WO2002074900A1

WO2002074900A1 - Culture system for embryos

Info

Publication number: WO2002074900A1
Application number: PCT/AU2002/000328
Authority: WO
Inventors: George Anthony Thouas; Gayle Maree Jones; Alan Osborne Trounson
Original assignee: Monash University
Priority date: 2001-03-21
Filing date: 2002-03-21
Publication date: 2002-09-26
Also published as: AUPR386101A0

Abstract

The present invention provides a culture system for an embryo, said system comprising: a tube having at least two open ends wherein one end is capable of receiving an embryo, said tube having a diameter capable of drawing and maintaining an embryo in the tube in a volume of medium suitable for culturing the embryo.

Description

CULTURE SYSTEM FOR EMBRYOS

The present invention relates to a culture system for embryos, methods of culturing embryos and to further production of organisms from the embryos cultured by the present method.

INTRODUCTION

In an emerging field of in vitro embryo production and especially in somatic cell nuclear transfer, embryo culture requires a special environment. Physical, chemical and biological factors influencing the development are only partially understood.

The culturing of early embryos to an implantation competent blastocyst has long been conducted in livestock and small animal species, and more recently in the human, by methods involving sterile cultivation within small volumes of physiological nutrient media based on fluids found in the reproductive tract.

The pregnancy rates resulting from these methods is highly variable but has generally been in the range of 20-30% for bovine and human embryos and over 50% for certain mouse strains. While it has managed to provide some success, the process of culturing embryos could be improved so that the chances of implantation of a healthy embryo are greatly improved.

At present, traditional culture methods employ multiple manipulation steps, each step subjecting the embryo to further potential damage which may severely reduce the chances of successful implantation at the blastocyst stage. The manipulation steps include sequential media changes, micromanipulation and morphological assessments for which embryos need to be displaced from their static culture environment. These steps impose small interruptions to pH, osmotic, temperature and atmospheric homeostasis that can result in cumulative metabolic and genetic damage that ultimately effects embryo growth and viability [McKiernan and Bavister, (1990), Biology of Reproduction, 43, 404- The embryo is a highly sensitive organism and whilst current laboratory equipment such as computer-controlled incubators and heated microscope stages have been developed to minimize environmental stresses, basic components such as plastic wear and mineral oil used in traditional culturing methods are still a potential source of embryo toxicity [Boone and Shapiro, (1990) Theriogenology 33, 23-50; Gorrill et al (1991), Fertility & Sterility, 55, 345-354].

During the culturing period, the embryo, generally cultured in a petridish, is subjected to fluctuations in temperature and evaporation of medium unless mineral oils are utilised as a direct medium overlay to act as a physical gas permeable barrier. These oils are routinely used in large quantities and in themselves pose problems to the embryo generally on a toxicity level.

Aggregation of embryos in standard cuWure conditions is often a further problem. To avoid aggregation, embryos should be physically separated from each other, a situation which results in decreased developmental rates, supposedly by the dilution of the autocrine and paracrine factors which support embryo development in vivo or when cultured in groups.

In other situations, before compaction, single embryos cultured without the zona pellucida may disaggregate and blastomeres may attach to the bottom of the culture dish (Wells and Powell, 2000). Several techniques have been developed for the problem including small drops of solutions covered with oil, agar embedding, or small holes (well of the well, WOW) made with a needle on the bottom of the tissue culture dish (Boediono et al., 1999; Vajta et al., 2000).

Moreover, some new, simplified nuclear transfer techniques require zona-free embryo culture after manipulation and fusion (Peura et al., 1998), a situation where the development of embryos is even more handicapped.

Accordingly, it is an object of the present invention to overcome or at least alleviate some of the problems of the prior art. SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a culture system for embryos, said system comprising: a tube having at least two open ends wherein one end is capable of receiving an embryo, said tube having a diameter capable of drawing and maintaining an embryo in the tube in a volume of medium suitable for culturing the embryo.

In another aspect of the present invention, there is provided a method of culturing embryos, said method comprising the steps of: obtaining an embryo in medium; obtaining a tube having at least two open ends and wherein one end is capable of receiving the embryo, said tube having a diameter capable of drawing and maintaining the embryo in the medium in the tube; drawing the embryo into the tube; and incubating and culturing the embryo in the tube.

In a further preferred aspect of the present invention, there is provided a blastocyst or preferably of two different species cultured by the methods described herein. The blastocyst may be cultured past the stage of hatching and show signs of trophectoderm cell outgrowth and attachment indicating a level of implantation competency. Accordingly, there is also provided a blastocyst ready for implantation that has been cultured using the methods provided above.

In a further aspect of the present invention, there is provided a method of obtaining an animal said method comprising the steps of: obtaining an embryo developed to a blastocyst by a method comprising culturing the embryo in a tube having at least two open ends wherein one end is capable of receiving an embryo, said tube having a diameter capable of drawing and maintaining an embryo in the tube in a volume of medium suitable for culturing the embryo; obtaining a receptive animal capable of incubating an embryo to term; implanting the blastocyst into the receptive animal; and allowing the receptive animal to incubate the blastocyst to term.

In another aspect of the present invention, there is provided an animal obtained by the methods described.

The present invention also contemplates an efficient storage of embryos, particularly those having developed to the blastocyst stage. The culturing of the embryo in the tubes also facilitates freezing and storage of embryos ready for implantation. This is particularly useful for embryo transfer into livestock.

FIGURES

Figure 1 shows an orientation of mouse embryos within a capillary tube.

Figure 2 shows a multiple culture system.

Figure 3 shows a possible design for a vertical disposable capillary rack which can be tilted horizontally on a microscope stage for embryo scoring and which can be labelled for human patient identification.

Figure 4 shows the orientation of the capillary rack on a microscope stage relative to the eyepiece of the microscope.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect of the present invention, there is provided a culture system for an embryo, said system comprising: a tube having at least two open ends wherein one end is capable of receiving an embryo, said tube having a diameter capable of drawing and maintaining an embryo in the tube in a volume of medium suitable for culturing the embryo. Disposable capillary tubes have only been routinely used as bench-top instruments to handle embryos outside of an incubator. There is no recorded published literature to date which claims to have used these capillaries for the purpose of pre-implantation embryo culture. Embryo culture vessels have classically been in the form of a dish, well or test-tube which is open-ended on one side and therefore acts as the most convenient mode for temporarily containing and visualizing embryos. The culture of somatic cell and microbial cell lines using capillaries as a means of visualizing and measuring growth rates of large cell populations has not been used to date.

The culture system is designed so that the embryo contained within the culture system can develop to a further advanced stage of development, preferably to the mature blastocyst stage. However, any stages such as early cleavage or morula may be selected after observing the development of the embryo directly in the tube. The embryo can be removed at any time depending on the desired developmental stage. This has many advantages since the embryo, once contained and matured in the culture system, is immediately available with minimal manipulation for implantation into an animal at a suitable stage of development. The stage of development may be chosen to coincide with an animal in a receptive stage of breeding.

An embryo, as used herein, is at a stage of development from the moment of conception or fertilisation of a female gamete such as an egg or ovum with a male gamete such as a spermatozoon. Therefore, the embryo may be any stage of development from the zygote to the blastocyst. The embryo may be a product of fertilisation of normal gametes or it may be a transgenic embryo that has been genetically manipulated by methods known to the skilled addressee. The system is suitable for culturing of any embryo of any animal or human.

The tube of the culture system may be of any diameter providing it can hold and maintain an embryo in the medium within the tube so that the embryo may develop within the drawn medium. Therefore, the tube must be capable of providing sufficient capillary action and surface tension to the medium to maintain the medium vertically within the tube and also to draw the embryo up the tube. Preferably, the tube has an internal diameter of 200-250 μm. These may be capillary tubes. Narrower ranges of internal diameter may be in the order of 200μm or less with the size of an embryo being the limiting factor. Narrower ranges such as 200μm or less may be of benefit for full promotion of development including zona pelucida thinning and full blastocyst expansion.

The effect that is achieved by these thin tubes is such that the embryos are exposed to a micro environment which is akin in physical terms to an oviduct where pre-implantation embryos are in close apposition to the luminal wall on all sides.

The external diameter of the tube will be dependent upon the material from which the tube is made. Preferably, the external diameter is kept to a minimum and as close in diameter to the internal diameter thereby providing a thin outer shell or wall for effective temperature transfer into the medium. The final effect, when incubated, will provide an artificial oviduct for development of the embryo.

Whilst the diameters (internal and external) may effect the holding capacity of the tube for maintaining the embryo and medium, the length of the tube may also contribute to the culture system. The length must be of a suitable length such that, in conjunction with the diameter of the tube, is capable of drawing and maintaining the embryo culture in medium in the tube. The length must also be of a suitable dimension to hold the embryo and medium in the tube so that the medium is exposed to air from either end of the tube. The correct length allows vertical culture without downward migration of the medium column.

The volume of medium will depend on the diameter of the capillary as well as the viscosity and temperature of the medium. These parameters may be considered with the teachings of general fluid mechanics to determine suitable volumes. However, sufficient fluid must be introduced into the tube to allow for culturing of an embryo. As little as one microliter in a tube of internal diameter of 200μm or less may be used. The tube is preferably of a grade which poses minimal toxicity to the embryo. Most preferably it is uncoated or treated, acid washed borosilicate laboratory grade glass. Plastic tubes are not ideal as the amount and type of plastic may be toxic to the embryo particularly since small volumes of medium are used and the plastic to medium ratio may be high. Another suitable type of tube includes quartz glass which, although less economical, is more pure than common commercially available sources of glassware.

Surface coatings on the inside of the tube may also be applied. Suitable coatings that enhance or promote development may be used. For instance, extracellular matrix macromolecules such as fibronectins, proteoglycans and collagens or polycarbonates to promote blastocyst growth and attachment competence during prolonged culture may be utilised. Examples of polycarbonates include several types of extruded polystyrenes or transparent nylons.

The volume of media including the embryo may be drawn into the tube by capillary action dependent upon surface tensions, or it may be aided by a suction means to draw the fluid up the tube. Such suction means may include a small rubber teat, or manual or power driven winding devices. However, providing the media and embryo are drawn up the tube and capable of being maintained in the tube for further development, it is within the scope of the present invention.

Any number of embryos may be cultured in the culture system. However, it is dependent upon the volume of media drawn into the tube. Multiple embryos may be cultured this way although will reflect the precise growth requirements of the species embryo that is grown. Preferably, two embryos are cultured per tube as this is the most conducive to blastocyst formation.

Figure 1 illustrates a preferred orientation of embryos in a capillary once the embryos are introduced into the capillary which is held upright. The embryos (1) are in close apposition to each other in a column of medium (3) and fall toward the air/medium interface (2) established by surface tension between the medium and the capillary wall (4).

Multiple cultures per tube may also be set up where each culture is separated by an air cushion (7) such that each culture is in close apposition to each other and cultured in parallel. Figure 2 illustrates a multiculture system where embryos (5) are cultured in multiple portions of media (6) separated by an air cushion (7) down the length of the tube.

The culture system may comprise one tube or multiple tubes wherein each tube contains one or more embryos as single or multiple cultures. The system may be mounted on a suitable holding means such as a stand or frame designed for direct observation for monitoring embryonic development. Figure 3 illustrates a holding means comprising a stand (8) having multiple perforations (9) through which a capillary tube (10) is inserted and mounted vertically. The stand may be transferred to a microscope stage (11) for further observation.

In another aspect of the present invention, there is provided a method of culturing an embryo, said method comprising the steps of: obtaining an embryo in medium; obtaining a tube having at least two open ends and wherein one end is capable of receiving the embryo, said tube having a diameter capable of drawing and maintaining the embryo in the medium in the tube; drawing the embryo into the tube; and incubating and culturing the embryo in the tube.

As described above, this method of embryo culturing may be suitable for any animal including humans. The embryo may be a normal embryo or it may be genetically modified to provide a transgenic embryo and ultimately a transgenic animal.

The embryo is best obtained just after conception or fertilisation and at the zygote stage. Most preferably, it is obtained at the one cell zygote stage. This stage may be obtained by methods available to the skilled addressee or it may be obtained by methods outlined by DK Gardner and Lane M, Chapter 5: Embryo Culture Systems in Handbook of In vitro Fertilisation, Trounson AO and Gardner DK, CRC Press, Boca Raton USA, 1993.

Once the embryo or zygote has been retrieved and washed in a holding medium, it is then allocated to a standard culture dish [Whitten and Biggers (1968), Journal of Reproduction and Fertility, 17, 399-413] comprising 20-30 μl drops of culture medium with an oil overlay. This culture dish may be pregassed by equilibration within a CO₂ incubator to a suitable pH and temperature for receiving embryos which are immediately drawn up from here into capillaries for culture.

The medium may be any medium suitable for culturing embryos to the blastocyst stage, and these are familiar to those skilled in the art. However, a simple medium without supplementation is sufficient. Simple media such as Mouse tubal fluid (MTF) medium [Gardner and Leese (1990), Journal of Reproduction and Fertility 88, 361-70] containing 4 mg/ml bovine serum albumin (BSA) is sufficient for mouse embryos, however other species which are more sensitive to environmental insults may require a complex medium with additional nutrient supplements. In these instances, media such as Gardner's media (G1 and G2) and Biggers' KSOM medium may be sufficient for use in livestock, primate and human culture systems. However, other media familiar to those skilled in the art are also applicable here.

The tube may be as described above for the culturing system. Most importantly, the tube must have holding capacity such that the medium surrounding the embryo is held in the tube generally by capillary action and surface tension so as to maintain the embryo within the tube. The media is cushioned in the tube by an air/media interface from either end of the tube, and occasionally by a small plug of oil.

The embryo or zygote may be drawn or taken up into the tube under passive capillary action or by an active pressure drawing the fluid up the tube. The second method may be employed providing the tube has sufficient capability to maintain and hold the medium and embryo or zygote in the tube.

Once the embryo or zygote is drawn into the tube, it is preferred that the tube is held vertically rather than horizontally so as to create a cushion on the air/medium interface. Horizontal incubation may also be employed although the vertical orientation is most preferred.

The embryos or zygotes may be cultured from this point with minimal manipulation until an appropriate developmental stage is reached. Preferably, the embryo is left to develop to the blastocyst stage. As described above, multiple incubations may also be employed.

Once the embryo has developed to an appropriate developmental stage, preferably the blastocyst stage in the tube, the tube may be used directly for embryo transfer into livestock or humans or it may be removed and introduced into the livestock or humans by conventional methods. However, the advantage of this invention is that multiple manipulations can potentially be avoided by transferring directly from the capillary tube.

In another aspect of the present invention, there is provided a method of inducing a pregnancy, said method comprising: obtaining an embryo by a method comprising culturing the embryo in a tube having at least two open ends wherein one end is capable of receiving an embryo, said tube having a diameter capable of drawing and maintaining an embryo in the tube in a volume of medium suitable for culturing the embryo; obtaining a receptive mother capable of incubating an embryo to term; and implanting the embryo into the mother.

The embryos developed by the methods of the present invention have a capacity to implant and induce pregnancy. The glass oviduct system is comparable to other systems but provides the added advantage of reduced manipulation for implanting the embryo into the mother. Preferably the embryo is a blastocyst and has been cultured in the glass oviduct system to this stage.

The receptive animal is an animal or mother capable of carrying a foetus to term and may be a female animal in a breeding cycle or artificially induced to accept an embryo and to carry the foetus to term. By "artificially induced" it is meant that pharmaceutical grade synthetic hormones such as follicle stimulating hormone (FSH) in conjunction with luteinizing hormone (LH), using prescribed stimulation protocols for a given species, be injected to prepare the womb for receiving the blastocyst.

This aspect of the invention includes the induction of pregnancy in an IVF process where the oocyte has been fertilised in vitro and cultured to a stage which can be implanted into the host mother. The mother may develop the embryo to term. In another aspect of the present invention, there is provided an animal obtained by the methods described.

There are several applications and advantages to the culture techniques described above. These may include:

(a) an improved culturing of embryos from domestic animal species and humans.

(b) an enhanced level of cost effectiveness resulting from a dramatic reduction in the amount of expensive plastic wear and mineral oil used in the traditional culturing techniques presently employed.

(c) elimination of potential sources of embryo toxicity such as derived from plastic wear and mineral oil.

(d) minimal embryo manipulations as are presently employed in traditional culturing methods. (e) greatly reduced evaporation rates with or without an oil or air bubble plug.

(f) reduced temperature equilibration times inside an incubator.

(g) adaptation of capillaries to use in conjunction with disposable plastic stand which can be designed for direct and efficient microscope observation when positioned horizontally.

(h) use of the capillary tube for embryo transfer into live stock species.

In another aspect of the present invention there is provided a method of storing an embryo, said method comprising a method of culturing an embryo, said method comprising the steps of: obtaining an embryo in medium; obtaining a tube having at least two open ends and wherein one end is capable of receiving the embryo, said tube having a diameter capable of drawing and maintaining the embryo in the medium in the tube; drawing the embryo into the tube; and storing the embryo in the tube.

Freezing techniques will be familiar to those skilled in the art. However, for freezing embryos in a plastic tube, it is desirable to utilise the methods outlined by JM Shaw, Oranratnachai A and Trounson A, Chapter 11 : Cryopreservation of Oocytes and Embryos in Handbook of In vitro Fertilisation, Trounson AO and Gardner DK, CRC Press, Boca Raton USA, 1993, and by Vajta et al, (1997), Molecular Reproduction and Development, 48(1), 9-17.

Another potential use for the present invention includes culture of blastocysts past implantation to obtain a differentiated fetus. This may potentially be cultivated and studied in vitro with applications to implantation physiology, developmental biology, fetal toxicology and transgenics.

The storage of embryos for further implantation is particularly useful for IVF treatments where a number of oocytes are successfully fertilised to embryos and cultured in the glass oviduct system. These may be stored ready for implantation and induction of pregnancy at a later stage.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above. EXAMPLES

Example 1 : Capillary Culture System

One cell zygotes were obtained by the method outlined by DK Gardner and Lane M, Chapter 5: Embryo Culture Systems in Handbook of In vitro Fertilisation, Trounson AO and Gardner DK, CRC Press, Boca Raton USA, 1993. The zygotes were transferred from holding medium to pre-gassed microdrops of mouse tubal fluid (MTF) medium containing 4mg/ml bovine serum albumin (BSA).

A sterile glass capillary tube was held between thumb and forefinger using sterile nitrile rubber gloves with care taken not to touch the sterile end of the capillary tube. The sterile glass capillaries were dispensed from prepackaged plastic tubes of Microcaps ® (product # 10-000-10) supplied by the Drummond Scientific Company (Broomall Pa, USA).

Two one cell zygotes were taken up into the capillary tube under capillary action.

The loaded capillary tube was then inverted and inserted vertically into a predrilled perspex block (Figure 3). The perspex block may be rested anywhere on a standard gas incubator shelf for incubation. Vertical culture is more optimal than horizontal culture because pairs of embryos are in close apposition to each other and are cushioned by the air/medium interface (Figure 1).

The zygotes are cultured with minimal manipulation within the incubator for four days until blastocyst development. The conditions of incubation were 37°C in humidified air containing 5% CO₂.

Results obtained using the above protocol are shown in Table 1. Table 1 : Growth of Blastocyst in a Glass Capillary

Control Test

%total blastocysts 78 (n=240) 84 (n=239)

%hatched 3.1 (n=240) 47.6** (n=239)

Mean total cell numbers 67.2 + 3 (n=61) 92.1 +3* (n=74)

*p<0.0001 , **p<0.001

The proportion of blastocysts which have hatched has increased significantly in comparison to the control. Both groups were cultured in simple MTF supplemented only with BSA. Blastocysts that were cultured past the hatching stage had undergone trophectoderm cell outgrowth and attachment, indicating that there was a proportion of hatched blastocysts that were potentially implantation competent. This period of prolonged culture could be extended for up to 3 weeks with maintenance of blastocyst morphology but no differentiation past this stage. Day 4 blastocysts were available for implantation into a receptive animal and were later found after blastocyst transfer to have implanted successfully 10 days after transfer.

A small number of bovine embryos were also successfully cultured as per the methods described above, with an average of 30% blastocyst formation.

Example 2 : Comparison of Embryo Culture Conditions

Somatic cell cloned one-cell embryos were produced according to the method of Peura et al (1998) with modifications and using primary granulosa cell monolayers as donors.

Immediately after activation, embryos were washed with SOFaaci medium (Holm et al, 1999) containing 5% bovine serum, then placed into the same medium with oil overlay (Sigma Mineral Oil, embryo tested), then distributed into three equal groups and cultured in (1) 5 ul drops of the above medium covered with oil, (2) WOWs (Vajta et al., 2000), or (3) Drummond microcaps. Loading into Drummond microcaps was performed by passing one end of the capillaries quickly through the oil layer and placing the end over the one-cell embryos. The capillary effect filled the microcaps first with a thin layer of oil, then with the medium which also contained the embryos. Subsequently, microcaps were turned upside down and cultured at 39°C in 5% CO2 and air with maximum humidity for 7 days.

Day 3 (Day O: day of reconstruction) cleavage rates and Day 7 blastocyst rates obtained in the three culture systems were the following

None of the WOW cultured embryos developed to the expanded blastocyst stage and their morphology was compromised. In contrast, 4 of 6 blastocysts grown in the microcaps expanded, and had an excellent or good quality with a well-defined inner cell mass.

On the basis of this result we conclude that the microcap model may be a very useful model for culturing nuclear transfer bovine embryos produced by zona- free somatic cell cloning.

Example 3: Micro-Culture of Mouse Zygotes to the Blastocyst Stage using the 'GO' Culture System

The quality and viability of mouse zygotes cultured to the blastocyst stage in vitro, in either the 'GO' culture system or the more traditional culture system of microdrops of culture medium under oil, were compared.

Inbred hybrid (C57BL6xCBa-Fι) mouse zygotes fertilized in vivo were retrieved from the oviducts of superovulated females into HEPES-buffered mouse tubal fluid (MTF) medium supplemented with 3mg/ml bovine serum albumin (BSA) approximately 22hr after human chorionic gonadotrophin (hCG) injection. Cumulus free zygotes were then cultured to the blastocyst stage in bicarbonate buffered MTF supplemented with 4mg/ml BSA. Control (microdrop) cultures comprised three replicate groups of thirty zygotes cultured in microdrops (10 zygotes/20μl) of culture medium under paraffin oil in sterile plastic Petri dishes. Test ('GO') cultures comprised an equal number of zygotes cultured vertically in pairs within sterile 1μl glass capillaries. Control and test groups were cultured in parallel at 37°C and a humidified atmosphere of 5% CO₂ in air. The endpoints of morula total cell number, blastocyst development, blastocyst hatching, blastocyst differential cell number, implantation, foetal formation, development and weight were assessed. Results were analyzed using Dunnet's ANOVA, with a p-value < 0.05 considered significant.

'GO' cultured morulae had a higher total cell number on day 4 of culture than control cultured morulae (29.4 vs15.7). The percentage blastocyst formation from 'GO' cultures was not different to microdrop cultures (82% vs 80%) however the percentage of zygotes that developed to blastocysts showing partial or complete hatching at the same time point was significantly higher in the 'GO' culture system (47.6% vs 3.3%). In comparison to microdrop cultures, the average 'GO' cultured blastocyst had significantly more cells in total (92 vs

75), in the inner cell mass (25 vs 21) and in the trophectoderm (67 vs 55).

Subsequent foetal analysis at day 15.5 (post coitum) showed no difference in implantation (74% vs 86%), foetal formation (55% vs 61.8%) and foetal wet weight (415mg vs 412mg) following transfer of blastocysts grown in vitro in the microdrop or 'GO' culture systems. Anatomical malformation in foetuses derived from either microdrop or 'GO' cultured blastocysts was not observed.

In conclusion, modulation of the culture microenvironment can significantly improve blastocyst quality without detriment to viability and foetal normality. The benefits of the 'GO' culture system may be due to the elimination of potential embryo toxins in plasticware or oil, or the more stable and physiological microenvironment offered by culture in a confined space in a very small amount of culture medium which would also enhance the action of potential autocrine/paracrine growth factors. The 'GO' culture system therefore represents a simple, cost-effective and beneficial alternative to traditional culture methods that may also benefit the development of human embryos in vitro.

Example 4: Human embryos cultured using the 'GO' culture system and establishment of pregnancy.

Patients who consented to have all of their embryos cultured in the "Glass Oviduct" system (test group) were recruited from those undergoing standard IVF treatment at Monash IVF Clayton (Monash Surgical Private Hospital) during the period indicated below. Patients included in this study were 40 years of age or less with a history of failure to achieve pregnancy following three or less gonadotrophin stimulated ART cycles. Zygotes (normally fertilised oocytes) produced using standard insemination or injection techniques were allocated singly to culture in glass microcapillaries as in Example 1 for a period of two days (post-insemination Days 1-3) in a humidified atmosphere of 5%CO₂, 5% O₂ and 90% N₂ at 37°C. Embryos were microscopically assessed and transferred by catheter into the uterus of the patient on day 3. Comparison was made between the test group and a control population of patients (control group) matched for age and cycle history who had undergone IVF treatment using standard culture protocols (microdrops of culture medium under an oil overlay in sterile, disposable plastic cultureware in temperature regulated, CO₂ regulated incubators) during the same trial period.

Clinical data: (Trial period: 29/10/01-11/03/02) Test group Control group

Patient sample size: 30 412

Number of completed cycles: 29

Number of cycles awaiting a pregnancy test: 1

Pregnancy rate per transfer: 9/29(30.9 %)114/412 (27.7%)

Live pregnancy rate (by ultrasound scan): 9/29 (30.9 %) Singleton: 6/29 (20.6 %) Twin: 3/29 (10.3 %) Embryo data:

Average number of eggs collected per patient: 10 11.7 Average number of normally fertilized oocytes: 6.16 6.76 Average number of cleavage stage embryos: 5.73 - Average number of embryos transferred: 1.9 1.8

In conclusion, the glass oviduct culture system is a suitable alternative to culture systems utilising microdrops of culture medium under oil in sterile, disposable plastic cultureware in temperature regulated, CO₂ regulated incubators. Viable human pregnancies can be established from embryos cultured from the zygote stage in the glass oviduct culture system.

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

REFERENCES

Boediono A, Suzuki T, Li LY, Godke RA (1999) Offspring born from chimeras reconstucted from parthenogenetic and in vitro fertilized bovine embryos. Molecular Reproduction and Development 53: 159-170.

Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H (1999) High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myoinositol with or without serum. Theriogenology 52: 683-700.

Peura TT, Lane M, Lewis IM, Trounson AO (1998) The effect of recipient oocyte volume on nuclear transfer in cattle. Molecular Reproduction and Development 50: 185-191. Vajta G, Peura TT, Holm P, Paldi A, Greve T, Trounson AO, Callesen H (2000) New method for culture of zona-included and zona-free embryos: the well of the well (WOW) system. Molecular Reproduction and Development 55: 256-264.

Wells KD, Powell AM (2000) Blastomeres from somatic cell nuclear transfer embryos are not allocated randomly in chimeric blastocysts. Cloning 2: 9-22.

Claims

CLAIMS:

1. A culture system for an embryo, said system comprising: a tube having at least two open ends wherein one end is capable of receiving an embryo, said tube having a diameter capable of drawing and maintaining an embryo in the tube in a volume of medium suitable for culturing the embryo.

2. A culture system according to claim 1 wherein the tube is a capillary tube.

3. A culture system according to claim 1 or 2 wherein the tube has an internal diameter of about 200-250 μm.

4. A culture system according to any one of claims 1 to 3 wherein the tube includes an internal coating to enhance embryo development.

5. A culture system according to claim 4 wherein the coating is selected from the group including extracellular matrix macro-molecules including fibronectins, proteoglycans, collagens or polycarbonates.

6. A method of culturing an embryo, said method comprising the steps of: obtaining an embryo in medium; obtaining a culture system according to claim 1 comprising a tube having at least two open ends and wherein one end is capable of receiving the embryo, said tube having a diameter capable of drawing and maintaining the embryo in the medium in the tube; drawing the embryo into the tube; and incubating and culturing the embryo in the tube.

7. A method according to claim 6 wherein the embryo is obtained soon after conception and at the zygote stage.

8. A method according to claim 6 or 7 wherein the embryo is drawn into the tube by passive capillary action.

9. A method according to any one of claims 6 to 8 wherein the embryo is incubated and cultured in the tube with the tube in a vertical orientation.

10. A method according to any one of claims 6 to 9 wherein at least two embryos are cultured in the same tube.

11. A method according to claim 10 wherein the embryos are in close apposition to each other in the same tube.

12. A method according to claim 10 wherein the embryos are cultured separately in media separated by an air pocket in the same tube.

13. An embryo cultured by a method according to any one of claims 6 to 12.

14. An embryo according to claim 13 which is a blastocyst.

15. A method of inducing a pregnancy, said method comprising: obtaining an embryo according to claim 13; obtaining a receptive mother capable of incubating an embryo to term; and implanting the embryo into the mother.

16. A method according to claim 15 wherein the embryo is a blastocyst.

17. A method of obtaining an animal said method comprising the steps of: obtaining an embryo according to claim 13; obtaining a receptive animal capable of incubating the embryo to term; implanting the embryo into the receptive animal; and allowing the receptive animal to incubate the embryo to term.

18. A method according to claim 17 wherein the embryo is a blastocyst.

19. A method according to claim 18 wherein the embryo is a blastocyst cultured past the stage of hatching and shows signs of trophectoderm cell outgrowth.

20. An animal obtained by the method according to any one of claims 17 to 19.

21. A method of storing an embryo, said method comprising; obtaining an embryo in media; obtaining a culture system according to claim 1 comprising a tube having at least two open ends and wherein one end is capable of receiving the embryo, said tube having a diameter capable of drawing and maintaining the embryo in the medium in the tube; drawing the embryo into the tube; and storing the embryo in the tube.

22. A method according to claim 21 wherein the embryos are stored in a frozen state.

23. A culture system according to claim 1 substantially as hereinbefore described in Example 1 and in the drawings.

24. A method of culturing according to claim 6 substantially as hereinbefore described in Example 3.

25. A method according to claim 15 substantially as hereinbefore described with reference to Example 4.