Media Solutions and Methods for Cryopreservation and Thawing of In Vitro Fertilization Specimens

Abstract

A medium solution which will increase the growth, survival and ultimately the live birth rate of oocytes and embryos which have been or will be subjected to cryopreservation. The solution contains varied amounts of glucose, pyruvates, amino acids, vitamins K5 and C, antioxidants, fatty acids to supply the specimens with the chemical ingredients and uptake requirements required to recover and prosper during and after the cryopreservation process. The solution supplies nutrients to the specimens that will replenish depletion and damage to the specimens and their mitochondria, spindles and structural features, such as cell walls. One formulation addresses the additional requirements of frozen specimens as opposed to the current media solutions and methods which treat the unfrozen specimens the same as the frozen specimens when recovering them from cryopreservation.

What is claimed is: 
     
         1 . A medium solution which will increase the growth, survival and ultimately the live birth rate of oocyte and embryo specimens which have been, or will be subjected to IVF cryopreservation and post-cryopreservation thawing, said solution comprising: effective amounts of glucose, pyruvates, amino acids, vitamins K5 and C, antioxidants, and fatty acids sufficient to supply the specimens with the chemical ingredients and uptake requirements required to recover and prosper during and after the cryopreservation process; said solution being operative to supply nutrients to the specimens which will replenish depletion and damage to the specimens and their mitochondria, spindles and structural features, such as cell walls. 
     
     
         2 . The medium solution of  claim 1  wherein said fatty acids include lipoic acid and linoleic acid. 
     
     
         3 . The medium solution of  claim 2  wherein said lipoic acid is present in a concentration range of about 0.000004 g/L to about 0.000008 g/L. 
     
     
         4 . The medium solution of  claim 3  wherein said linoleic acid is present in a concentration range of about 0.0000025 g/L to about 0.000010 g/L. 
     
     
         5 . The medium solution of  claim 1  which includes a HEPES or MOPS buffer. 
     
     
         6 . A method for thawing frozen or vitrified embryo specimens which are contained in liquid nitrogen (LN), said method comprising:
 a) a first step of removing a specimen from said LN and placing said specimen in a first solution comprising a base saline medium and a first amount of sucrose as an energy source;   b) a second step of removing said specimen from said first solution and placing said specimen in a second solution comprising a base saline medium and a second amount of sucrose which is less than said first amount;   c) a third step of removing said specimen from said second solution and placing said specimen in a third solution comprising a base saline medium and a third amount of sucrose which is less than said second amount; and   d) a fourth step of removing said specimen from said third solution and placing said specimen in a rejuvenation medium comprising effective amounts of glucose, pyruvates, amino acids, vitamins K5 and C, antioxidants, and fatty acids sufficient to supply the specimen with the chemical ingredients and uptake requirements required to recover and prosper during and after the cryopreservation process; said solution being operative to supply nutrients to the specimen which will replenish depletion and damage to the specimen and its mitochondria, spindles and structural features, such as cell walls.   
     
     
         7 . The method of  claim 6  further comprising a fifth step of removing said specimen from said rejuvenation medium and placing said specimen in a generic culturing medium which will prepare said specimen for reimplantation. 
     
     
         8 . A method for freezing or vitrifying embryo or oocyte specimens for storage in liquid nitrogen (LN), said method comprising:
 a) a first step of removing a specimen from a culturing medium and placing said specimen in a first rejuvenation medium comprising effective amounts of glucose, pyruvates, amino acids, vitamins K5 and C, antioxidants, and fatty acids sufficient to supply the specimen with chemical ingredients and uptake requirements required to recover and prosper during and the cryopreservation process; said medium being operative to supply nutrients to the specimen which will protect said specimen from depletion and damage to the specimen and its mitochondria, spindles and structural features, such as cell walls during cryopreservation, said first rejuvenation medium being devoid of HEPES and MOPS;   b) a second step of removing said specimen from said first rejuvenation medium placing said specimen in a second rejuvenation medium comprising effective amounts of glucose, pyruvates, amino acids, vitamins K5 and C, antioxidants, and fatty acids sufficient to supply the specimen with chemical ingredients and uptake requirements required to recover and prosper during and the cryopreservation process; said medium being operative to supply nutrients to the specimen which will protect said specimen from depletion and damage to the specimen and its mitochondria, spindles and structural features, such as cell walls during cryopreservation, said second rejuvenation medium further including HEPES or MOPS;   c) a third step of removing said specimen from said second rejuvenation medium and placing said specimen in a first solution comprising a first amount of one or more cryoprotectant;   d) a fourth step of removing said specimen from said first solution and placing said specimen in a second solution comprising a second amount of one or more cryoprotectant which is greater than said first amount; and   e) a fifth step of removing said specimen from said third solution and placing said specimen in a liquid nitrogen storage vessel.   
     
     
         9 . The medium solution of  claim 8  wherein said fatty acids include lipoic acid and linoleic acid. 
     
     
         10 . The medium solution of  claim 9  wherein said lipoic acid is present in a concentration range of about 0.000004 g/L to about 0.000008 g/L. 
     
     
         11 . The medium solution of  claim 10  wherein said linoleic acid is present in a concentration range of about 0.0000025 g/L to about 0.000010 g/L.