In vitro fertilisation

#The present invention relates to in vitro co-culturing of oocytes and spermatozoa, in vitro fertilisation (IVF), and in vitro culturing of fertilised gametes, wherein the culturing conditions comprises lowered oxygen tension in the entire culturing period or lowered oxygen tension in periods of the culturing period or the culturing conditions comprises changed oxygen tension throughout the culturing period or changed oxygen tension in periods of the culturing period. The invention further relates to in vitro maturation (IVM) of immature oocytes or spermatozoa, co-culturing the matured oocytes with sperm cells to fertilise the eggs.

#In nature, fertilisation occurs by sperm cells being deposited into the female of warm-blooded animal species (including humans) and then binding to and fusing with an oocyte. This fertilised oocyte then divides to form an embryo. Over the last several decades, the use of assisted reproduction techniques has allowed scientists and clinicians to intervene in these events to treat poor fertility in some individuals or to store sperm, oocytes or embryos for use at other locations or times. In vitro fertilisation (IVF) involves collection of eggs e.g. by laparoscopy, followed by mixing eggs and sperm and culture of the embryos before transferring them to the uterine cavity of the female to continue their development. A recent innovation has been the introduction of the technique of intracytoplasmic sperm injection (ICSI), in which sperm are microinjected into the egg cytoplasm.

#At each step of the way, in vitro intervention decreases the normal survival and function of sperm, oocytes, and embryos. Much research has been dedicated towards improving these procedures; however, overall success remains limited. For example, less than 20% of IVF attempts result in the birth of a child. Oocytes and embryos also show significantly disrupted function after culture. Thus, in spite of several decades of work, much room remains for improvement in the field of assisted reproduction technologies and especially in gamete and embryo handling, culture, and storage.

#FIG. 1 illustrates an embryo scoring system based on Van Abbel et al.

#FIG. 2 illustrates a cumulative embryo scoring (CES) system.

#FIG. 3 illustrates an incubator.

#The following description discloses a method for in vitro producing a mammalian pre-embryo comprising the steps of a) providing gametes selected from a mammalian oocyte and a mammalian spermatozoa, b) culturing the oocyte and the spermatozoa, c) fertilising the oocyte with the spermatozoa obtaining a fertilised oocyte, and either d) allowing cell-division of the fertilised oocyte obtaining a multicellular pre-embryo, wherein at least one of the steps a), or b) is conducted at an oxygen tension below 15% or e) allowing cell-division of the fertilised oocyte obtaining a multicellular pre-embryo, wherein the culture is performed at an oxygen tension allowing cultivation of the cells and wherein at least one of the steps comprises a change in the oxygen tension. Said change in the oxygen tension comprises culturing the cell culture in at least two different oxygen tensions within a single step. The change in the oxygen tension is performed in accordance to the development stage of the cell culture as described elsewhere. In particular handling or observing the cell culture at the atmospheric oxygen tension is not the change in oxygen tension, which is the intention of the present invention.

#By changing or lowering the oxygen tension of certain steps of the IVF procedure or changing or lowering the oxygen tension of all the IVF steps, an improvement of the quality of the gametes and of the embryos is obtained.

#Culturing mammalian gametes, in vitro fertilised oocytes or embryos in conditions including the atmosphere with an oxygen concentration of 20-21%, is thought to hamper the gametes and eggs. Lowering the oxygen level to a level between 1% and 15%, or lowering the oxygen level to a level between 1% and 19% and changing the oxygen tension in one or all steps a), b), c) and e), it is the intention that a higher amount of the embryos may reach the blastocyst stage. One of the problems with human in vitro maturation and human in vitro fertilisation techniques is thought to be the activation and concentration of free reactive oxygen species preliminary generated from the high oxygen tension and supplemented during sperm preparation and in vitro fertilisations. By handling and culturing the gametes and embryos at lowered or changing oxygen tension, in combination with co-culturing the oocytes and spermatozoa we obtained an improvement of the IVF method, surprisingly the improvement of the IVF method was obtained without utilising co-culturing with feeder-cells.