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In-vitro culture method and culture medium for embryos containing IGF2 (insulin-like growth factor 2)

A technology of in vitro culture and culture medium, which is applied in the field of assisted reproduction of mammals, especially humans, and can solve problems such as slow development, insufficient development of oocytes and embryos, and pregnancy failure

Active Publication Date: 2020-02-21
SHANDONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing technology has insufficient development ability of oocytes and embryos, which causes slow development and even pregnancy failure after early embryo transfer

Method used

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  • In-vitro culture method and culture medium for embryos containing IGF2 (insulin-like growth factor 2)
  • In-vitro culture method and culture medium for embryos containing IGF2 (insulin-like growth factor 2)
  • In-vitro culture method and culture medium for embryos containing IGF2 (insulin-like growth factor 2)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Patient recruitment and ethics review

[0044] The research for this application was approved by the Review Committee of the Institute of Reproductive Medicine, Shandong University. All methods described in this application were performed in accordance with the approved guidelines and regulations of the Institute of Reproductive Medicine, Shandong University. The in vitro cultured oocytes used came from clinically discarded immature eggs (GV stage) in the Reproductive Hospital Affiliated to Shandong University. A formal informed consent was obtained from each patient before the experiments related to human beings.

Embodiment 2

[0045] Embodiment 2 experimental method and reagent

[0046] Oocyte and Embryo Collection and Microinjection

[0047] 24-28 day old mice were superstimulated for 44 hours with 5 IU pregnant mare serum gonadotropin (PMSG) and 5 IU human chorionic gonadotropin (hCG). Oocytes were harvested and cultured in small drops of M16 medium (M7292; Sigma-Aldrich), overlaid with mineral oil and maintained at 37°C in 5% CO 2 middle. For collection of fertilized eggs and embryos, control and Imp2 - / - Females were mated with adult WT males after hCG injection. For fertilized egg collection, fallopian tubes were punctured, while for embryo collection, the uterus was flushed at indicated time points after hCG administration. For microinjection, mRNA was transcribed in vitro using the mMESSAGE mMACHINE SP6 Transcription Kit (Invitrogen, AM1450). The siRNAs were obtained from RiboBio and the sequences are given in Table 2.

[0048] Fertilized egg culture, embryo transfer and fertility asses...

Embodiment 3

[0085] High expression of embodiment 3Imp2 in mouse oocyte and early embryo

[0086] The protein and mRNA profiles of IMP2 in mouse oocytes and early embryos were determined by Western blotting and quantitative real-time PCR (qRT-PCR), respectively. Transcripts for the mRNA-binding protein IMP2 were found to be highly expressed in mouse oocytes and early embryos. Expression is strongest at the germinal vesicle (GV) stage and is significantly lower in MII oocytes. The expression decreased further after fertilization, and completely absent at the blastocyst stage ( figure 1 a).

[0087] Immunofluorescence staining showed that IMP2 was localized in the cytoplasm of oocytes and preimplantation embryos ( figure 1 b). IMP2 expression is uniformly distributed at the oocyte stage but undergoes dynamic changes during zygote development. Morula and blastocyst stages showed IMP2 expression at the outer edge of the blastomere ( figure 1 b), and Western blot analysis further confirme...

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Abstract

The invention provides an in-vitro culture method for embryos of mammals, particularly human beings. An insulin-like growth factor 2, namely IGF2, is added in a culture medium. The invention further provides the culture medium which contains the insulin-like growth factor 2, namely IGF2, for the embryos of mammals, particularly human beings. By adopting the in-vitro culture method for the embryosand the in-vitro culture medium for the embryos, the velocity of forming blastocyst of mammals, particularly human beings, can be increased, in addition, the quality of the embryos can be improved, and thus the success rate of artificial insemination can be increased.

Description

technical field [0001] The invention relates to the field of assisted reproduction of mammals, especially humans. Specifically, the present invention relates to a method and medium for in vitro cultivation of embryos of mammals, especially humans. Background technique [0002] In the past few decades, in vitro fertilization-embryo transfer (IVF-ET) has provided an important solution to the problem of infertility in humans and other species. Embryos need to be cultured between fertilization and embryo transfer. The existing technology has insufficient development ability of oocytes and embryos, which causes the problems of slow development and even pregnancy failure after early embryo transfer. About half of human preimplantation embryos develop arrest in vitro before reaching the blastocyst stage, which is the stage used for embryo transfer in vivo. Miscarriage and recurrent pregnancy loss, characterized by poor embryonic growth, are a common disorder of human reproductio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2501/105A01K2217/075A01K2217/206A01K2227/105
Inventor 陈子江刘洪彬赵跃然马金龙耿玲路钢刘辉张传鑫李孟静
Owner SHANDONG UNIV
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