMWHA are encompassed by the present invention, as are any number of changes of the ratio or timings of changes.

The oocyte, zygote, embryo, and/or blastocyst may also be exposed to the same combination of VLMWHA and LMWHA throughout the entirety of the in vitro culture step.

The above compositions comprising different ratios or LMWHA and VLMWHA may in one embodiment not comprise any further active agents, or in another embodiment may comprise further active agents, such as PH20 or any other additional agent as described herein or as will be apparent to the skilled person.

The amount of VLMWHA and LMWHA may be measured in grams or miligrams or micrograms for example, i.e. an equal amount of VLMWHA and LMWHA may be considered to be an equal weight of each hyaluronic acid. However, the skilled person will appreciate that the smaller size of VLMWHA will result in much greater molar amount of VLMWHA to LMWHA, if the same amount in weight of each agent is used. Therefore, in another embodiment, it is the molar amounts of each sized fragment that are required, i.e. the ratio of VLMWHA to LMWHA is assessed based on the molar amount of each fragment, rather than physical weight. The agent or composition of the invention may be supplied to the culture medium, or may already be present in the culture medium, in such amounts as to result in any concentration of the LMWHA and/or VLMWHA and/or PH20. For example, the final concentration of LH WH and/or VLMWHA in the culture medium may be from about 10ug/ml to about 5mg/ml, for example from about 50ug/ml to about 4mg/ml, for example from about 100ug/ml to about 3mg/ml, for example from about 200ug/ml to about 2.5 mg/ml, for example from about 300ug/ml to about 2 mg/ml, for example from about 400ug/ml to about 1.75mg/ml, for example from about 500ug/ml to about 1.5mg/ml, for example from about 600ug/ml to about 1.25mg/ml, for example from about 700ug/ml to about 1 mg/ml, for example from about 800ug/ml to about 900ug/ml. In a preferred embodiment, the final concentration of LMWHA and/or VLMWHA in the culture medium is between 500ug/ml and 1000ug/ml.

In one embodiment the final concentration of PH20 in the culture medium may be from about 1-30 ng/ml. It is considered advantageous if the concentration of PH20 in the culture medium is less than 30 ng/ml. For example, the final concentration of PH20 in the culture medium may be from about 1-30 ng/ml, for example from about 2-25 ng/ml, for example from about 3-22 ng/ml, for example from about 4-20 ng/ml, for example from about 5-18 ng/ml, for example from about 6-16 ng/ml, for example from about 7-14 ng/ml, for example from about 8-12 ng/ml for example from about 9-11 ng/ml for example about 10 ng/ml.

The activity of PH20 is measured by its ability to hydrolyze S-35 labelled bovine cartilage chondroitin sulfate. <20 ng of recombinant human SPAM1 is required for the 50% hydrolysis of 10 pg chondroitin sulfate, as measured under the described conditions (information from R&D website).

It will be appreciated that where a protein has enzymatic activity, that protein may be expressed in ways other than a physical weight. The invention encompasses such proteins, such as Hyal-2 and PH20 as measured in units of activity, i.e enzymatic units as well as physical weight. For example, the physical weight of PH20 and hyal-2 may be for example the same, or the weight of PH20 may be higher or lower than that of hyal-2. However, a more accurate method of ensuring the correct amount of activity is achieved is to ensure the relevant amount of each enzyme is used based on the specific activity of each enzyme. For example, in one embodiment the amount of PH20 that the oocyte, zygote, embryo and/or blastocyst is exposed to results in the same specific activity as the amount of hyal-2 that the oocyte, zygote, embryo and/or blastocyst is exposed to, i.e. the actual weight may be different, but the activity, or enzymic units, is the same. Similarly, in a situation where it is considered advantageous to have a greater activity of PH20 than hyal-2, the skilled person will be aware of the specific activity of each enzyme and will be able to adjust the actual weight of each enzyme accordingly, The amount of Hyal-2 may be any amount, but is preferably between about 50 and 500 units/ml, for example between 100 and 450 units/ml, for example between 150 and 400 units/ml, for example between 200 and 350 units/ml, for example between 250 and 350 units/ml, for example about 300 units/ml. The hyal-2 may be from any source, and may be naturally occurring or may be a recombinantly expressed hyal-2. In one embodiment, the hyal-2 is hyaluronidase II from sheep testis (Sigma H2126), lyophilized powder, >300 units/mg.

Whilst culturing an oocyte, a zygote, embryo or a blastocyst, it is considered important to renew the culture medium at particular stages. For example in one embodiment the culture medium may be renewed every 12 hours, or every 24 hours, or every 36 hours or every 48 hours or every 60 hours or every 72 hours, for example. In one embodiment the culture medium is renewed every 48 hours. The agent or composition of the invention may be renewed at the same time as the culture medium is renewed, particularly if the fresh culture medium already contains an appropriate amount of the agent or compositions of the invention. Alternatively, the agent or composition of the invention may be added to the culture of the oocyte, zygote, embryo and/or blastocyst at intervals corresponding to the time when the media is refreshed, or at times other than when the culture medium is refreshed. For example, the agent or composition of the invention may be added every 1 hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 8 hours, every 10 hours, every 12 hours, every 18 hours, every 24 hours, every 36 hours, every 48 hours or more, independently of when the culture medium is refreshed.

The LMWHA and/or VLMWHA and/or PH20 or compositions of the invention disclosed herein may be added prior to the commencement of culturing the oocyte, zygote, embryo and/or blastocysts, or may be added after 1 hour or more of culture of the oocyte, , embryo zygote and/or blastocyst, for example after 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 28 hours, 72 hours or more. In one embodiment, the oocyte, embryo zygote and/or blastocyst is exposed to an agent or composition of the invention at day 0, day 1 , day 2, day 3, day 4, day 5, day 6 or day 7. Preferably the oocyte, zygote, embryo and/or blastocyst is exposed to an agent or composition after cleavage, for example on day 2 post fertilisation.

Any suitable culture medium may be used. The culture medium may be a commercial culture medium, and may already comprise the composition or compositions of the invention. Alternatively, the composition or compositions of the invention may be added separately to the culture medium. Conventional embryo culture media do not contain HA of any size. Recently, new HA contained embryo culture media has been produced commercially which contain HMWHA. However, it is only the surprising data of the present invention that reveals the important benefits conferred by LMWHA and VLMWHA media supplementation. In a particular embodiment, cleaved (4+ cell) embryos are cultured in a well-defined serum free medium, for example synthetic Oviductal Fluid; SOF, in the presence of an agent or composition of the invention, for example a composition comprising LMWHA and/or VLMWHA and/or PH20. In a further embodiment, PH20, or a composition comprising PH20 may be applied to the fertilisation media, particularly if the sperm are considered to be deficient to some degree in PH20 production.

In all embodiments and aspects, by the culture of an oocyte, a zygote, embryo and/or a blastocyst we include the meaning of the culture of at least one oocyte, at least one zygote, at least one embryo and/or at least one blastocyst. It will be appreciated that where the method of culture may begin with the culture of an oocyte, as the oocyte develops following fertilisation, the oocyte will become a zygote, embryo and a blastocyst. As such the methods of culture may include the culture of more than one developmental stage. We also include the meaning of any period of in vitro growth of any stage of fertilisation and growth of the embryo, from the separate gamete stage to the implantation of the embryo into the host, unless otherwise specifically stated. The oocyte(s), zygote(s), embryo(s) and/or blastocyst(s) may be for use in a method of in vitro fertilisation and may be for implantation into a host.

The culture of an oocyte, zygote, embryo and/or blastocyst may be for any number of reasons. In one embodiment the culture of an oocyte, zygote, embryo and/or blastocyst may be for the purposes of producing a viable embryo for implantation into a host as part of a method of assisted reproduction.

The culture may also be for the production of an embryo from which to take embryonic stem cells. An oocyte, zygote, embryo and/or blastocyst may also be cultured for research purpose to investigate embryos metabolism and test effects of different compounds on blastocyst development and quality.

In one embodiment, the oocyte, zygote, embryo and/or blastocyst is of human origin.

In another embodiment, the oocyte, zygote, embryo and/or blastocyst is not of human origin, for example may be of horse, camel, cow, sheep, goat, pig, dog or cat or chicken origin; or may be a rare or endangered species. The present invention is considered to be useful for use with an oocyte, zygote, embryo and/or blastocyst from any species as hyaluronan is a highly conserved molecule across animal species. Consistent data on beneficial effects of hyal-2 (in sheep and cow) and hyaluronan in several other species have been reported. For example, the composition may find use in the production of agricultural or other high value animals. In a further embodiment, the oocyte, zygote, embryo and/or blastocyst is from a species that is rare or endangered.

As used herein the term embryo refers to a zygote or post-zygotic derivatives of a fertilized egg. The term embryo therefore refers to any entity in the pre-implantation stages following fertilization of the egg. The term thus includes a fertilized egg and a zygote, pro-nucleus stage, cleaved 2-cell, 4-cell, 8-16 cell, morula or compacted morula, early blastocyst, expanded and fully expanded blastocyst and hatched blastocyst.

As used herein, the term "blastocyst" refers to the structure formed in the early embryogenesis of mammals, after the formation of the morula. It possesses an inner cell mass (ICM), or embryoblast, which subsequently forms the foetus, and an outer layer of cells, or trophoblast or trophectoderm cells, which later forms the placenta. The trophoblast surrounds the inner cell mass and a fluid-filled blastocyst cavity known as the blastocoele. The human blastocyst comprises 70-100 cells. Blastocyst formation begins at day 5 after fertilization in humans when the blastocoele opens up in the morula. We also include the meaning of the culture of the product of nuclear transfer and zygotes, blastocysts and embryos derived from more than one species, or which comprise genetic material from more than one species. Therefore the composition according to this and all aspects is for the use in culturing the embryo at any stage. The composition according to any aspect or embodiment may be employed during all stages of development and culture, or only at specific stages of development and culture.

In one embodiment, as disclosed above, the agents or compositions of the invention may be added to standard commercial, or otherwise, media as and when the presence of LMWHA and/or VLMWHA and/or PH20 is required, for example may be added to the culture medium prior to the commencement of culture of an oocyte, zygote, embryo and/or blastocyst, or may be added to the culture medium following the commencement of culture of the oocyte, zygote, embryo and/or blastocyst.

In another embodiment, the agents or compositions of the invention are already present in the culture media, in whatever the desired concentration and/or ratio is. That is, it is envisaged that the relevant culture media, for example culture media for use in assisted fertilisation, for example to culture the products of the fertilisation step of IVF or the injection step of ICSI, may be produced with the LMWHA and/or VLMWHA and/or PH20 as a fundamental component of the media recipe.

Accordingly an assisted fertilisation or conception media, such as an IVF medium or an ICSI medium may be produced with the LMWHA and/or VLMWHA and/or PH20 as a fundamental component of the medium recipe. Therefore, in a second aspect of the invention, the invention provides a culture medium comprising LMWHA and/or VLMWHA and/or PH20. Preferences for the LMWHA, VLMWHA, PH20, such as the concentrations and various combinations thereof are as defined according to the first aspect of the invention. The culture medium is typically for the culture of an oocyte, a zygote, embryo and/or a blastocyst and may be used in a method of in vitro fertilisation and/or assisted reproduction. For the avoidance of doubt, the culture medium may typically be for the culture of a zygote, a blastocyst and/or an embryo, for example a fertilized egg, pronucleus stage, cleaved 2-cell, 4-cell, 8-16 cell, morula or compacted morula, early blastocyst, expanded and fully expanded blastocyst and hatched blastocyst, rather than for the culture of an oocyte, as will be apparent from elsewhere herein. It is preferred that the pH of the culture medium is buffered, for example buffered to between about pH 5 and 8, for example between about pH 5 and 7.5, for example between about pH 7.2 and 7.4. Generally, the media of a neutral pH, for example between pH 7.2 and 7.4. PH20 and Hyal-2 are considered to be active at a neutral pH, whilst other hyals are considered to be active only at acidic pH.

The medium can be any suitable medium for embryo culture, including, for example, a sequential medium or a single medium that is replenished at least once during the culture process, or a non-sequential medium or a medium that is not replenished (e.g., uninterrupted or continuous culture) during the culturing of the embryo. Existing media that are known to those of skill in the art or that are commercially available can be modified to have a LMWHA or VLMWHA concentration or ratio as described herein. Some non- limiting examples of existing media that can be utilized in the methods or that can be modified for use include, without limitation, Continuous Single Culture™ media (Irvine Scientific), any Global® media (LifeGlobal), Gl medium (Vitrolife), G2 medium (Vitrolife), a Human Tubal Fluid (HTF) medium, HTF medium with or without glucose or phosphate, Origio Sequential Series and Sage 1-Step (Origio), Whitten's Medium, Ham's F-10 Medium, Sage Media, and the like, with or without protein supplementation. In some embodiments the medium is a homemade medium to which the agents or composition or compositions of the invention may be added. For example, a homemade media may comprise the ingredients as listed in Example 2.

It is considered that a culture media of the invention encompasses any suitable culture media further comprising an agent or composition according to the first aspect of the invention.

A third aspect of the invention provides a method for the culture of an oocyte, a zygote, embryo and/or a blastocyst. Preferences for the agents, compositions, timings and other features are as defined above and further defined below. In one embodiment the method comprises culture in media according to the second aspect of the invention.

The invention also provides, in a fourth aspect, a method of assisted fertilisation optionally in vitro fertilisation or ICSI, comprising culturing an oocyte, a zygote, embryo and/or a blastocyst according to the method of the third aspect. Preferences for the agents, the compositions, the combinations of different lengths, timings of exposure and other features are as described in relation to earlier aspects of the invention. The method of culture or in vitro fertilisation comprises, in one embodiment, culturing an oocyte, a zygote, embryo and/or a blastocyst in the presence of LMWHA and/or VLMWHA and/or PH20, or the compositions of the invention described herein. For example, the method may comprise culturing the oocyte, zygote, embryo and/or blastocyst in hyaluronic acid that is 25kDa or less in size. The method may comprise culture in the presence of hyaluronic acid that is 4-8 disaccharides or tetrasaccharides or hexasaccharides in length, or may comprise culture in a combination of both sizes of hyaluronic acid, as described in earlier aspects of the invention. For example, in the claimed method of in vitro fertilisation, the oocyte, zygote, embryo and/or blastocysts may be exposed to different forms or different ratios of hyaluronic acid at different times. For example, the ratio of VLMWHA to LMWHA may be higher during fertilisation and/or early development than the ratio towards the later stages of development, prior to implantation. The above methods may also comprise PH20, wherein the oocyte, zygote, embryo and/or blastocyst may be exposed to the PH20 at different stages of development, or periods of culture and wherein the concentration of PH20 may vary over the course of time.

In one embodiment, the oocyte is exposed to PH20 following stripping of the surrounding cells, or wherein the zygote and/or blastocyst is exposed to PH20. PH20 is commonly used prior to fertilisation. However, PH20 has not, until now, been considered to have a further role in the fertilisation and development procedure. The novel finding that the products of PH20 catalysis are beneficial in the development of the embryo lead to the inventive use of the enzyme in later stages of development. The invention also provides a composition comprising PH20 and hyal-2 for use in a method of assisted fertilisation, optionally in vitro fertilisation or ICSI. Preferences for these aspects of the invention, including culture conditions, and timings of administration are as described above in relation to all aspects of the invention. The invention therefore also provides LMWHA and/or VLMWHA and/or PH20 for use in a method of assisted fertilisation, optionally in vitro fertilisation or ICSI. Accordingly, it will be apparent to the skilled person that the invention provides a method of assisted fertilisation optionally in vitro fertilisation or ICSI, comprising culturing a zygote, an embryo, for example a cleaved embryo and/or a blastocyst according to the method of the third aspect; typically comprising culturing an embryo and/or a blastocyst according to the method of the third aspect.

It will also be apparent to the skilled person that the invention provides a composition comprising PH20 and hyal-2 for use in the culturing of a zygote, an embryo, for example a cleaved embryo and/or a blastocyst as part of a method of assisted fertilisation, optionally in vitro fertilisation or ICSI. Preferences for these aspects of the invention, including culture conditions, and timings of administration are as described above in relation to all aspects of the invention as noted above. It will also be apparent that the invention also provides LMWHA and/or VLMWHA and/or PH20 for use in the culturing of a zygote, embryo and/or a blastocyst as part of a method of assisted fertilisation, optionally in vitro fertilisation or ICSI.

The method of culture or in vitro fertilisation may also further comprises the addition of one or more additional agents relevant to the culture of the oocyte, zygote, embryo and/or blastocyst, for example wherein the factors comprise one or more of one or more hyaluronase enzymes, for example hyal-2. Similarly to PH20, the Hyal-2 may be from the same or a different species to the species of the oocyte, zygote, embryo and/or blastocyst. Preferably the Hyal-2 is from the same species as the oocyte, zygote, embryo and/or blastocyst.

For example, hyal-2 from sheep testis has been shown to be effective in both cow and sheep. The embryo can be cultured in the medium, for example the medium as described in earlier aspects of the invention for any desired period of time. For example, the embryo can be cultured for between 1 hour and 7 days, for example between 6 hours and 6 days, for example between 12 hours and 5 days, for example between 18 hours and 4 days, for example between 24 hours and 3 days, for example between 24 hours up to 7 days or any time period or range there between after fertilization. Preferably, the embryo is cultured for 3-6 days before transfer. The embryo can be cultured until developmental milestones are met such as achieving a certain number of cells (e.g., 6-8 cells) or attaining a stage (e.g., the blastocyst stage). In some embodiments, a cryopreserved embryo is thawed and cultured using the methods described herein before transfer. In some embodiments, the embryo was cultured using methods as described herein prior to cryopreservation. In some embodiments, the embryo was not cultured using methods as described herein prior to cryopreservation. The invention also provides LMWHA and/or VLMWHA and/or PH20 or the compositions of the invention for use in a method of in vitro fertilisation or assisted fertilisation. The invention also provides LMWHA and/or VLMWHA and/or PH20 or the compositions of the invention for use in a method of in vitro fertilisation or other assisted fertilisation including intra-cytoplasmic sperm injection (ICSI). The term "assisted conception" may also be used herein in place of "assisted fertilisation".

Similarly to as noted above, it will also be apparent to the skilled person that the invention provides LMWHA and/or VLMWHA and/or PH20 or the compositions of the invention for use in the cuituring of a zygote, an embryo, for example a cleaved embryo and/or a blastocyst as part of a method of assisted fertilisation, optionally in vitro fertilisation or ICSI.

The invention further provides the use of LMWHA and/or VLMWHA and/or PH20 or the compositions of the invention in the manufacture of a medicament for the treatment of infertility or for use in a method of assisted fertilisation, optionally in vitro fertilisation or ICSI; for example (as will be apparent) for use in the cuituring of a zygote, embryo and/or a blastocyst as part of a method of assisted fertilisation, optionally in vitro fertilisation or ICSI.

As detailed above in relation to the simultaneous or sequential exposure of the oocyte, zygote, embryo and/or blastocysts to LMWHA and/or VLMWHA, in any of the methods or uses the exposure to PH20 and hyal-2 may also be varied.

The oocyte, zygote, embryo and/or blastocyst may also be exposed to a combination of both PH20 and hyal-2, and the ratio of the two enzymes may vary over time. For example, during fertilisation or early development, the amount of PH20 may be higher than the amount of hyal-2 in the culture medium. This may be varied such that later in development, the amount of PH20 is lower than the amount of hyal-2.

In one embodiment, in any of the methods or uses, the oocyte is exposed to PH20 following removal of the cumulus cells. In a further embodiment, the oocyte is only exposed to PH20 during removal of the cumulus cells, and PH20 is not added to the culture media until the zygote is formed, or until the blastocyst is formed, or at any stage in between.

The inventors also surprisingly found that hyal-2 is expressed by the developing embryo starting at morula and blastocyst stages and is detectable in the culture medium, importantly without disturbing the integrity of the embryo. The level of hyal-2 in the culture medium correlates with blastocyst/embryo quality and pregnancy rates, and thus provides a non-invasive means for aiding in the identification of the most suitable embryos for implantation. Thus, in a further aspect, the invention provides a method for selecting an embryo, or aiding in selecting an embryo, for example an embryo generated through assisted fertilisation, optionally in vitro fertilisation or ICSI,, as suitable for implantation, wherein the method comprises determination of the level of hyal-2 in the culture medium used to culture an oocyte, zygote, embryo and/or blastocyst. Methods and techniques to assess the level of hyal-2 in the culture medium will be immediately apparent to those skilled in the art, and include routine methods such as ELISA. The inventors have found that embryos produced through IVF produce more hyal-2 than those produced by ICSI, and are generally of a higher quality. The method of the invention is therefore considered to be particularly beneficial to aid selection of an embryo produced via ICSI, for example wherein ICSI is the preferred or only option for generating an embryo. The method of the invention allows the selection of the most appropriate embryo(s) for implantation, which is particularly important when the pregnancy rate from ICSI embryos is low, or lower than that of embryos generated by IVF.

The culture medium may be tested for the presence of hyal-2 at any point during culture. However, it is considered that the developing embryo does not express hyal-2 until at least the morula stage or the blastocyst stage. Therefore in one embodiment, the culture medium is tested for the presence of hyal-2 when the embryo is at the morula stage, or at the blastocyst stage. In another embodiment the culture medium is tested for the presence of hyal-2 when the embryo has been developing in the medium for between 2 days and 10 days, for example between 3 days and 9 days, for example between 4 days and 8 days, for example between 5 days and 7 days, for example 6 days. It will be appreciated that if the culture medium is changed for any reason, then the level of any hyal-2 present will decrease until such time as it is able to accumulate.

In a preferred embodiment, the level of hyal-2 is monitored in the culture medium of several developing embryos, produced by the same male and female, and produced in the same way at the same time. In this way, a direct comparison between the amount of hyal-2 made by each embryo can be made, and the embryo or embryos with the highest amount of hyal-2 production can be selected for implantation, or can be scored as being of good quality when other criteria are taken into account, such as development pattern and number of trophectoderm and inner cell mass cells. The skilled person will be well aware of techniques that can be used to monitor the amount of hyal-2 produced by an embryo into the culture medium. In the first instance, the level of hyal-2 protein may be assayed through the use of, for example, immune assays, such as an ELISA test or a western blot, utilising anti-hyal-2 antibodies.

Alternatively, the skilled person will appreciate that the level of mRNA produced by the embryo and released into the culture medium may also correlate with the production of hyal-2, and as such the amount of mRNA may be assayed by routine techniques, for example by reverse transcriptase PCR, or quantitative rt-PCR. The detection of mRNA often involves the use of specific primers allowing hybridization and/or amplification of the mRNA. The skilled person would readily be able to design and use appropriate primers to detect the relevant mRNA in a range of species.

In either case, the method comprises simply taking an amount of culture medium, for example between 0.1 μΙ and 500 μΙ, for example between 0.5 μΙ and 450 μΙ, for example between 1 μΙ and 400 μΙ, for example between 1.5 μΙ and 350 μΙ, for example 2 μΙ and 300 μΙ, for example between 3 μΙ and 250 μΙ, for example between 4 μΙ and 200 μΙ, for example between 5 μΙ and 150 μΙ, for example between 6 μΙ and 100 μΙ, for example between 7 μΙ and 75 μΙ, for example between 8 μΙ and 50 μΙ, for example between 0 μΙ and 40 μΙ, for example between 15 μΙ and 35 μΙ, for example between 20 μΙ and 25 μΙ, and performing the standard assays that are common in the art, completely avoiding contact with the developing embryo.

In one embodiment, the assay to detect the level of hyal-2 in the culture medium is an enzymic assay, preferably an ELISA assay that results in a visual colour change, or a change that can be measured by routine lab equipment such as a spectrophotometer. In one embodiment the assay may comprise an anti-hyal-2 antibody conjugated to a reporter moiety, for example conjugated to HRP or any other known marker. Such markers are commonplace in the field. Where the reporter moiety is HRP, the substrate may be, for example 3,3',5,5'-Tetramethylbenzidine (TMB) which act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase and production of 3,3',5,5'-tetramethylbenzidine diimine. The resulting diimine causes the solution to take on a blue colour, and this colour change can be read on a spectrophotometer at a wavelength of 650 nm. In a further embodiment, the substrate may be OPD (o-phenylenediamine dihydrochloride) that produces a yellow-orange product detectable at 492nm by ELISA plate readers. In other embodiments the reporter moiety may be a moiety that gives of a fluorescent signal, such as GFP or FITC or other known fluorescent markers.

In other embodiments the assay may comprise a primary anti-hyal-2 antibody, such as from goat or sheep or rabbit (for example rabbit anti human Hyal-2 antibody; Abeam ab68608), and a secondary antibody directed towards the primary antibody, and conjugated to a reporter moiety as described above.

In one embodiment, the assay produces a colour change which may be observed on a coated paper.

In a further embodiment, the level of hyal-2 in the culture media is detected via detection of the enzymatic activity of hyal-2 itself. The activity of Hyal is determined by comparing HA substrate levels post reaction to a standard curve of pre-coated HA substrate amounts. For example, a hyaluronidase activity ELISA kit is commercially available by Echelon Bioscience Inc. www.echelon-inc.com. Product number: K-6000. The HA substrate may also be biotinylated and bound to Sepharose beads with the aid of 1 -ethyl-3-(3- dimethylaminopropyl-)carbodiimide and N-hydroxysulfosuccinimide. For example, the amount of biotinylated HA released from the beads over 5h incubation with Hyal-2 under pH 4.5, may be measured by alkaline phosphatase-conjugated avidin in the presence of p-nitrophenyl phosphate and recorded by plate reader at a wavelength of 405 nm (Bourguignon et al 2004, The Journal of Biological Chemistry, 279, 26991-27007.

In a further embodiment, a turbidimetric assay may be used to measure Hyal activity. In a specific embodiment, the enzyme may be added to 100 pL of a solution containing 200 mmol/L sodium acetate buffer, 150 mmol/L NaCI, pH 5.5, with 10 pg of hyaluronan (0.5 mg/mL in acetate buffer). The unhydrolyzed hyaluronan may be precipitated with 200 μΙ_ of 2.5% cetyltrimethylammonium bromide (w/V) dissolved in 2% NaOH (w/V). The turbidity may be monitored at 400 nm (until 10 min) in a microplate reader (Sunrise™, Tecan). Pucca et al 2014, Toxicon 90:326-336.

Although an enzymatic test is the preferred method to detect the presence of Hyal-2 in the culture medium, other techniques exist and would be known to the skilled person, such as GC-MS, HPLC, and MRI to detect the protein, or microarray and RNAseq for mRNA.

In one embodiment, the embryo is deemed to be suitable for implantation if the level of hyal-2 in the culture medium is detectable. By the meaning of detectable we include the meaning of any level that is above that of an appropriate control sample, such as culture media which has not been used to culture an oocyte, a zygote, embryo or a blastocyst. In a further embodiment, where the level of hyal-2 in the culture medium from a number of developing embryos or blastocysts that have been cultured individually is determined, the embryo(s) or blastocyst(s) with the highest level of hyal-2 in the culture medium are selected for implantation.

In another embodiment, the embryo is deemed to be suitable for implantation if the embryo is in the top 1 , top 2 or top 3, or top 4, or top 5, or top 6 hyal-2 producing embryos tested, wherein the embryos have all been subjected to the same conditions. Alternatively, the top 1 or top 2 hyal-2 producing embryos may be implanted, and the remaining embryos stored, for example cryopreserved, as reserve embryos should they be required.

In one embodiment, it is the rate of increase in the level of hyal-2 that is considered to be important in assessing the quality of the blastocyst or embryo. Accordingly, in one embodiment, the level of hyal-2 is monitored in the culture medium from a number of developing embryos or blastocysts that have been cultured individually, at more than one time point, and the blastocyst(s) or embryo(s) which shows the greatest increase in hyal- 2 over a given time period are selected for implantation, or further scrutiny. For example, the level of hyal-2 may be monitored at regular or irregular time intervals of 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours or 72 hours or any interval in between.

In a further embodiment, the embryo or blastocyst is deemed to be suitable for implantation if the level of hyal-2 in the media is above 2ng/ml, for example above 2.25ng/ml, for example above 2.5 ng/ml, for example above 2.75ng/ml for example above 3.0ng/ml, for example above 3.25ng/ml, for example above 3.5ng/ml, 3.75ng/ml, for example above 4.0ng/ml, for example above 4.25ng/ml, for example above 4.5ng/ml, for example above 4.75ng/ml, for example above 5.0, for example above 5.25ng/ml, for example above 5.5ng/ml, for example above 5.75ng/ml, for example 6.0ng/ml, for example 6.25ng/ml, for example above 6.5ng/ml, for example above 6.75ng/ml, for example above 7.0ng/ml, for example above 7.25ng/ml, for example above 7.5ng/ml, for example above 7.75ng/ml, for example above 8.0ng/ml, for example above 8.5ng/ml, for example above 8.75ng/ml, for example above 9.0ng/ml, for example above 9.25ng/ml, for example above 9.5ng/ml, for example above 9.75ng/ml, for example above 10ng/ml, for example above 10.25ng/ml, for example above 10.5ng/ml, for example above 10.75ng/ml, for example above 11.0ng/ml, for example above 11.25ng/ml, for example above 11.5ng/ml, for example above 11.75ng/ml, for example above 12.0ng/ml, for example above 12.25ng/ml, for example above 12.5ng/ml, for example above 12.75ng/ml, for example 13.0ng/ml, for example 13.5ng/ml, for example 13.75ng/ml, for example above 14ng/ml, for example above 14.25ng/ml, for example above 14.5ng/ml, for example 14.75ng/ml, for example above 15.0ng/ml, for example above 15.25ng/ml, for example above 15.5ng/ml, for example above 15.75ng/ml, for example above 16.0ng/ml, for example above 16.25ng/ml, for example above 16.5ng/ml, for example above 16.75ng/ml, for example above 17.0ng/ml, for example above 17.25ng/ml, for example above 17.5ng/ml, for example above 17.75ng/ml, for example above 18.0ng/ml, for example above 18.25ng/ml, for example above 18.5ng/ml, for example above 18.75ng/ml, for example above 19.0ng/ml, for example above 19.25ng/ml, for example above 19.5ng/ml, for example above 19.75ng/ml, for example above 20ng/ml.

The skilled person will appreciate that the concentration of hyal-2 in the media used to culture a blastocyst or embryo will be dependent upon not only the blastocyst or embryo itself, but also the length of time that the blastocyst or embryo has been cultured, and the volume of culture media used. Accordingly, in one embodiment, the embryo or blastocyst is deemed to be suitable for implantation if the level of hyal-2 in the media is above 2ng/ml, for example above 2.25ng/mi, for example above 2.5 ng/ml, for example above 2.75ng/ml for example above 3.0ng/ml, for example above 3.25ng/ml, for example above 3.5ng/ml, 3.75ng/ml, for example above 4.0ng/ml, for example above 4,25ng/ml, for example above 4.5ng/ml, for example above 4.75ng/ml, for example above 5.0, for example above 5.25ng/ml, for example above 5.5ng/ml, for example above 5.75ng/ml, for example 6.0ng/ml, for example 6.25ng/ml, for example above 6.5ng/ml, for example above 6.75ng/ml, for example above 7.0ng/ml, for example above 7.25ng/ml, for example above 7.5ng/ml, for example above 7.75ng/ml, for example above 8.0ng/ml, for example above 8.5ng/ml, for example above 8.75ng/ml, for example above 9.0ng/ml, for example above 9.25ng/ml, for example above 9.5ng/ml, for example above 9.75ng/ml, for example above 10ng/ml, for example above 10.25ng/ml, for example above 10.5ng/ml, for example above 10.75ng/ml, for example above 1 1.0ng/ml, for example above 1 1.25ng/ml, for example above 1 1.5ng/ml, for example above 1 1.75ng/ml, for example above 12.0ng/ml, for example above 12.25ng/ml, for example above 12.5ng/ml, for example above 12.75ng/ml, for example 13.0ng/ml, for example 13.5ng/ml, for example 13.75ng/ml, for example above 14ng/ml, for example above 14.25ng/ml, for example above 14.5ng/ml, for example 14.75ng/ml, for example above 15.0ng/ml, for example above 15.25ng/ml, for example above 15.5ng/ml, for example above 15.75ng/ml, for example above 16.0ng/ml, for example above 16.25ng/ml, for example above 16.5ng/ml, for example above 16.75ng/ml, for example above 17.0ng/ml, for example above 17.25ng/ml, for example above 17.5ng/ml, for example above 17.75ng/ml, for example above 18.0ng/ml, for example above 18.25ng/ml, for example above 18.5ng/ml, for example above 18.75ng/ml, for example above 19.0ng/ml, for example above 19.25ng/ml, for example above 19.5ng/ml, for example above 19.75ng/ml, for example above 20ng/ml, when the blastocyst or embryo has been cultured in 25μΙ of culture media for 48h from day 3 (16 -cell stage) to day 5 (blastocyst stage).

Embryos typically are cultured in 25μΙ of culture media. In the samples analysed by us, the embryos were cultured for 48h from day 3 (16 -cell stage) to day 5 (blastocyst stage). The volume of the culture media has been optimised to accommodate metabolic needs of the developing embryos and protect the embryos against the accumulation of waste (toxic) products of embryo metabolic activity. Therefore, reducing the volume is not considered a desirable option. This may particularly be the case in view of many IVF centres now moving towards adopting a single step culture system which does not need to change the culture medium on day 3.

As noted above, a culture volume of 25μΙ is considered optimal to accommodate metabolic needs of the developing embryos and protect the embryos against the accumulation of waste (toxic) products of embryo metabolic activity. However, the skilled person will appreciate that the culture volume may vary, and as the culture volume varies, the expected concentration of hyal-2 in the media may vary. For example, if the culture volume was 50μΙ it may be considered that the values of hyal-2 which represent an embryo or blastocyst deemed to be suitable for implantation listed above are reduced by one half. The skilled person is equipped to carry out such routine calculations based on the culture volume used.

Similarly, if the blastocyst or embryo is cultured for a longer or shorter period of time, the concentration of hyal-2 found in the media may be greater or smaller than those values given above. The skilled person is capable of taking any differences in culture period into account when determining the concentration of hyal-2 that would be considered to render a blastocyst or embryo suitable for implantation.

In a further embodiment, a blastocyst or embryo is deemed to be unsuitable for implantation if the level of hyal-2 in the culture media is undetectable. For example, if the level of hyal-2 in the culture media is undetectable when the embryo has been cultured in 25μΙ of culture media for 48h from day 3 (16 -cell stage) to day 5 (blastocyst stage). In yet a further embodiment, a blastocyst or embryo is deemed to be unsuitable for implantation if the level of hyal-2 in the culture media is 2ng/ml or less. For example if the level of hyal-2 in the culture media is 2ng/ml or less when embryo has been cultured in 25μΙ of culture media for 48h from day 3 (16 -cell stage) to day 5 (blastocyst stage).

The invention also provides a method of assisted fertilisation, for example in vitro fertilisation or ICSI, comprising: i) determination of the level of hyal-2 expressed by a cultured oocyte (though typically not an oocyte, as will be apparent), zygote, embryo and/or blastocysts (preferences for the determination are as defined above),

ii) selection of a suitable embryo for implantation (method of selection and criteria for suitability are as defined above), and

iii) implantation of one or more suitable embryos into a host.

Preferences for the culturing, for example media, inclusion of LMWHA and/or VLMWHA, and/or hyal-2, and/or PH20, time of incubation etc are as defined above

Further, the level of hyal-2 in the culture medium of an embryo can be used in conjunction with other signs of blastocyst quality, for example the presence of a favourable trophectoderm to inner cell mass cell ratio, so that the skilled person can better assess the suitability of an embryo for implantation. For example, the trophectoderm to inner cell mass ratio is a common marker of embryo quality, as is development speed, blastocyst development rate and hatching rate from a zona pellucida, and the like Developmentally competent blastocyst are considered to have an ICM:TE ratio of 0.3-0.4. (Fouladi-Nashta A.A., Alberio R., Kafi M., Nicholas B., Campbell K.H.S., Webb R. (2005) Differential staining combined with TUNEL labelling (DST) to detect apoptosis in preimplantation embryos. Reproductive Biomedicine Online 10: 497-502). Other signs of a high quality embryo include a favourable morphokinetic analysis, and/or a favourable morphological assessment.

The skilled person will be well aware of techniques to assess the suitability of the derived embryo for implantation, for example a proportion of the blastocysts may be stained by differential staining to count the total cell number, number of cells in inner cell mass (ICM) and trophoectoderm (TE) compartment of the blastocysts. The ratio of ICM to TE is a known indicator of blastocyst quality. In addition, this staining can be combined with TUNEL staining to detect apoptotic cells in the ICM or TE as indicator of embryo health. There are in addition other factors which can be used to indicate blastocyst quality, and which may be used in conjunction with the present methods, including favourable metabolomics markers, such as amino acid turn over, oxygen consumption and changes in pyruvate consumption. A range of techniques are used to analyse these including HPLC, LC-MS, liquid chromatography mass spectrometry; H-NMR, proton nuclear magnetic resonance; NIR, near infrared.

The skilled person will be well aware of such techniques and the parameters which would be considered to indicate a high quality blastocyst or embryo.

It will be appreciated that if the level of hyal-2 in the culture medium is being used to assess the embryo, then hyal-2 typically is not added to the culture medium, or is added at a time or in such a way that its contribution to the overall hyal-2 level can be distinguished from that produced by the embryo. It may be possible, for example, to add a hyal-2 from a different species to that of the embryo and to use a detection method that can distinguish between the two, for example a species-specific antibody. Alternatively, the hyal-2 that is added to the culture may have a marker, for example a tag, for example a his or myc tag, which would allow the determination of the level of added Hyal-2 which may be subtracted from the overall level of hyal-2. However, typically, hyal-2 would not be added if the level of hyal-2 produced by the embryo was to be assessed.

Thus the agents and compositions of the invention and the ability to screen for high quality embryos produces a synergist effect. Whilst it was known that hyal-2 was useful in improving the quality of blastocysts, supplementation with hyal-2 would have precluded the assessment of hyal-2 produced by the blastocyst. No one could have predicted that replacement of hyal-2 in the media with LMWHA and/or VLMWHA would reproduce the useful effects of hyal-2. However, this inventive finding allows the subsequent analysis of the hyal-2 produced by the embryo. Thus the combined effects of the invention are considered to be very useful in the production and selection of embryos for implantation.

Accordingly, the invention also provides a method of assisted fertilisation, such as in vitro fertilisation or ICSI, or a method for the growth and selection of high quality blastocysts or embryos for implantation into a host, the method comprising:

the culture of an oocyte, zygote, embryo and/or blastocyst according to any aspect or embodiment of the invention; and the selection of high quality blastocysts or embryos by a method which comprises the determination of the level of hyal-2 in the culture media used to culture the zygote, blastocyst or embryo. The inventors of the present invention have also surprisingly found embryos cultured in the presence of hyaluronic acid and/or LMWHA and/or VLMWHA and/or hyal-2 and/or PH20 showed increased survival following cryopreservation. It is considered that hyaluronan improves the cell membrane integrity such that it is able to more easily recover following cryopreservation. Alternatively, or additionally, the hyaluronic acid and/or LMWHA and/or VLMWHA and/or hyal-2 and/or PH20 may protect nuclear or cytoplasmic components. Accordingly, the invention provides hyaluronic acid and/or LMWHA and/or VLMWHA and/or hyal-2 and/or PH20 for use in promoting the recovery of biological organisms or tissue following cryopreservation. For example, the agents of the invention may be used to preserve developing embryos, oocytes or sperm. Thus, the invention also provides a method of storing biological tissue, for example developing embryos, oocytes or sperm, wherein the method comprises pre-incubation in the presence of hyaluronic acid and/or LMWHA and/or VLMWHA and/or hyal-2 and/or PH20 prior to cryopreservation.

The agents of the invention may be used to promote the recovery of any type of biological tissue following cryopreservation, for example the tissue may have a human origin, or may not have a human origin. The tissue may, for example, be from any of a horse, camel, cow, sheep, pig, dog, cat or any other animal. In a further embodiment the agents are useful in the storage of biological tissue from a species that is endangered. The invention also provides various kits for use in any of the methods of the invention. For example, in one embodiment the invention provides a kit comprising at least any two of LMWHA, VLMWHA and PH20 in separate containers.

A second kit provided by the invention comprises at least any two of LMWHA, VLMWHA and PH20; and means to detect the level of hyal-2, for example an anti-hyal-2 antibody or primers for the detection of hyal-2 mRNA.

The kits may also further comprise cryo tubes. A third kit comprises Hyaluronic acid/LMWHA/VLMWHA hyal-2/PH20 and cryo-tubes. Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For the avoidance of doubt, any of the above described compositions, or agents, such as LMWHA, VLMWHA and PH20 are for use in any of the claimed methods, for example a method of culture, a method of in vitro fertilisation etc. Preferences described for one aspect, for example culture medium, culture time, time of administration to the oocyte, zygote, embryo and/or blastocyst apply to all aspects unless otherwise stated. Each agent or composition is also claimed for its use in such a method, and the use of an agent or composition of the invention in the manufacture of a medicament is also claimed.

Any of the culture methods and media are also suitable for use in a method of assisted fertilisation, such as in vitro fertilisation or ICSI, for example use in the culturing of a zygote, embryo and/or a blastocyst as part of a method of assisted fertilisation, optionally in vitro fertilisation or ICSI.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

References ALANIZ, L, RIZZO, M., GARCIA, M. G., PICCIONI, F., AQUINO, J. B., MALVICINI, M., ATORRASAGASTI, C, BAYO, J., ECHEVERRIA, I., SAROBE, P. & MAZZOLINI, G. 2011. Low molecular weight hyaluronan preconditioning of tumor-pulsed dendritic cells increases their migratory ability and induces immunity against murine colorectal carcinoma. Cancer Immunol Immunother, 60, 1383-95.

BASTOW, E. R., BYERS, S., GOLUB, S. B., CLARKIN, C. E., PITSILLIDES, A. A. & FOSANG, A. J. 2008. Hyaluronan synthesis and degradation in cartilage and bone. Cell Mol Life Sci, 65, 395-413.

BOURGUIGNON, L. Y., ZHU, H., CHU, A., IIDA, N., ZHANG, L. & HUNG, M. C. 1997. Interaction between the adhesion receptor, CD44, and the oncogene product, p 85HER2, promotes human ovarian tumor cell activation. J Biol Chem, 272, 27913-8.

CAMENISCH, T. D., SPICER, A. P., BREHM-GIBSON, T., BIESTERFELDT, J., AUGUSTINE, M. L, CALABRO, A., JR., KUBALAK, S., KLEWER, S. E. & MCDONALD, J. A. 2000. Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J Clin Invest, 106, 349-60.

CHERR, G. N., YUDIN, A. I. & OVERSTREET, J. W. 2001. The dual functions of GPI- anchored PH-20: hyaluronidase and intracellular signaling. Matrix Biol, 20, 515-25.

CRATER, D. L. & VAN DE RUN, I. 1995. Hyaluronic acid synthesis operon (has) expression in group A streptococci. J Biol Chem, 270, 18452-8.

DELMAGE, J. M., POWARS, D. R., JAYNES, P. K. & ALLERTON, S. E. 1986. The selective suppression of immunogenicity by hyaluronic acid. Ann Clin Lab Sci, 16, 303-10. DOUGHERTY, B. A. & VAN DE RUN, I. 1993. Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose dehydrogenase activity. J Biol Chem, 268, 71 18-24.

FEINBERG, R. N. & BEEBE, D. C. 1983. Hyaluronate in vasculogenesis. Science, 220, 1 177-9.

GMACHL, M. & KREIL, G. 1993. Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc Natl Acad Sci U S A, 90, 3569-73.

HOFINGER, E. S., HOECHSTETTER, J., OETTL, M., BERNHARDT, G. & BUSCHAUER, A. 2008. Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases. Glycoconj J, 25, 101 -9.

ITANO, N. & KIM ATA, K. 2002. Mammalian hyaluronan synthases. IUBMB Life, 54, 195- 9.

KIM, E„ BABA, D., KIMURA, M., YAMASHITA, M„ KASHIWABARA, S. & BABA, T. 2005.

Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass. Proc Natl Acad Sci U S A, 102, 18028-33.

LAURENT, T. C. 1998. The Chemistry, Biology and Medical Applications of Hyaluronan and Its Derivatives. Wenner-Gren INternationai Series. London: Portland Press Ltd,.

LAZZARI, G., COLLEONI, S., LAGUTINA, I., CROTTI, G., TURINI, P., TESSARO, I.,

BRUNETTI, D., DUCHI, R. & GALLI, C. 2010. Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species. Theriogenology, 73, 748-57.

LEPPERDINGER, G., MULLEGGER, J. & KREIL, G. 2001. Hyal2-less active, but more versatile? Matrix Biol, 20, 509-14.

MAREI, W. F„ SALAVATI, M. & FOULADI-NASHTA, A. A. 2013. Critical role of hyaluronidase-2 during preimplantation embryo development. Mol Hum Reprod.

MCBRIDE, W. H. B., J.B. 1979. Hyaluronidase-sensitive halos around adherent cells: Their role in blocking lymphocyte-mediated cytolysis. J Exp Med, 149, 9. OHNO-NAKAHARA, M., HONDA, K., TANI OTO, K., TANAKA, N., DOI, T., SUZUKI, A., YONENO, K., NAKATANI, Y., UEKI, M., OHNO, S., KNUDSON, W., KNUDSON, C. B. & TANNE, K. 2004. Induction of CD44 and MMP expression by hyaiuronidase treatment of articular chondrocytes. J Biochem, 135, 567-75.

SALUSTRI, A., YANAGISHITA, M. & HASCALL, V. C. 1989. Synthesis and accumulation of hyaluronic acid and proteoglycans in the mouse cumulus cell-oocyte complex during follicle-stimulating hormone-induced mucification. J Biol Chem, 264, 13840-7.

SHIMADA, M., YANAI, Y., OKAZAKI, T., NOMA, N., KAWASHIMA, I., MORI, T. & RICHARDS, J. S. 2008. Hyaluronan fragments generated by sperm-secreted hyaiuronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization. Development, 135, 2001-11.

STERN, R. 2003. Devising a pathway for hyaluronan catabolism: are we there yet? Glycobiology, 13, 105R-1 5R.

STERN, R. 2004. Hyaluronan catabolism: a new metabolic pathway. Eur J Cell Biol, 83, 317-25.

STERN, R. 2005. Hyaluronan metabolism: a major paradox in cancer biology. Pathol Biol (Paris), 53, 372-82.

STERN, R., ASARI, A. A. & SUGAHARA, K. N. 2006. Hyaluronan fragments: an information-rich system. Eur. J. Cell Biol., 85, 699-715.

THOMPSON, C. B., SHEPARD, H. M., O'CONNOR, P. M., KADHIM, S., JIANG, P., OSGOOD, R. J., BOOKBINDER, L H., LI, X., SUGARMAN, B. J., CONNOR, R. J., NADJSOMBATI, S. & FROST, G. I. 2010. Enzymatic depletion of tumor hyaluronan induces antitumor responses in preclinical animal models. Mol Cancer Ther, 9, 3052-64. TOOLE, B. P. 2001. Hyaluronan in morphogenesis. Semin Cell Dev Biol, 12, 79-87.

WEST, D. C, HAMPSON, I. N., ARNOLD, F. & KUMAR, S. 1985. Angiogenesis induced by degradation products of hyaluronic acid. Science, 228, 1324-6.

ZHENG, Y., DENG, X., ZHAO, Y., ZHANG, H. & MARTIN-DELEON, P. A. 2001. Spaml (PH-20) mutations and sperm dysfunction in mice with the Rb(6.16) or Rb(6.15) translocation. Mamm Genome, 12, 822-9.

Figure legends Figure 1 - Details HA catabolism

Figure 2 - This diagram shows how we are missing Hyal-2 action in vitro. The upper image show an ovary next to the oviduct. Ovulated oocytes from the ovary is caught by the infandibulum of the oviduct and transferred to the Ampulla. Fertilisation happens in the Ampulla-Isthmus junction. Different parts of oviducts are labelled.

The lower panel shows that in an in vitro culture system, oocytes are collected from ovaries, and cultured in the presence of hormones (LH, FSH or EGF) to achieve maturation. Matured oocytes show expansion of cumulus cells which surround the oocyte. The expansion occurs by the production of large size HA by the cumulus cells. Large sized HA is water absorbent and results in expansion of cumulus cells (see oocyte in the middle circle). For in vitro fertilisation, the matured oocytes are pipetted gently to remove the loose cumulus cells before co-incubation with sperm. This washing process removes HA produced by the cumulus cells. Fertilised embryos are then cultured in synthetic oviductal fluid (SOF) medium for development to blastocyst stage. The culture media is replaced every 2 days to remove toxic metabolites. Our data show that the embryos produce HA which can be detected in the spent culture media. Washing embryos removes this important substrate.

Figure 3 - This diagram show the mechanism of HA function. Large size HA is produced by HAS present in cell membrane. The presence of Hyal-2 which is co-localised with CD- 44 results in initial fragmentation of HA into 20KD HA molecules. This is essential for internalisation of HA and eventual recycling. However, small HA fragments function as a signalling molecule through MAPK and PI3K/AKT to induce cell survival and proliferation.

Figure 4 - This diagram shows the role of Hyal-2 in the oviduct and its mechanism of action through heat shock proteins (HAS) and interleukin 1 a (IL-1 a). The role of other hyals such as PH-20 produced by sperm is also hypothesised. PH-20 is sperm hyaluronidase. It cleaves HA to smaller fragments of 4-8 disaccharides.

Figure 5 -Supplementation of embryo culture media with hyaluronidases cleaves the endogenously produced HA by the developing embryos to VLMWHA fragments resulting in increased blastocyst yield and quality through HA receptors-mediated upregulation of HSPs and IL-1 a, which have higher potential for pregnancy establishment after transfer. Figure 6 - Treatment with anti-hyall (rabbit polyclonal anti human Hyal1 (Abeam ab85375)) does not influence ovine blastocyst development. This shows that Hyal-1 is not crucial for blastocyst formation. Apart from CD-44, another receptor for HA, RHAMM also seems to be critical for embryo development. Treatment of embryo culture media with anti-RHAMM completely blocks blastocysts rate. Even addition of Hyal-2 cannot rescue the embryos. This shows that RHAMM is even more important HA receptor in the embryos. Figure 7 - Supplementation of embryo culture media with bovine PH-20 at lower doses significantly increases ovine blastocyst rate and quality.

Figure 8. Effect of HA fragments on blastocyst formation and hatching rate. Day 2 cleaved in vitro produced sheep embryos were cultured in the absence (control) or presence of 300IU ovine Hyal-2 or 10ng/ml bovine PH-20 or 500 or 1000 ug/ml LMWHA (15-40kDa) or VLMWHA (<10kDa). The culture media was renewed every 48h till day 8 after fertilisation when total number of blastocysts and hatched blastocysts were counted. The data are average blastocyst rate from 5 independent culture repeats. Figure 9 - Level of hyal-2 in an embryo spent culture media can be used as a biomarker predictor of pregnancy outcome after transfer of that embryo, a) Spent culture media from human IVF were supplied from Coventry Centre for Reproductive Medicine (CRM), Walsgrave Hospital by Professor Geraldine Hartshorne. Sample of 1-3ul of the media were used for detection of Hyal-2 by dot blot technique, similar to Western blot. Positive control was lysate from sheep uterus. Negative control contains unused human embryo culture medium. The data show that human blastocysts produce Hyal-2 which is detectable in the spent culture medium. This provided a reliable target for development of non-invasive biomarker of embryo quality or predictor of pregnancy outcome after embryo transfer which is highly desirable in human ART. b) the level of hyal-2 in the medium correlates with blastocyst or embryo quality. Human blastocyst embryos which resulted in positive clinical pregnancy after transfer, released significantly higher levels of hyal-2 in the culture medium. This was measured by ELISA and was on average over 6ng/ml as compared 2ng/ml in embryos which failed to result in pregnancy after transfer. A significant difference was found between the hyaluronidase concentration in media from transferred embryos which resulted in a pregnancy, and those that did not (p=0.009) c) The level of hyal-2 produced by human embryos increases as the embryo develops from day 3 (8-16 cell embryo) to blastocyst stage on day 5. In addition, blastocyst embryos produce higher hyal- 2 as compared to compacting and cavitating embryos, d) Good (top) quality embryos produce and release significantly higher levels of hyal-2 as compared to poor quality embryos.

Figure 10 - Effect of Hyal-2, PH-20 and LMWHA fragments on survival of embryos after cryopreservation.

Figure 11 - Analysis of gene transcripts for Hyal-2 and PH-20 in human, sheep and cow. High similarities between the species are noted. The PH-20 gene in sheep has not been fully mapped, and that used is based on predicted alignment. A) Similarity of PH20 orthologue genes in Cow vs Sheep vs Human; B) Similarity of hyal-2 orthologues compared between cow and human v sheep.

Figure 12 - Supplementation with Hyal-2 + PH20 + HA has the same effect on blastocyst rate in sheep as Hyal-2.

Figure 13 - Cleaved sheep embryos cultured in the presence of 300IU/ml Hyal-2 resulted in higher pregnancy rate (b) (on day 35) after transfer (83%) and live birth rate (c) (63%) as compared to control blastocysts with 55% pregnancy rate and 27% live birth rate. Similarly, cleaved sheep embryos cultured in the presence of 10ng/ml PH20 resulted in 73% pregnancy and 41% live birth rates and embryos supplemented with 500ug/ml small size HA (15-50KDa) resulted in 75% pregnancy and 38% live birth rates. Safety works; no adverse effect of in vitro culture was observed in the lambs born from this experiment. These include normal weights of placenta (0.5kg on average) and normal range of lambs' weights at birth (3-7kg), time to stand (3-30minutes) and time to start suckling (5- 50minut.es).

Figure 14 - The level of Hyal-2 produced by IVF embryos is higher than ICSI embryos. More top quality embryos were produced in the IVF group than the ICSI group and there was a higher pregnancy rate in IVF group.

Figure 15 - Hyal-2 and RHAMM proteins are present in human embryos. A) shows Hyal2 staining (green) in a human blastocyst. This is consistent with cell surface expression of the Hyal2 enzyme, and its presence within endocytic vesicles for the degradation of high mw HA. Differences in staining intensity may reflect cell fate, although that has not been determined. B) shows the presence of the RHAMM receptor (green) within a human blastocyst, with intense staining concentrated in of the ICM of the embryo. It is unknown whether this relates to cell fate.

Figure 16 - Shows the presence of RHAMM and Hyal-1 in different stage sheep embryos. Hyal-1 level reduces as the embryo reaches to blastocyst stage. RAHAMM is present in all stage embryos and in blastocyst is highly expressed in the inner cell mass (ICM) which is the origin of embryo germ cell layers and development to embryo proper. The surrounding trophectoderm cells develop to placenta during implantation to support development of the embryo. A) Immunofluorescence shows positive cell surface RHAMM staining (green) in embryos at all stages of preimplantation embryo development. Negative; IgG control, hpf; hours post-fertilisation. B) Immunofluorescence shows positive Hyal-1 staining (green) in early 2, 4, 8 and 16-32 -cell stage embryos, but decreases in morula and blastocyst. Negative; IgG control, hpf; hours post-fertilisation. C) presence of mRNAfor Hyal-1 and HA receptors RHAMM and CD44 in immature sheep oocytes, in vitro matured oocyte at metaphase II stage (Mil), cleaved 2-4 cell and 16 cell stage embryos and blastocysts. RHAMM is present at all stages from immature oocyte to blastocyst. Hyal- 1 is expressed in Mil oocyte, had highest expression level in 16-cell embryo and then had remarkable reduction of expression in the blastocyst. CD44 is present in all stage oocytes and embryos.

Examples

Example 1 - Ovine in vitro embryo production

Ovine embryos are produced in vitro as previously described in Marei et al. 2012. Briefly, ovaries are collected from abattoir and transported to the lab where cumulus oocyte complexes (COCs) are aspirated from 3-5 mm antral follicles. Grade 1 and 2 COCs characterized by dark homogenous ooplasm and more than 4 layers of compact cumulus cells are washed twice in TCM199 supplemented with 20 mM HEPES and 0.4% (w/v) BSA and once in maturation medium. Groups of up to 50 COCs are then cultured in four-well dishes containing 500 μΙ/well maturation medium; TCM199 medium supplemented with 10% (v/v) fetal bovine serum, 5 g/ml follicle stimulating hormone (Folltropin; Bioniche Animal Health, Belleville ON), 5 pg/ml luteinising hormone (Lutropin; Bioniche Animal Health), 1 g/ml oestradiol, 10ng/ml Epidermal growth Factor, 100μΜ Cysteamine, 2mM L-glutamine, 200μΜ sodium pyruvate and 50 pg/rnl gentamycin. COCs are incubated for 24 h at 38.5°C under 5% C02 in humidified air. This provides a suitable environment for nuclear maturation of the oocytes to metaphase II stage. The COCs are then fertilized in 4-well dishes containing 400μΙ F-TALP containing 1 x 106 motile sperm/ml selected by swim-up in S-TALP from a proven fertile bull. COCs were cultured for 18 h at 38.5oC in a humidified incubator of 5% C02 in air. At 20h after fertilisation (day 0), presumptive zygotes are denuded from cumulus cells by gentle pipetting and cultured in 500 μΙ of serum-free SOFaaci supplemented with 0.4% (w/v) fatty acid-free BSA at 38.5°C in a humidified incubator with 5% 02, 5% C02, and 90% N2. The culture medium is renewed every two days until Day 8 when the number of expanded and hatched blastocysts is counted.

Example 2 SOFaaci culture medium (AH Fouladi-Nashta)

SOF culture medium

Ultrapure water 100ml

Nad 629.0mg

KCI 53.4mg

KH2P04 16.2mg

MgS04 18.2mg

CaCL2 2H20 26.2mg

Sodium Lactate 75.7ul

NaHC03 210. Omg

Sodium Pyruvate 80.0mg

L-Glutamine 2.9mg

Phenol-Red 1 mg

Tri-Sodium Citrate 10. Omg

Myo-inositol 49.9mg

BME amino acid (50X) 22.5ul/ml

MEM amino acid (100X) 5,0ul/ml

BSA 4mg/ml or PVA 3mg/ml Adjust pH: 7.4, Osmolality: 270-280mOsm

How to make SOFaaciBSA culture medium

100ml 50ml 25ml

Stock S3 10.0ml 5.0ml 2.5ml

Stock B 10.0ml 5.0ml 2.5ml

Stock C 3.0ml 1.5ml 750ul

Stock D 1.0ml 0.5ml 250ul

Stock L 3.0ml 1.5ml 750ul

Stock M 1.0ml 0.5ml 250ul

Stock G 2.5ml 1.25ml 625ul

Stock GLN 10.0ml 5.0ml 2.5ml

BME 2.250ml 1.125ml 562ul

MEM 500ul 250ul 125ul

Stock T 2.0ml 1.0ml 0.5ml

Stock 1 2.0ml 1.0ml 0.5ml

BSA 400mg 200mg 100mg

Water 52.75ml 26.375 13.185

Adjust the pH to 7.4, Osmolarity should be between 270-280 mOsmol. Filter and store fridge for up to one week

Stock solutions

Stock C (33mM pyruvate)

Pyruvate Na 36mg

Dissolve pyruvate to 10ml MQ water. Filter and store at 4°C for up to one week.

Stock GLN (10mM glutamine)

Glutamine 73mg

Dissolve glutamine in 50mls MQ water, filter and store at 4°C for up to one month Stock B (250mM NaHC03)

NaHC03 1.0505g

Phenol red 5mg (make 1 mg/ml solution stock)

Dissolve NaHC03 in 45ml MQ water and add 5ml of 1 mg/ml phenol red solution.. Filter and store at 4°C for up to two weeks.

Stock T

Tri-Sodium Citrate

Dissolve 50mg Tri-sodium Citrate in 10ml MQ water. Filter and store for up to two weeks.

Stock L (330mM Na Lactate)

Na Lactate (60% syrup) 1.41 mis Dissolve sodium lactate in 30ml MQ water. Filter and store at 4°C for up to one month.

Stock S3 g/IOOml g/200ml

NaCI 6.294 12.588

KCL 0.534 1.068

KH2P04 0.162 0.324

Add the above to the desired amount of MQ water, filter and store in fridge for up to 3 months.

Stock D (171 mM CaCI2.2H20)

CaCI2.2H20 1.260g

Dissolve in 50ml MQ water, filter and store in fridge for up to 3 months.

Stock M (49mM MgCL2.6H20)

MgCI2.6H20 0.5g Dissolve in 50ml MQ water, filter and store in fridge for up to 3 months.

Stock G (60mM Glucose)

Glucose 0.54g

Dissolve in 50m MQ water, filter and store in fridge for up to 3 months.

Stock I

Dissolve 499mg myo-lnositol in 20ml MQ water. Filter and store for up to 3 months. Example 3 - LMWHA, VLMWHA Hyal-2 or PH-20 as a supplement for human embryo culture

• Oocytes are collected by aspiration of ovarian follicles after ovarian stimulation treatment

· The oocytes are cultured in maturation medium to achieve nuclear maturation to metaphase II stage (duration of culture up to 24h)

• The oocytes are fertilised in vitro with freshly collected or frozen semen

• Early cleaved embryos and presumptive zygotes are transferred to embryo culture medium 24h after fertilisation

• Cleaved embryos (4 cell plus; 48h after culture) are collected and transferred to embryo culture medium supplemented with 300IU/ml Hyal-2 or 10ng/ml PH-20

• The culture media with supplemented Hyal-2 or PH-20 is renewed every 48h

• Blastocysts are produced on day 6 after fertilisation

• Good quality blastocysts show well expansion and distinguished inner cell mass

• One good quality blastocyst is transferred into the uterus of the patient (depending on the female patient's age two blastocysts may be transferred). The remainder of blastocysts (if any) will be cryopreserved with patient's consent.

Example 4 - Supplementation with hyal-2, PH-20, LMWHA and/or VLMWHA increases the blastocyst rate and quality Figure 7 details how supplementation of embryo culture media with PH-20 at lower doses significantly increases blastocyst rate and quality. Figure 8 shows a similar effect for supplementation with hyal-2, LMWHA and/or VLMWHA.

Ovine embryos are produced after in vitro maturation and fertilisation of COCs as described in Example 1. Cleaved embryos (48h after fertilisation; 4 cell stage) were cultured in SOFaaciBSA medium in the absence or presence of 300U/ml Hyal-2 or 10ng/ml PH-20 or 500ng/ml or 1000ng/ml low molecular weight HA (LMWHA; 10-40Kda) or very low molecular weight HA (VLMWHA; <10kDa) . The culture media including the treatments were renewed every 2 days until day 8 when the number of expanded and hatched blastocysts was counted.

Due to high similarity in HA, Hyal-2 and PH-20 structure between human and cow and sheep, these effects are expected to be observed on human embryos. Example 5 - Hyal-2 released into the culture medium of the developing blastocyst and/or embryo is indicative of blastocyst and/or embryo quality

Spent culture media of human blastocyst embryos were collected. The culture media did not contain any supplemented HA or Hyal. Production of Hyal-2 by human embryos was determined by dot-blot techniques as detailed below. Fresh human embryo culture was used as negative control. Sheep uterine lysate was used as the positive control.

A polyvinyl difluoride (PVDF) membrane is soaked in absolute methanol for 5 min on 100RPM vortex. While letting the membrane dry, a table is drawn using a pencil on the membrane to mark the positions where the samples will be loaded. The samples are loaded on the membrane according to their total concentration in 2-4 μΙ volume (dots) of the membrane. The dots are applied slowly to allow penetration to the membrane (3-5mm diameter). Keep the membrane in the 37 °C incubator and wait for the dots to dry out completely.

The membrane is then blocked with 5% skim milk in PBS -T (PBS 1x containing 0.05% Tween) for 90min in RT. Keep the membrane in plastic sealed bags. Wash 3 times in PBS- T every 10 min (30min Total) before Incubating with primary antibody (0.1 -10 pg/ml for purified antibody, 1 :1000 to 1 :100000 dilution for, antisera, 1 :100 to 1 :10000 for hybridoma supernatant) dissolved in PBS-T for 30 - 60min at RT according to the manufacturer's instructions. Rabbit polyclonal anti human hyal-2 antibody (ab68608; 1 in 250 dilution) was used for this analysis. The membrane is then washed for 1 h (6 times every 10 min) with PBS-T before incubation with HRP conjugated secondary antibody 1 :30000 for 1h at RT.

The membrane is then washed for 1h:30min (6 times every 15min) with PBS-T. It will be dried by holding it up. Visualisation of dots is done by adding 1ml from each A and B reagents of ECL advance Kit (2ml total) on the membrane and incubating it for 5 min RT and the dark.

The ECL is then removed from the membrane and dried completely before obtaining the membrane image using a Gel Documentation facility in the dark by chemiluminescence option. It will be appreciated that the above represents standard immunoblotting techniques, the exact parameters of which the skilled person will understand to be variable dependent upon the actual reagents used.

Figure 9 shows that hyal-2 produced by a developing embryo or blastocyst is detectable in the culture medium, providing a totally non-invasive new marker of embryo and/or blastocyst quality.

Figure 9 b) - d) shows data from human embryos cultured in 25μΙ of culture media. The embryos were cultured for 48h from day 3 (16 -cell stage) to day 5 (blastocyst stage). The volume of the culture media has been optimised to accommodate metabolic needs of the developing embryos and protect the embryos against the accumulation of waste (toxic) products of embryo metabolic activity.

Example 6 - Hyal-2, PH-20 and LMWHA fragments promote survival of embryos after cryopreservation

In vitro produced cleaved (4 cell stage) sheep embryos were cultured in SOFaaci medium in the absence or presence of 300U/ml Hyal-2 or 10ng/ml PH-20 or 1000ng/ml low molecular weight HA (LMWHA; 10-40Kda). The culture media including the treatments were renewed every 2 days until day 6 when expanded blastocysts were harvested and processed for cryopreservation by open pulled straw verification method. The embryos were then defrosted in the warming media, washed thoroughly in the SOFaaci medium and the cultured for 48h in this medium in the absence of the treatments. Embryos survival was recorded by the number of embryos re-expanded and hatched from zona pellucida.

Claims

1. Low molecular weight hyaluronic acid (LMWHA) or very low molecular weight hyaluronic acid (VLMWHA) or PH20 for use in the culture of an embryo, zygote, blastocyst and/or an oocyte.

2. A composition comprising low molecular weight hyaluronic acid (LMWHA) and/or very low molecular weight hyaluronic acid (VLMWHA) and/or PH20 for use in the culture of an embryo, zygote, blastocyst and/or an oocyte.

3. The LMWHA of claim 1 of the composition of claim 1 wherein the LMWHA is between 15kDa and 40kDa in size.

4. The VLMWHA of claim 1 or the composition of any of claims 1 or 2 wherein the VLMWHA is <10kDa, optionally wherein the VLMWHA is a tetrasaccharide or a hexasaccharide, optionally wherein the VLMWHA is 4-8 monosaccharides or less in size.

5. The composition of claim 2 wherein the composition comprises LMWHA and does not comprise VLMWHA and PH20.

6. The composition of claim 2 wherein the composition comprises VLMWHA and does not comprise LMWHA and PH20.

7. The composition of claim 2 wherein the composition comprises PH20 and does not comprise LMWHA and VLMWHA.

8. The composition of claim 2 wherein the composition comprises LMWHA and VLMWHA and does not comprise PH20.

9. The composition of claim 2 wherein the composition comprises LMWHA and PH20 does not comprise VLMWHA.

10. The composition of claim 2 wherein the composition comprises VMWHA and PH20 and does not comprise LMWHA.

11. The composition of any of claims 2, 3, 4, and 8 wherein the composition comprises a mixture of hyaluronic acid molecules of approximately 10-40 kDa in size, optionally 15- 40 kDa in size, and molecules of approximately 10 kDa or less in size.

12. The composition of any of claims 2, 3, 4, 8 and 11 wherein the ratio of low molecular weight hyaluronic acid to very low molecular weight is 1 :1.

13. The composition of any of claims 2, 3, 4, 8, 11 and 12 wherein the ratio of low molecular weight hyaluronic acid to very low molecular weight hyaluronic acid is greater than 1 :1.

14. The composition of any of claims 2, 3, 4, 8, and 11-13 wherein the ratio of low molecular weight hyaluronic acid to very low molecular weight hyaluronic acid is less than 1 :1.

15. The LMWHA or VLMWHA or PH20 according to claim 1 , or the composition of any of claims 2 to 14 wherein the composition is added to the culture media prior to the commencement of culture.

16. The LMWHA or VLMWHA or PH20 according to claim 1 or the composition of any of claims 2 to 14 wherein the composition is added to the culture media following the commencement of culture.

17. The LMWHA or VLMWHA or PH20 according to claim 1 or the composition of any of claims 2-16 wherein the culture of the embryo, zygote, blastocyst and/or oocyte is part of a method of assisted fertilisation, optionally in vitro fertilisation or intra-cytoplasmic sperm injection (ICSI).

18. The LMWHA or VLMWHA according to claim 1 , or the composition according to any of claims 2-17 wherein the hyaluronic acid is cationic hyaluronic acid or N-acetylated hyaluronic acid.

19. The composition of any of claims 2-18 wherein the composition further comprises one or more additional agents, optionally wherein the composition further comprises hyal- 2.

20. Culture medium comprising the LMWHA or VLMWHA or PH20 as defined in claim 1 or the composition as defined in any of claims 2-19.

21. The culture medium of claim 20 for use in the culture of an embryo, zygote, blastocyst and/or an oocyte.

22. The culture medium of any of claims 20 to 22 for use in a method of assisted fertilisation, optionally in vitro fertilisation or ICSI, optionally for use in the culturing of a zygote, embryo and/or a blastocyst as part of a method of assisted fertilisation, optionally in vitro fertilisation or ICSI.

23. The culture medium of any of claims 20 to 22 wherein the total concentration of hyaluronic acid is 500-1000 pg/ml.

24. The culture medium of any of claims 20-23 wherein the culture medium is buffered to a pH of between about pH 5-8, optionally between about 5-7.5, optionally between about 7.2-7.4

25. A method for the culture of an embryo, zygote, blastocyst and/or an oocyte wherein the method comprises culture in medium wherein the culture medium is as defined in any of claims 20 to 24.

26. A method of assisted fertilisation, optionally in vitro fertilisation or ICSI comprising culturing an embryo, zygote, blastocyst and/or an oocyte according to the method of claim 25 .

27. A method of assisted fertilisation, optionally in vitro fertilisation or ICSI, comprising culturing an embryo, zygote, blastocyst and/or an oocyte in the presence of the composition as defined in any of claims 2-19.

28. The method of any of claims 25-27 wherein the embryo, zygote, blastocyst and/or oocyte is exposed to different forms of hyaluronic acid at different times.

29. The method of any of claims 25-27 wherein the embryo, zygote, blastocyst and/or oocyte is exposed to a combination of LMHWA and VLMWHA, optionally wherein the ratio of low molecular weight hyaluronic acid to very low molecular weight hyaluronic acid varies over time, optionally wherein the ratio of LMWHA:VMWHA decreases over time.

30. The method of any of claims 25 to 29 wherein the LMWHA or VLMWHA or PH20 according to claim 1 , or the composition according to any of claims 2-19 is already present in the culture media.

31. The method of any of claims 25 to 29 wherein the LMWHA or VLMWHA or PH20 according to claim 1 , or the composition according to any of claims 2-19 is added to the culture media, optionally added to the culture media following initial culture of the embryo, zygote, blastocyst and/or oocyte.

32. The method of any of claims 25-31 wherein the embryo, zygote, blastocyst and/or oocyte is exposed to the LMWHA or VLMWHA or PH20 according to claim 1 , or the composition according to any of claims 2-19 , after 1 hour or more of culture of the embryo, zygote, blastocyst and/or oocyte, optionally after 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 28 hours, 72 hours or more.

33. The method of any of claims 25-32 wherein the zygote, embryo or blastocyst is exposed to PH20 only after cleavage, optionally around 48 hours after fertilisation.

34. The method of any of claims 25 to 33 wherein the method further comprises exposing the embryo, zygote, blastocyst and/or oocyte to other factors, optionally wherein the factors comprise one or more of one or more hyaluronase enzymes, optionally hyal-2; inhibitors of hyaluronic acid, optionally inhibitors of hyaluronic acid synthase, optionally 4- methylumbelliferon; and inhibitors of hyaluronase enzymes, optionally ascorbic acid.

35. The method of any of claims 25 to 34 wherein the embryo, zygote, blastocyst and/or oocyte is for implantation into a host.

36. The method of any of claims 25 to 35 wherein the embryo, zygote, blastocyst and/or oocyte is of human origin.

37. The method of any of claims 25 to 35 wherein the embryo, zygote, blastocyst and/or oocyte is not of human origin.

38. The method of claim 37 wherein the embryo, zygote, blastocyst and/or oocyte is of horse, camel, cow, sheep, pig, dog or cat or chicken origin.

39. The method of any of claims 37-38 wherein the origin of the embryo, zygote, blastocyst and/or oocyte is from a species that is rare or endangered.

40. LMWHA for use in a method of assisted fertilisation, optionally in vitro fertilisation or ICSI, optionally for use in the culturing of a zygote, embryo and/or a blastocyst as part of a method of assisted fertilisation, optionally in vitro fertilisation or ICSI.

41. VLMWHA for use in a method of assisted fertilisation, optionally in vitro fertilisation or ICSI, optionally for use in the culturing of a zygote, embryo and/or a blastocyst as part of a method of assisted fertilisation, optionally in vitro fertilisation or ICSI.

42. A method for selecting an embryo or blastocyst generated through assisted fertilisation, optionally in vitro fertilisation or ICSI, as suitable for implantation, wherein the method comprises determination of the level of hyal-2 in the culture medium used to culture the embryo or blastocyst.

43. The method of claim 42 wherein the determination of the level of hyal-2 in the culture medium comprises the determination of the level of the protein of hyal-2 or the determination of the level of the mRNA encoding hyal-2.

44. The method of any of claims 42 and 43 wherein the embryo or blastocyst is deemed to be suitable for implantation where the level of hyal-2 protein in the medium is: a) detectable; or b) above 2ng/ml, optionally above 2.25ng/ml, optionally above 2.5 ng/ml, optionally above 2.75ng/ml optionally above 3.0ng/ml, optionally above 3.25ng/ml, optionally above 3.5ng/ml, 3.75ng/ml, optionally above 4.0ng/ml, optionally above 4.25ng/ml, optionally above 4.5ng/ml, optionally above 4.75ng/ml, optionally above 5.0ng/ml, optionally above 5.25ng/ml, optionally above 5,5ng/ml, optionally above 5.75ng/ml, optionally 6.0ng/ml, optionally 6.25ng/ml, optionally above 6.5ng/ml, optionally above 6.75ng/ml, optionally above 7.0ng/ml, optionally above 7.25ng/ml, optionally above 7.5ng/ml, optionally above 7.75ng/ml, optionally above 8.0ng/ml, optionally above 8.5ng/ml, optionally above 8.75ng/ml, optionally above 9.0ng/ml, optionally above 9.25ng/ml, optionally above 9.5ng/ml, optionally above 9.75ng/ml, optionally above l Ong/ml, optionally above 10.25ng/ml, optionally above 10.5ng/ml, optionally above 10.75ng/ml, optionally above 1 1.0ng/ml, optionally above 1 1.25ng/ml, optionally above 1 1.5ng/ml, optionally above

11.75ng/ml, optionally above 12.0ng/ml, optionally above 12.25ng/ml, optionally above 12.5ng/ml, optionally above 12.75ng/ml, optionally 13.0ng/ml, optionally 13.5ng/ml, optionally 13.75ng/ml, optionally above 14ng/ml, optionally above 14.25ng/ml, optionally above 14.5ng/ml, optionally above 14.75ng/ml, optionally above 15.0ng/ml, optionally above 15.25ng/ml, optionally above 15.5ng/ml, optionally above 15.75ng/ml, optionally above 16.0ng/ml, optionally above 16.25ng/ml, optionally above 16.5ng/ml, optionally above 16.75ng/ml, optionally above 17.0ng/ml, optionally above 17.25ng/ml, optionally above 17.5ng/ml, optionally above 17.75ng/ml, optionally above 18.0ng/ml, optionally above 18.25ng/ml, optionally above 18.5ng/ml, optionally above 18.75ng/ml, optionally above 19.0ng/ml, optionally above 19.25ng/ml, optionally above 19.5ng/ml, optionally above 19.75ng/ml, optionally above 20ng/ml, optionally wherein the blastocyst or embryo has been cultured in 25μΙ of culture media for 48h from day 3 (16 -cell stage) to day 5 (blastocyst stage).

45. The method of any of claims 42 to 44 wherein the embryo or blastocyst is deemed to be suitable for implantation where the level of hyal-2 mRNA in the medium is detectable.

46. The method of any of claims 42 to 45 wherein the level of hyal-2 in the culture medium from a number of developing embryos or blastocysts that have been cultured individually is determined.

47. The method of claim 46 wherein the embryo or embryos, or blastocyst or blastocysts with the highest level of hyal-2 in the culture medium are selected for implantation.

48. The method of any of claims 46 and 47 wherein the most suitable embryo or embryos for implantation are those expressing a high level of hyal-2 and which have at least one other favourable sign of embryo quality, optionally a favourable trophectoderm to inner cell mass cell ratio, and/or a favourable embryo morphokinetic analysis, and/or a favourable morphological assessment, and/or a favourable metabolomics marker, optionally 02 consumption, amino acid turnover and/or pyruvate usage.

49. The method of any of claims 42 to 48 wherein the determination is carried out by an assay that is one or more of an immunological assay, optionally an ELISA assay; an enzymatic assay; or rt-PCR; and optionally results in a visible colour change.

50. A method of assisted fertilisation, optionally IVF or ICSI, comprising: i) determination of the level of hyal-2 expressed by a cultured embryo or blastocyst according to any of claims 42 to 49,

ii) selection of a suitable embryo or blastocyst for implantation, optionally wherein the selection is carried out according to any of claims 44-48

iii) implantation of one or more suitable embryos or blastocysts into a host.

51. The method of claim 50 wherein the embryo or blastocyst is cultured in the presence of one or more of the compositions according to any of the preceding claims, or cultured in the media according to any of claims 20-24.

52. A kit comprising at least any two of: LMWHA, VL WHA and PH20 in separate containers.

53. The kit according to claim 52 further comprising means to detect the level of hyal- 2, optionally wherein the means comprise any one or more of:

an anti-hyal-2 antibody, optionally conjugated to a reporter moiety;

a secondary anti-body conjugated to a reporter moiety;

substrates for the reporter moiety; and

primers for the detection of hyal-2 mRNA,

54. Hyaluronic acid and/or LMWHA and/or VLMWHA and/or hyal-2 and/or PH20 for use in promoting the recovery of biological organisms or tissue following cryopreservation.

55. The hyaluronic acid and/or LMWHA and/or VLMWHA and/or hyal-2 and/or PH20 according to claim 54 for promoting the recovery of developing embryos, oocytes or sperm.

56. A method of storing biological tissue wherein the method comprises pre-incubation of the biological tissue in hyaluronic acid and/or LMWHA and/or VLMWHA and/or hyal-2 and/or PH20, or in a compositions comprising hyaluronic acid and/or LMWHA and/or VLMWHA and/or hyal-2 and/or PH20.

57. The method of claim 56 wherein the biological tissue comprises one or more oocytes, zygotes, blastocysts, sperm or developing embryos.

58. The method of any of claims 56 and 57 wherein the biological tissue is of human origin.

59. The method of any of claims 56 and 57 wherein the biological tissue is not of human 5 origin.

60. The method of claim 59 wherein the biological tissue is of horse, camel, cow, sheep, pig, dog or cat origin; and/or the biological tissue is from a species that is endangered.

o

61. A composition comprising at least any two of LMWHA, VLMWHA and PH20, optionally wherein the composition is as defined in any of the preceding claims.