Patent Application
20120252119
The present invention relates generally to methods for the collection and maturation of oocytes. In particular, the present invention relates to in vitro methods that utilise improved collection and maturation media, which promote maturation of oocytes prior to fertilisation.
In mammals, immature eggs (oocytes) grow and develop in follicles within the ovary. Immature oocytes are metabolically coupled to somatic granulosa cells, which surround the oocyte and nurture the development of the oocyte until ovulation. Essentially, maturation of the oocyte depends on its association with its companion somatic granulosa cells which not only support its growth and development, but also regulate the progression of meiosis.
The cytoplasmic and nuclear maturation of the oocyte during pre-ovulatory development are closely related but differentially distinguishable processes crucial for successful fertilisation, development of the embryo, and possibly also for the ability of the embryo to implant, ultimately affecting pregnancy outcome.
During cytoplasmic development, the diameter of the oocyte substantially increases from ˜15 to 100 μm, corresponding to a 300-fold increase in volume. At this stage the oocyte is both transcriptionally and translationally very active. For example, a mature mouse oocyte contains ˜200-fold more RNA and ˜50-60-fold more protein than an average somatic cell. The content of mRNA in the oocyte is also high, ˜15-20%, compared to that of ˜2-3% in a somatic cell.
Nuclear maturation of the oocyte occurs after the gonadotropin luteinising hormone surge, and involves the dissolution of the nuclear membrane, chromosome condensation followed by orientation in the equatorial plate, and organisation of the microtubules in a spindle.
A significant proportion of children in western countries are now born using assisted reproduction technologies, including the use of in vitro fertilization (IVF). IVF generally takes the form of stimulating the ovaries of women to produce multiple growing follicles, collecting the ova from these large, growing follicles that are preparing to ovulate, contacting collected ova with sperm in vitro and introducing the resultant embryo into the uterus. In vitro maturation (IVM) of oocytes is an adjunct therapy to IVF, which greatly reduces the requirement for gonadotropin administration during treatment. IVM involves the removal of ova from smaller follicles in patients who receive either low levels of gonadotropin or even no gonadotropin. The procedure used to obtain eggs requires a modified patient management system and ova pick-up procedure.
Large doses of gonadotropin used in standard IVF procedures can lead to a condition of ovarian hyperstimulation syndrome (OHSS), which occurs in approximately 5% of women undertaking IVF cycles. OHSS is usually mild and self-limiting. In some cases, urgent medical attention is required. When severe, the condition can be potentially life threatening requiring hospitalization, intravenous fluids, pain relief, and other medication. Pulmonary embolism from a clot in the leg or complications of severe dehydration may occur in rare cases.
Women with the condition of polycystic ovarian syndrome require IVM in preference to IVF to avoid ovarian hyperstimulation caused by the administration of gonadotropin, or any other ovarian follicle stimulating agent. IVM is also applied to women whose preference is to minimise follicle stimulation during infertility treatment. IVM is also more convenient to the patient as it requires less drug administration, which is usually performed by the patients themselves. IVM also has cost advantages, as the cost of drug use is minimised.
Nevertheless, the efficiency of IVM relative to IVF in establishing pregnancies and live births are reduced. Although there have been some improvements in recent times to patient management, there has been little advance in laboratory techniques.
In vitro production (IVP) of animal embryos has a variety of purposes, such as genetic improvement in livestock and domesticated breeds, genetic rescue in rarer breeds, as well as a platform technology for manipulations, such as production of sexed embryos from sexed sperm, or cloning by somatic cell nuclear transfer. An essential technique in the production of embryos in vitro is the maturation of oocytes in vitro (IVM). IVP has the potential to replace current conventional techniques such as multiple ovulation and embryo transfer (MOET), where (similarly to human clinical application) gonadotropin treatment is required. However, adoption of IVP for breeding and other uses has been hampered by the poor efficiencies of producing transferable stage embryos, the poor results following embryo transfer of such embryos and the poor results following freezing and thawing (storage) of such embryos.
Accordingly, new methods and media for culturing oocytes would be desirable. In particular, new methods and media for collecting and maturing oocytes to improve assisted reproductive technologies would be particularly desirable.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
The present invention arises out of studies of oocyte in vitro culture media, and the constituents thereof, which enhance the maturation of oocytes once harvested from the ovary.
In one aspect, the present invention provides a method of producing an embryo from an oocyte by an assisted reproduction technology, the method comprising:
In another aspect, the present invention provides a method of in vitro maturation of an oocyte, the method comprising:
In another aspect, the present invention provides an oocyte maturation medium, the medium comprising:
In another aspect, the present invention provides a combination product comprising the following components:
In another aspect, the present invention provides a method of inducing oocyte maturation, the method comprising culturing an oocyte in a maturation medium comprising a phosphodiesterase inhibitor and a ligand for inducing maturation of the oocyte, wherein the concentration of the ligand in the maturation medium overcomes cAMP-induced meiotic arrest of the oocyte, thereby maturing the oocyte.
In another aspect, the present invention provides a method of inducing maturation of an oocyte which is in a state of meiotic arrest, the method comprising contacting the oocyte with a ligand at a concentration sufficient to overcome the meiotic arrest.
Where the terms “comprise”, “comprises”, “comprised” or “comprising” are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other feature, integer, step, component or group thereof.
As used in this specification, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise.
Where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all values in between these limits.
The present invention relates to an improved method of collection and maturation of oocytes in vitro. It has been determined that maturation of oocytes harvested from the ovary in a number of species, including human and bovine species, is significantly improved when the harvested oocyte is placed in a collection medium comprising a phosphodiesterase inhibitor and an agent that increases intracellular cAMP concentration in the oocyte and/or in cumulus cells associated with the oocyte, followed by culturing the oocyte in a maturation medium also comprising a phosphodiesterase inhibitor. With respect to assisted reproduction technologies, the improved methodology allows fertilisation of the oocyte to be delayed until the maturity of the oocyte is closer to that occurring naturally during the reproductive cycle (when compared to the maturity of oocytes collected in known media).
Accordingly, in a first aspect the present invention provides a method of producing an embryo from an oocyte by an assisted reproduction technology, the method comprising:
Furthermore, in a second aspect the present provides a method of in vitro maturation of an oocyte, the method comprising:
As discussed above, the success of the assisted reproductive technology depends to a large extent on the maturity of the oocyte prior to fertilisation. Oocytes harvested from ovaries typically undergo spontaneous resumption of meiosis, i.e. proceed to nuclear maturation, when placed in culture. This nuclear maturation may often occur before the oocyte has undergone complete cytoplasmic maturity. This is believed to ultimately affect the success of fertilisation and possibly subsequent embryo implantation and development.
In this regard, it will be understood that the term “oocyte” includes an oocyte alone or an oocyte in association with one or more other cells, such as an oocyte as part of a cumulus oocyte complex.
Accordingly, the methods according to the first and second aspects of the invention utilise a media for collecting an oocyte from an ovary of a subject, also referred to herein as “an oocyte collection medium”, “collection medium”, or variations thereof, which comprises a first phosphodiesterase inhibitor and an agent that increases intracellular cAMP concentration in the oocyte.
Furthermore, the methods according to the first and second aspects of the invention utilise a media for subsequent culturing and maturing of the collected oocyte, also referred to herein as “an oocyte maturation medium”, “maturation medium”, or variations thereof, which comprises a second phosphodiesterase inhibitor.
As set out above, both the collection and maturation media contain a phosphodiesterase inhibitor. The presence of a phosphodiesterase inhibitor in the collection and maturation media, and the further presence of an agent which increases oocyte intracellular cAMP in the collection medium, provides the advantage of preventing the harvested oocyte from undergoing spontaneous resumption of meiosis. Therefore the respective media promote cytoplasmic maturation of the oocyte before the nuclear maturation and subsequent fertilisation process begins.
A “phosphodiesterase inhibitor” is to be understood to mean an agent which blocks or inhibits phosphodiesterases (PDEs) directly or indirectly and whose action results in inactivation of the cyclic nucleotide targets (for example, cAMP and cGMP) by hydrolytic cleavage of the 3′-phosphodiester bond, resulting in passive accumulation of specific cyclic nucleotides. Inhibitors can be non-selective for all phosphodiesterase isoforms or selective for specific isoforms.
“Phosphodiesterase isoforms” refer to a family of isozymes or isoforms responsible for the metabolism or degradation of the intracellular second messengers, cAMP and cGMP. Specific isoforms can have highly selective cellular and subcellular localizations. Examples of phosphodiesterase isoforms include PDE3 and PDE4.
PDE inhibitors which can be used in the methods of the invention include any non-toxic inhibitor of PDE, whether selective or non-selective in nature. PDE inhibitors may be in the form of proteins, antibodies, aptamers, antisense nucleic acids, antisense oligonucleotides, siRNAs, polypeptides, peptides, small molecules, drugs, polysaccharides, glycoproteins, and lipids. For example, suitable PDE inhibitors include, but are not limited to, isobutylmethylxanthine (IBMX), cilostamide, theophylline, AH-21-132, Org-30029 (Organon), Org-20241 (Organon), Org-9731 (Organon), Zardaverine, vinpocetine, EHNA (MEP-1), Milrinone, Siguazodan, Zaprinast, SK+F 96231, Tolafentrine (Byk Gulden), and Filaminast (Wyeth-Ayerst Pharmaceuticals). Other PDE inhibitors are also known in the art.
The PDE inhibitor in the collection medium (“the first PDE inhibitor”) may be the same as, or different to, the PDE inhibitor in the maturation medium (“the second PDE inhibitor”).
In one embodiment, the PDE inhibitor in the collection medium is IBMX.
In another embodiment, the PDE inhibitor in the maturation medium is cilostamide.
In one specific embodiment, the PDE inhibitor in the collection medium is IBMX and the PDE inhibitor in the maturation medium is cilostamide.
As set out above, the collection media also contains an agent that increases the concentration or level of intracellular cAMP in the collected oocyte and/or increases the concentration or level of cAMP in cumulus cells associated with the oocyte. The agent may do so directly or indirectly by increasing cAMP synthesis or production, or by decreasing its degradation, or both, within the oocyte and/or in cumulus cells associated with the oocyte. Methods for measuring the level of cAMP synthesis, production or degradation are known in the art.
In one embodiment, cAMP synthesis or production may be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 fold, 50-fold, or 100-fold, relative to an untreated oocyte. Similarly, in another embodiment, cAMP degradation may be decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100, relative to an untreated oocyte.
Agents that increase the concentration or level of intracellular cAMP may be in the form of proteins, antibodies, aptamers, antisense nucleic acids, antisense oligonucleotides, siRNAs, polypeptides, peptides, small molecules, drugs, polysaccharides, glycoproteins, and lipids. For example, agents which increase the synthesis or production of cAMP include activators of adenyl cyclase, such as forskolin. Examples of modulators which decrease cAMP degradation include inhibitors of phosphodiesterases such as theophylline. In one embodiment, the agent which increases the concentration or level of intracellular cAMP is one or more of forskolin, invasive adenylate cyclase and prostaglandin E2.
The method according to the first and second aspects of the invention may further include the step of exposing the oocyte to a ligand which induces nuclear maturation of the oocyte. In one embodiment, the concentration of the ligand is sufficient to overcome cAMP-induced meiotic arrest of the oocyte.
The ligand may be included as a component of the oocyte maturation medium, or may be a separate component or part of a separate media to which the oocyte is contacted with. In the latter case, the ligand may be added after step (b) of the methods according to the first and second aspects of the invention.
The ligand may be in the form of proteins, antibodies, aptamers, antisense nucleic acids, antisense oligonucleotides, siRNAs, polypeptides, peptides, small molecules, drugs, polysaccharides, glycoproteins, and lipids. For example, the ligand may include, but is not limited to, follicle stimulating hormone (FSH), epidermal growth factor (EGF) (including the EGF-like peptides amphiregulin and epiregulin), or functional isoforms thereof. It is contemplated that one or more ligands may be used in the methods of the invention.
In one embodiment, the ligand is FSH.
In one embodiment, the concentration of FSH is greater than 10 mlU/ml. For example, the concentration of FSH may be in the range between 10-200 mlU/ml.
In a further embodiment, the ligand is EGF.
In one embodiment, the concentration of EGF is greater than 1 ng/ml.
The first aspect of the invention contemplates an “assisted reproductive technology”. The term “assisted reproduction technology” as used throughout the specification is to be understood to mean any fertilization technique in humans and animals involving isolated oocytes and/or isolated sperm, including a technique using an oocyte or embryo cultured in vitro (for example in vitro maturation of an oocyte), in vitro fertilization (IVF; aspiration of an oocyte, fertilization in the laboratory and transfer of the embryo into a recipient), gamete intrafallopian transfer (GIFT; placement of oocytes and sperm into the fallopian tube), zygote intrafallopian transfer (ZIFT; placement of fertilized oocytes into the fallopian tube), tubal embryo transfer (TET; the placement of cleaving embryos into the fallopian tube), peritoneal oocyte and sperm transfer (POST; the placement of oocytes and sperm into the pelvic cavity), intracytoplasmic sperm injection (ICSI), testicular sperm extraction (TESE), and microsurgical epididymal sperm aspiration (MESA); or any other in vitro technique for producing embryos in humans and/or animals, such as nuclear transfer, parthenogenic activation and the use of totipotent cells.
In one embodiment, the assisted reproduction technology is used to produce a human embryo.
In another embodiment, the assisted reproduction technology is used to produce a bovine embryo.
In a method according to a first and second aspect of the invention an oocyte is first harvested or collected from an ovary of a subject. Oocyte collection can be performed according to standard techniques long known in the art. For example, see Textbook of Assisted Reproduction: Laboratory and Clinical Perspectives (2003) Editors Gardner, D. K., Weissman, A., Howles, C. M., Shoham, Z. Martin Dunits Ltd, London, UK; and Gordon, I. (2003) Laboratory Production of Cattle Embryos 2nd Edition CABI Publishing, Oxon, UK.
Most oocyte collection techniques involve the insertion of an aspirating needle into an ovarian follicle using transvaginal ultrasound. The aspirating needle is connected by tubing to a material collection trap and the collection trap, in turn, is connected to a suction source such as a manually operated syringe or an electromechanical vacuum source. Oocytes are typically isolated from multiple follicles. As such, the harvested oocytes represent a heterogenous population with regard to their developmental potential.
In one embodiment of the methods of the invention, the assisted reproduction technology comprises IVF. IVF relates to the fertilization of an oocyte in vitro, wherein the oocyte is isolated from the subject and incubated in liquid media to allow fertilization of the oocyte.
As indicated above, methods are well known in the art for collecting oocytes from suitable females and fertilizing the oocytes in vitro. It is contemplated that fertilisation of the oocyte will ideally occur greater than 24 hours, but no later than 60 hours, after the oocyte collection step, such that maturity of the oocyte is at a sufficient stage to maximise the success of subsequent steps in the IVF procedure.
Typically, the oocyte is held in the collection medium for 15-120 minutes at 35-39° C. The oocyte is then incubated in the maturation medium for 20-60 hours, typically in a 37-39° C. incubator with a suitable gas mixture. An example of a suitable gas mixture includes, but is not limited to, a gas mixture comprising of CO2 (1-10% by volume), balanced with air or with mixtures of O2 and N2 in proportions that sustain biological activity.
The oocyte is then held in the maturation medium for between 16 and 60 hours of maturation, typically between 24-50 hours of maturation and in some cases between 28-44 hours of maturation.
As will be appreciated, the time for maturation may differ between species. Generally the time of maturation will be the time that the meiotic stage of metaphase II is reached in these systems, and as such the time for maturation will typically be from 12 hours before to 18 hours after the median time to metaphase II stage of meiosis is reached. A suitable time would be from 3 hours before and 6 hours after the meiotic stage of metaphase II is reached. For example in the bovine setting the IVM time will generally be in the range from 18-24 hours in the absence of compounds that specifically inhibit meiosis progression to metaphase II.
In the human setting, the IVM time will generally be greater than 30 hours, and most usually between 30-50 hours.
In one embodiment, the IVM time in human is equal to or greater than 36 hours, for example between 36-48 hours.
In one specific embodiment, the IVM time in the human is equal to or greater than 40 hours, for example between 40-48 hours.
In one specific embodiment, the IVM time in the human is equal to or greater than 48 hours, for example between 48-50 hours.
The term “subject” as used throughout the specification is to be understood to include any female subject including a female human or a female mammal. Examples of suitable mammals include a primate, a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g. a dog or a cat), a laboratory test animal (e.g. a mouse, a rat, or a guinea pig), or any animal of veterinary or economic significance.
In one embodiment, the subject is a Bos indicus cow. In another embodiment, the subject is a Bos taurus cow.
In these and other aspects of the invention, the oocyte may be, for example, an oocyte that is part of a follicle, part of a cumulus oocyte complex (COO) or may be a denuded oocyte.
In one embodiment of the methods of the invention, the subject is a female human, the oocyte is a human oocyte, and the embryo is a human embryo.
In another embodiment of the methods of the invention, the subject is a cow, the oocyte is a bovine oocyte, and the embryo is a bovine embryo.
It will be appreciated that a phosphodiesterase inhibitor, an agent that increases intracellular cAMP concentration in the oocyte, and a ligand which induces nuclear maturation of the oocyte, may be used as supplements in oocyte culture media, including oocyte maturation media.
Accordingly, in a third aspect the present invention provides an oocyte culture medium comprising:
In one embodiment, the oocyte culture medium is an oocyte maturation medium.
In a fourth aspect the present invention provides an oocyte maturation medium comprising:
In some embodiments, the phosphodiesterase inhibitor in the maturation medium according to the third or fourth aspect of the invention may be as hereinbefore described. In one specific embodiment, the phosphodiesterase inhibitor may be cilostamide.
In some embodiments, the ligand according to the third or fourth aspect of the invention may be as hereinbefore described. In specific embodiments, the ligand may be FSH or EGF. The concentration of each ligand may be as hereinbefore described.
In some embodiments of the third and fourth aspects of the invention, the oocyte maturation medium is a human oocyte maturation medium or a bovine oocyte maturation medium.
In a fifth aspect, the present invention provides a combination product comprising the following components:
In one embodiment of this aspect of the invention, the agent increases intracellular cAMP production in the oocyte. For example, the agent may be forskolin. In another embodiment, the agent decreases intracellular cAMP degradation in the oocyte.
In some embodiments, the ligand in the maturation medium is FSH or EGF. The concentration of FSH is generally greater than 10 mlU/ml and the concentration of EGF is generally greater than 1 ng/ml.
In some embodiments of the fifth aspect of the invention, the first phosphodiesterase inhibitor and the second phosphodiesterase inhibitor are different. For example, the first phosphodiesterase inhibitor may be IBMX and the second phosphodiesterase inhibitor may be cilostamide.
In some embodiments, the first phosphodiesterase inhibitor and the second phosphodiesterase inhibitor are the same.
In some embodiments, the oocyte collection and maturation medium, or combination product thereof, of the invention may be used according to any one of the methods of the invention. For example, they may be used for collection and maturation of a human or a bovine oocyte.
The oocyte collection medium of the present invention may also be used for flushing, washing and holding the oocyte during the process of harvesting the oocyte from an ovary of the subject.
The oocyte collection medium may be comprised of one or more of NaCl, KCl, Mg2SO4, KH2PO4, Ca[lactate], NaHCO3, amino acids and derivatives, a protein such as serum albumin, glucose, pyruvic acid and an antibiotic. The medium includes a PDE inhibitor and an agent which increases the intracellular concentration of cAMP in the oocyte, as hereinbefore described.
In some embodiments, the PDE inhibitor in the oocyte collection medium is IBMX. In one embodiment, the concentration of IBMX is in the range between 5-5000 μM. Generally the concentration is in the range between 50-1000 μM.
In some embodiments, the agent which increases the intracellular concentration of cAMP in the oocyte is forskolin. In one embodiment, the concentration of forskolin is in the range from 1-2000 μM. Generally the concentration is in the range between 10-200 μM.
The oocyte maturation medium of the present invention allows maturation of the collected oocyte to a physical stage prior to fertilisation which simulates the maturity of an oocyte which is released by the ovary during ovulation in the reproductive cycle in vivo.
One example of a situation where application of this medium may be desired arises when it is necessary to treat the oocytes collected from the subject with oocyte-maturation hormones in vitro due to the subject's intolerance of such hormones. The invention contemplates holding the oocytes in the maturation medium for a period of at least 24 hours, but no more than 60 hours, following collection of the oocytes, to promote development prior to fertilization.
The oocyte maturation medium may be comprised of one or more of NaCl, KCl, Mg2SO4, KH2PO4, Ca[lactate], NaHCO3, amino acids and derivatives, a protein such as serum albumin, glucose, pyruvic acid and an antibiotic and includes a PDE inhibitor. The maturation medium may also include a ligand for inducing nuclear maturation of the oocyte, wherein the concentration of the ligand in the medium overcomes cAMP-induced meiotic arrest of the oocyte. However, as described above, it would be understood that the oocyte maturation medium need not contain the ligand, the ligand may be a separate component or part of a separate media.
The PDE inhibitor and ligand may be as hereinbefore described.
In one embodiment, the PDE inhibitor in the maturation medium is cilostamide. Generally, cilostamide is used at a concentration in the range from 0.01-100 μM and typically is in the range of 0.01-50 μM. For example in the bovine setting, a suitable concentration is between 10-30 μM, and in the human setting between 0.1-1.0 μM.
In a further specific embodiment, the ligand which induces nuclear maturation of the oocyte is FSH and/or EGF and the concentration of FSH and EGF is greater than 10 mlU/ml and 1 ng/ml, respectively and less than 500 mlU and 50 ng/ml, respectively, and preferably greater than 50 mlU and 5 ng/ml, respectively and less than 200 mlU and 20 ng/ml, respectively.
The components of the oocyte collection and maturation medium, and combination product thereof, of the present invention may be packaged separately in suitable containers (preferably sterilized) such as ampoules, bottles, or vials, either in multi-use or in unit forms. The containers may be hermetically sealed after being filled. The components may be in isolated form, or in purified or semi-purified form, and may contain additional additives for the stability and/or use of the components. Methods for packaging the various components are known in the art.
The collection and maturation media, and combination product thereof, of the present invention is suitable not only for use in humans, but also for culturing oocytes and embryos from other mammals. Thus, not only does the present invention have application for assisted reproduction technologies in humans, but it is also applicable to assisted reproduction techniques in non-human mammals, and other technologies of producing embryos in non-human mammals, such as the use of parthenogenic activation, somatic cell nuclear transfer and the use of totipotent stem cells.
In a sixth aspect the present invention provides a method of inducing oocyte maturation, the method comprising culturing an oocyte in a maturation medium comprising a phosphodiesterase (PDE) inhibitor and a ligand for inducing maturation of the oocyte, wherein the concentration of the ligand in the maturation medium overcomes cAMP-induced meiotic arrest of the oocyte, thereby maturing the oocyte.
The PDE inhibitor and the ligand, and concentrations thereof, according to this aspect of the invention may be as hereinbefore described. In one embodiment of this aspect of the invention, the phosphodiesterase inhibitor is cilostamide. In some embodiments, the ligand is FSH or EGF. Generally, the concentration of FSH is greater than 10 mlU/ml and the concentration of EGF is greater than 1 ng/ml.
In a seventh aspect, the present invention provides a method of inducing maturation of an oocyte which is in a state of meiotic arrest, the method comprising contacting the oocyte with a ligand at a concentration sufficient to overcome the meiotic arrest.
In one embodiment of this aspect of the invention the meiotic arrest is cAMP induced. The ligand, and concentration thereof, according to this aspect of the invention may be as hereinbefore described. In some embodiments, the ligand is FSH or EGF. Generally, the concentration of FSH is greater than 10 mlU/ml and the concentration of EGF is greater than 1 ng/ml.
In one embodiment of the second, sixth and seventh aspect of the invention, the method is part of an assisted reproduction technology. For example, the assisted reproduction technology comprises in vitro fertilisation.
Reference will now be made to the following experimental examples which embody the above general principles of the invention. However, it is to be understood that the following description is not to limit the generality of the above description. Thus, the invention encompasses any and all variations which become evident as a result of the teaching provided herein.
Oocyte quality plays an important role on embryonic development. For example, the inventors have shown that in vivo matured oocytes lead to higher blastocyst percentage than in vitro matured oocytes.
Unfortunately, current in vitro maturation techniques are suboptimal. In vivo, oocyte developmental competence is acquired gradually during the growth and development of the follicle. However, the inventors have shown that oocytes which have been retrieved from the follicle are capable of spontaneously overcoming meiotic arrest, thereby progressing to metaphase II before the cytoplasm has achieved full maturity.
Although immature oocytes can resume meiosis following isolation from the follicle, cytoplasmic maturation lags behind nuclear maturation. The inventors surmise that allowing more time for the immature oocyte to complete cytoplasmic maturation will improve oocyte developmental competence in vitro.
A possible strategy to improve developmental competence of oocytes is to keep them meiotically arrested in vitro for a prolonged period of time rather than allowing them to resume meiosis. Without wishing to be bound by theory, the inventors hypothesise that this delay gives the oocyte time to undergo cytoplasmic modifications (e.g. storage of mRNA and proteins, morphological changes, ultra structural remodeling) and might enhance synchronization of the starting population of immature oocytes to be used for downstream assisted reproduction applications.
In this regard, the inventors have tested the effect of including cAMP modulating agents in collection and handling (maturation) media on oocyte maturation kinetics such as intracellular cAMP levels, oocyte-cumulus cell gap junctional communications, nuclear maturation and embryonic development of cattle oocytes.
Unless otherwise specified, all chemicals and reagents were purchased from Sigma (St. Louis, Mo., USA).
Bovine ovaries were collected from local abattoirs and transported to the laboratory in warm saline (30-35° C.). All ovaries collected on a day were pooled and used at random. Antral follicles (2 to 8 mm in diameter) were chosen for aspiration by using an 18-gauge needle and a 10 ml syringe.
Oocytes were aspirated with their cumulus vestments intact (COCs). A subset of the COCs were denuded of their cumulus vestment prior to assay (DO). Aspiration and subsequent selection procedures (pre-IVM phase) were performed for 2 hours where oocytes (COCs and DOs) were processed in various media (termed “pre-IVM phase media” or “pre-IVM media”). These included processing of oocytes either in follicular fluid, or in two types of collection medium. The collection medium for oocyte aspiration and selection included: (1) a bovine oocyte collection medium (termed “Bovine VitroMat”, Cook Australia, Eight Mile Plains, Old, Australia) supplemented with 50 μg/ml gentamycin and 0.2 mg/ml fatty acid-free bovine serum albumin (FAF-BSA; ICPbio Ltd, Auckland, NZ); or (2) the same medium supplemented with two cAMP modulators, namely an adenylate cyclase activator, forskolin (100 μM), and a non-specific PDE inhibitor, 3-isobutyl 1-methylxanthine (IBMX) (500 μM).
Millimolar stock concentrations of the cAMP modulators were stored at −20° C. dissolved in anhydrous dimethyl-sulphoxide (DMSO). Solutions containing modulators were diluted fresh for each experiment.
At the end of pre-IVM phase, intact COCs with compact cumulus vestments greater than five cell layers and evenly pigmented cytoplasm were selected under a dissecting microscope. Before in vitro maturation (IVM), COCs were washed twice in respective pre-IVM phase media, followed by two washes in IVM medium (see below).
The basic oocyte maturation medium (also referred to as the “IVM medium” or “IVM phase medium”) used for the IVM phase was a bovine maturation medium (termed Bovine VitroMat, Cook Australia), a medium formulated to closely replicate the ionic composition of bovine follicular fluid. Where indicated, the type-3 PDE specific inhibitor cilostamide (20 μM; Biomol Plymouth Meeting, Pa.) or the epidermal growth factor receptor (EGFR) kinase inhibitor, AG1478 (Alexis Biochemicals, San Diego, Calif.), were added to the IVM medium from a millimolar stock solution stored at −20° C. dissolved in DMSO. All IVM treatments were supplemented with 0.1 lU/ml follicle stimulating hormone (FSH) (Puregon, Organon, Oss, Netherlands). COCs were cultured in pre-equilibrated 300 μl drops overlaid with mineral oil and incubated at 39° C. with 5% CO2 in humidified air.
At 24 or 30 hours after IVM, COCs were washed twice using Bovine VitroWash (Cook Australia), and transferred to insemination dishes containing in vitro fertilisation (IVF) medium (Bovine VitroFert, Cook Australia) supplemented with penicillamine (0.2 mM; Sigma), hypotaurine (0.1 mM; Sigma), and heparin (2 mg/ml; Sigma). Frozen semen from a single bull of proven fertility was used for insemination. Briefly, thawed semen was layered over a discontinuous (45%: 90%) Percoll gradient (Amersham Bioscience) and centrifuged for 20-25 minutes at 700 g. The supernatant was removed and the sperm pellet was washed with 500 μl Bovine VitroWash and centrifuged for a further 5 minutes at 200 g. Spermatozoa were resuspended with IVF medium (Bovine VitroFert), then added to the fertilization media drops (Bovine VitroFert, supplemented with 0.01 mM heparin, 0.2 mM penicillamine and 0.1 mM hypotaurine) at a final concentration of 1×106 spermatozoa/ml. COCs were inseminated at a density of 10 μl of IVF medium per COC for 24 hours, at 39° C. in 6% CO2 in humidified air. COCs were removed by gentle pipetting 23-24 hours post insemination and five presumptive zygotes were transferred into 20 μl drops of pre-equilibrated Cook Bovine VitroCleave medium (Cook Australia) and cultured under mineral oil at 38.5° C. in 7% O2, 6% CO2, balance N2, for five days (day 1 to day 5). On Day 5, embryos in groups of 5-6 were transferred to 20 μl drops of pre-equilibrated Bovine VitroBlast (Cook Australia) at 38.5° C. overlaid with mineral oil and cultured to Day 8. Embryos were assessed for quality at Day 8 according to the definitions presented in Stingfellow and Seidel, 1998, Manual of the International Embryo Transfer Society. In. (IETS: Savoy, Ill., USA) and were performed independently and blinded by an experienced bovine embryologist.
Blastocysts were placed into 0.5% pronase at 37° C. to remove the zona, followed by a brief wash in 4 mg/ml poly-vinyl alcohol (PVA) in phosphate-buffered saline (PBS/PVA). Zona-free blastocysts were then incubated in 10 mM trinitrobenzene sulfonic acid in PBS/PVA at 4° C. for 10 minutes. Blastocysts were subsequently incubated with 0.1 mg/ml anti-dinitrophenol-BSA antibody (Molecular Probes, Eugene, Oreg., USA) at 37° C. for 10 minutes, and then placed in guinea pig serum with propodium iodide for 5 minutes at 37° C. Blastocysts were washed and incubated in 10 μg/ml propidium iodide for 20 minutes at 37° C. (to stain the trophectoderm), followed by 4 μg/ml bisbenzimide (Hoechst 33342; Sigma-Aldrich) in 100% ethanol at 4° C. overnight (to stain both the inner cell mass (ICM) and trophectoderm). Blastocysts were then whole mounted in a drop of 80% glycerol in PBS on microscope slides and coverslips were sealed with nail polish. Blastocysts were then examined under a fluorescence microscope (Olympus, Tokyo, Japan) at 400× equipped with an ultraviolet filter and a digital camera attached to determine total and compartment cell counts where inner cell mass (ICM) nuclei appeared blue and trophectoderm (TE) nuclei stained pink.
Cyclic AMP content of COCs and denuded oocytes (DOs), i.e. those oocytes cultured as COCs but denuded of their cumulus vestment prior to assay, were measured using a radioimmunoassay method described and validated previously (Reddoch et al., 1986, Endocrinology 119: 879-886). After time end-points, 6-10 COCs and 21-24 DOs were washed in VitroCollect (Cook Australia), transferred to 0.5 ml of ethanol (100%) and stored at −20° C. Before cAMP measurements, samples were vortex agitated for 30 seconds and then centrifuged at 3000 g for 15 minutes at 4° C. Briefly, supernatants were collected, evaporated, resuspended in assay buffer (50 mM sodium acetate, pH 5.5) and acetylated by the addition of triethylamine (AJAX Chemicals, Sydney, Australia) and acetic anhydride (BDH Laboratory Supplies, Poole, England) 2:1 v/v. cAMP was measured in duplicates after appropriate dilution. 125I-labelled cAMP (specific activity of 2175 Ci/mM) and cAMP antibody (as prepared by Reddoch et al supra) were added to samples and left overnight at 4° C. The following day, 1 ml cold 100% ethanol was added and the samples were centrifuged at 3000 g. The supernatant was removed and the pellet dried and counted using a gamma counter. Duplicates of known concentration samples were used to produce a standard curve (4-1024 fmol cAMP).
At the end of the incubation, COCs were denuded and oocytes were fixed for 30 min in 4% paraformaldehyde in PBS (pH 7.4). Oocytes were then permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 1 hour, then transferred to 0.001% 4′,6-diamidino-2-phenylindole (DAPI), a fluorescence stain for nuclear material, for 15 min. Oocytes were rinsed 3 times in PBS+0.03% BSA, mounted on slides and evaluated for nuclear status at 400× using an Olympus fluorescence microscope. Stages of germinal vesicle (GV) and subsequent meiotic development were assessed based on known techniques (Chohan and Hunter, 2003, Anim. Reprod. Sci. 76: 43-51). In brief, GV chromatin of bovine oocytes was classified into: GV I—condensed filamentous chromatin around the nucleolus and nuclear membrane; GV II—filamentous chromatin surrounding the nucleolus; GV III—filamentous chromatin clumps are distributed in the nucleus and the nucleolus has disappeared; GV IV—chromatin has condensed into a thick clump; Early diakenesis-chromatin beginning to condense into a single lump; Diakenesis-chromatin has condensed into a single lump; Metaphase I—tetrads are aligned on the spindle; and Metaphase II—the metaphase chromatin is evident as well as a small chromatin-containing polar body.
Cumulus-oocyte gap junctional communication (GJC) was measured by quantitative fluorescence microscopy of calcein transfer to the oocyte as described previously (Thomas et al., 2004, In “Biol. Reprod.”, pp 1142-1149). GJC was measured either after 2 hours of pre-IVM phase or followed by an additional 3 hours of IVM with or without cilostamide (20 μM). A mean number of 10-12 oocytes were used in each treatment group in each of four replicate experiments. After culture, COCs were transferred to a solution of 1 μM calcein-AM (3′,6′-di(O-acetyl)-2′,7′-bis[N,N-bis(carboxymethyl)amino methyl]-fluorescein, tetraacetoxy methyl ester; C-3100; Molecular Probes; Eugene, Oreg., USA) freshly prepared in a BSA-free Bovine VitroCollect (Cook Australia) supplemented with polyvinyl alcohol (PVA; 0.3 mg/ml). COCs were cultured with the dye for 15 minutes, and then unincorporated dye was removed by three washes in calcein-AM-free Bovine VitroCollect (Cook Australia) and incubated for a further 25 minutes to allow transfer from the cumulus cells to the oocyte. Prior to fluorescence microphotometry, COCs were completely denuded of their surrounding cumulus cells using vigorous pipetting so that only dye confined within the denuded oocyte after transport via gap junctions was measured. Within 30 minutes of denuding, the intra-oocyte fluorescence emission of calcein in oocytes was measured using a fluorophotometric-inverted microscope (Leica, Wetzlar, Germany).
Statistical analyses were conducted using Prism 5.00 GraphPad for Windows (GraphPad Software, San Diego, Calif., USA). Statistical significance was assessed by ANOVA followed by either Dunnett's or Bonferroni's multiple-comparison post-hoc tests to identify individual differences between means. All values are presented with their corresponding standard error of the mean (SEM).
cAMP Content of Bovine COCs and Dos During Pre-IVM Phase
The aim of the experiment in
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Denuded oocytes (DOs) were used to evaluate the effect of the surrounding cumulus cells on intra-oocyte cAMP levels (
Effect of cAMP Modulating Agents in Pre-IVM Phase and Type-3 PDE Inhibition on Spontaneous Oocyte Maturation (GV/GVBD)
Bovine COCs were incubated for 2 hours in the pre-IVM phase media as hereinbefore described, followed by 7 hours culture in the presence of FSH, and in the presence or absence of cilostamide (20 μM). Oocytes were then fixed and assessed for meiotic progression and classified as GV (germinal vesical intact—still under meiotic arrest) or GVBD (germinal vesical breakdown—resumption of meiosis). A mean number of 45 oocytes were used in each treatment group and time-point from four replicate experiments. The presence of letters (a or b) above each column indicate a statistical difference between the means as determined by ANOVA analysis followed by Bonferronni's post hoc test, P<0.05.
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After 5 hours (2 hours of pre-IVM and 3 hours of IVM+cilostamide) collected oocytes were already arrested at their destined GV stages prior to the IVM incubation (
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Effect of cAMP Modulating Agents During Pre-IVM and IVM on Oocyte-Cumulus Cell Gap Junctional Communication
The oocyte-cumulus gap junctional communication (GJC) assay was performed at the end of pre-IVM phase and at the end of 5 hours of oocytes culture (2 hours of pre-IVM and 3 hours of IVM±cilostamide). As seen in the columns 1 to 3 of
Moreover, levels of gap junctional communication dramatically decreased after 3 hours of IVM culture (post pre-IVM phase) when COCs were collected either in pure follicular fluid, or collection medium without cAMP modulators. The exception were COCs which were processed in collection media supplemented with cAMP modulators during pre-IVM phase, regardless of the presence or absence of cilostamide during IVM (P<0.05) (
The presence of letters (a, b, g, h or x) above each column in
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Intracellular Concentration of cAMP in Oocytes after Pre-IVM and IVM Phases
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In this regard,
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Effect of cAMP Modulating Agents During Pre-IVM and IVM Phases on Cleavage and Development to the Blastocyst Stage
As shown in Table 1, inclusion of cAMP modulators in collection media during the pre-IVM phase improves cleavage rates compared to absence of the modulators (89±2.0% vs. 78±2%, respectively, P<0.05), and improves blastocyst development (32±3% vs. 26±3%, P<0.05) when oocytes are matured in standard IVM medium in the presence of FSH for 24 hours.
a,bValues with different superscripts within the same column represent a statistically significant difference (P < 0.05). Values are expressed as (mean ± SEM).
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However, oocytes fertilised at 30 hours yielded a dramatic increase in blastocysts when pre-treated with cAMP modulating agents and matured with FSH+cilostamide (increase from 42% to 69% blastocysts)(
Effect of cAMP Modulating Agents During Pre-IVM and IVM on Blastocyst Cell Numbers
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The current study was undertaken to test the hypothesis that developing a process for inducing oocyte maturation in vitro by manipulating intracellular cAMP levels in the oocyte immediately following collection and during the maturation process, synchronizes nuclear and cytoplasmic compartments, thereby leading to improved developmental competence of maturing oocytes.
The participation of cAMP in controlling oocyte maturation in mammals has been shown in the past few years. Nevertheless, the molecular mechanisms of the series of coordinate events guided by cAMP have not been clearly defined. The experimental data as hereinbefore described showed that the efficacy of PDE inhibitors may be enhanced by modulating cAMP levels during the period preceding IVM. In the first part of this study (
The different collection conditions used in this study have been found to exert a profound effect on oocyte intracellular cAMP levels, oocyte meiotic progression, oocyte-cumulus cells gap junctional communication and finally oocyte developmental competence. The present results demonstrated that modulating cAMP for a short period of time from the time of COC isolation can lead to sustained gap junctional communication between cumulus cells and oocyte (
The increase in gap junctional communication observed in this study, when cAMP levels were modulated during the pre-IVM phase, was due to prolonged gap junction conductance and/or the prevention of gap junction removal from the cumulus cell-derived meiosis-modulating factors to the oocyte, thereby delaying GV configurations or GVBD after 7 hours of oocyte culture (
An intriguing question was how the exogenous modulation of cAMP can evoke a stimulatory or inhibitory response in germinal and somatic compartments. In the described experiments, the intra-oocyte cAMP level was 15 fold higher when cAMP modulators were present in culture media during the pre-IVM and IVM phases compared to other treatments (
Therefore, the aim of one of the experiments, the results of which are shown in
This observation may be the first model described in a ruminant species for induced oocyte maturation, as opposed to spontaneous maturation where mechanically removing oocytes from their follicle results in spontaneous meiotic resumption. However, although it is well established that in vivo, meiotic resumption occurs due to the pre-ovulatory surge of gonadotrophins, the mechanism(s) by which this occurs is not fully understood.
When oocytes were cultured in the presence of cilostamide, FSH treatment resulted initially in an inhibitory effect that later becomes stimulatory (
The experiments reported here also show that modulating oocyte cAMP levels during pre-IVM and IVM increased the capacity to support early development. As shown in Table 2, modulating cAMP during pre-IVM alone, or during IVM alone, generated a blastocyst percentage around 30%. However, including cAMP modulators in both phases led to a significant increase (up to 3 fold) in blastocyst percentage (69%). It is interesting to note that in the absence of cAMP modulators in the pre-IVM, and culturing the oocytes in the presence of cilostamide decreased blastocyst percentage (29%) compared to their presence in the pre-IVM media (69%). These current results are in parallel with previous reports on coasting in vivo, in which 80% blastocysts have been developed, when animals received leutinising hormone 6 hours before oocyte aspiration.
Collectively, the evidence presented in this example demonstrated, for the first time, that intracellular cAMP levels during the pre-IVM phase has a profound effect on oocyte developmental competence and blastocyst quality, starting from COC removal from the follicle to the end of maturation. A stable modulation of intracellular cAMP levels within a physiological range in the oocyte and cumulus cells during in vitro maturation can create an induced maturation in vitro that in turn can lead to acquisition of high developmental potential.
The figure shows that there was a significant increase in cAMP concentration in in vivo matured oocytes following the hCG treatment, whereas spontaneously matured in vitro matured oocytes had low levels of cAMP, which fall even further. Such a loss in cAMP with spontaneously matured oocytes appeared to be associated with reduced developmental competence.
This figure demonstrates that the concentration of both PDE inhibitor and FSH together can regulate the timing of induced maturation.
=MEM+FSH) following 18 and 22 hours of culture. Oocytes were then assessed for meiotic progression and classified as MII stage.
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This improved oocyte developmental competence is also reflected in blastocyst quality as shown in
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The concept of Induced-IVM developed using animal oocytes was then examined using human oocytes.
Adaptation of the technology to human requires determination of: (1) optimal concentration of cilostamide to use during a human IVM phase; (2) optimal concentrations of forskolin and IBMX to use during a human pre-IVM phase; (3) the interacting effects of these agents on the duration required to complete oocyte maturation; and (4) the effect of denuding time (somatic cell removal) on post-fertilisation embryo development.
Immature human cumulus-oocyte complexes (COC) were collected from young, healthy women (not from IVF patients). These women received minimal ovarian stimulation (typically 300-500 lU FSH/cycle, no hCG), as is typically conducted in one variant of a routine clinical IVM cycle. Oocyte pick-up was performed when the lead ovarian follicle reached 12 mm. Immature oocytes were then stratified to various treatments during either the Pre-IVM or IVM periods, as detailed below. In addition, time of removal of somatic cells from oocytes (denuding) during the oocyte maturation period was examined. The following trials were conducted:
At the oocyte pick-up, COC were collected immediately into pre-IVM treatments in VitroCollect medium (Cook Australia, Brisbane, Australia) and held for one hour. Following pre-IVM, COC were washed and then transferred to IVM treatments (e.g. dose of cilostamide) in VitroMat medium (Cook) and matured under standard conditions. At the various times post-denuding, oocytes were monitored for maturation (polar body [PB] extrusion). At the end of maturation (48 hours) a final meiosis score was given. As oocytes matured they were inseminated using standard intra-cytoplasmic sperm injection (ICSI) procedures using donor sperm. 24 hours later fertilisation rates were determined. Embryos were cultured for up to 6 days until developmental arrest using standard procedures.
The results are shown below in Table 3.
Germinal Vesicle Breakdown (GVBD) is a marker that oocytes have resumed meiosis. These results show that 1.0 μM is an inhibiting dose of cilostamide for human oocytes. 0.1 μM is non-inhibiting so this dose was used during IVM for the study reported immediately below.
Effect of Pre-IVM cAMP Modulating Agents with 0.1 μM Cilostamide During Human IVM
The results are shown below in Tables 4 to 6.
These results demonstrate the principal of Induced-IVM using human oocytes. The results show that, when oocytes are treated during the IVM phase with 0.1 μM cilostamide in the presence of FSH, Pre-IVM with IBMX+FSK increases the proportion of mature (64.9% PB-stage) oocytes relative to standard (the control; 41.7% PB-stage) pre-IVM oocyte collection conditions.
The results are shown below in Table 7.
Here we have generated human embryos from oocytes matured using the Induced-IVM system. Despite low numbers so far, the system is proving highly efficient at generating embryos with between 35-40% progressing to the blastocyst stage. This is substantially higher than the standard clinical rates. A critical factor in the Induced-IVM system is the duration of IVM or the time of insemination. The results here suggest that denuding between 44-48 hours adversely affects embryo development.
Finally, it will be appreciated that various modifications and variations of the methods and compositions of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the present invention.
This international patent application claims priority from U.S. provisional patent application 61/178,318 filed on 14 May 2009, the contents of which are herein incorporated by this reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU2010/000569 | 5/14/2010 | WO | 00 | 6/20/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61178318 | May 2009 | US |
