CNP based biphasic in vitro maturation technique of human immature oocytes

Abstract

The invention relates to a technique for promoting in vitro maturation of human immature oocytes. A CNP based biphasic in vitro maturation (CNP based biphasic IVM, C-IVM) technique for human immatureoocytes simulates an in vivo development process of oocytes to realize in vitro maturation of the oocytes in a phased manner: after immature cumulus-oocyte complexes (COCs) are obtained from small antral follicles, firstly (1) preculture (pre-IVM) is performed, meiosis of the oocytes is restrained through C-type natriuretic peptide (CNP), and besides, cytoplasm maturing of the oocytes is promoted;and (2) in vitro maturation (IVM) culture is performed, maturing of oocyte nuclei is promoted through amphiregulin (AREG) and FSH, first polar bodies are discharged, and MII oocytes are formed. Through the technique disclosed by the invention, the human oocytes low in maturity can be promoted to form mature oocytes having high development potential in vitro, the state of low IVM efficiency at present is significantly improved, and the technique has the characteristics of being flexible, safe and efficient.

Description

Technical field

[0001] The present invention relates to an in vitro maturation (in vitro maturation, IVM) technology of human immature oocytes, in particular to a technical scheme for promoting in vitro maturation of human immature oocytes with low maturity. Background technique

[0002] IVM technology refers to a technology that takes the immature cumulus-oocyte complex (COC) out of antral follicles and promotes its development and maturity in vitro. At present, the IVM program widely adopted at home and abroad is: ovulation stimulation of patients, egg collection 36 hours after HCG injection, and immature COCs → place immature COCs (or oocytes) with FSH, LH / HCG, etc. Incubate the ingredients in the IVM medium for 24-48 hours to obtain MII eggs → ICSI insemination of MII eggs to form embryos for use. We call this in vitro maturation program the standard IVM (standard IVM). The quality and developmental potential of mature eggs obtained through the current standard IVM technolo...

Examples

Embodiment Construction

[0005] 1. Preparation of culture medium

[0006] Transport fluid: 10mg / ml human serum albumin (HSA), 50nM CNP and 20IU / ml heparin are added to human fallopian tube fluid.

[0007] Pre-culture medium (pre-IVM): α-MEM medium is supplemented with rFSH 2.5mIU / ml, insulin 5ng / ml, E 2 10nM, CNP 25nM and HAS 10mg / ml. Place the pre-IVM medium in CO on the day before egg collection 2 Equilibrate overnight in the incubator.

[0008] In vitro maturation medium (IVM): IVM medium is supplemented with rFSH 100mIU / ml, E 2 10nM, insulin 5ng / ml and rhAREG 100ng / ml. Before IVM culture, place IVM culture medium in CO 2 Equilibrate in the incubator for at least 3-4 hours.

[0009] 2. Use a 17G needle to puncture the small antral follicles in the ovarian cortex and aspirate the follicular fluid containing COCs. Put the aspirated follicular fluid (containing COCs) in the transfer fluid, store it in a 37°C constant temperature cell transfer box, and quickly transfer it to the embryo culture room (complet...

Technical field

[0001] The present invention relates to an in vitro maturation (in vitro maturation, IVM) technology of human immature oocytes, in particular to a technical scheme for promoting in vitro maturation of human immature oocytes with low maturity. Background technique

[0002] IVM technology refers to a technology that takes the immature cumulus-oocyte complex (COC) out of antral follicles and promotes its development and maturity in vitro. At present, the IVM program widely adopted at home and abroad is: ovulation stimulation of patients, egg collection 36 hours after HCG injection, and immature COCs → place immature COCs (or oocytes) with FSH, LH / HCG, etc. Incubate the ingredients in the IVM medium for 24-48 hours to obtain MII eggs → ICSI insemination of MII eggs to form embryos for use. We call this in vitro maturation program the standard IVM (standard IVM). The quality and developmental potential of mature eggs obtained through the current standard IVM technolo...

Examples

Embodiment Construction

[0005] 1. Preparation of culture medium

[0006] Transport fluid: 10mg / ml human serum albumin (HSA), 50nM CNP and 20IU / ml heparin are added to human fallopian tube fluid.

[0007] Pre-culture medium (pre-IVM): α-MEM medium is supplemented with rFSH 2.5mIU / ml, insulin 5ng / ml, E 2 10nM, CNP 25nM and HAS 10mg / ml. Place the pre-IVM medium in CO on the day before egg collection 2 Equilibrate overnight in the incubator.

[0008] In vitro maturation medium (IVM): IVM medium is supplemented with rFSH 100mIU / ml, E 2 10nM, insulin 5ng / ml and rhAREG 100ng / ml. Before IVM culture, place IVM culture medium in CO 2 Equilibrate in the incubator for at least 3-4 hours.

[0009] 2. Use a 17G needle to puncture the small antral follicles in the ovarian cortex and aspirate the follicular fluid containing COCs. Put the aspirated follicular fluid (containing COCs) in the transfer fluid, store it in a 37°C constant temperature cell transfer box, and quickly transfer it to the embryo culture room (complet...