Determination of biological characteristics of embryos fertilized in vitro by assaying for bioactive lipids in culture media

Abstract

The present invention provides methods for determining various biological characteristics of in vitro fertilized embryos, including overall embryo health, implantability, and increased likelihood of developing successfully to term. More specifically, the present invention concerns analyzing media specimens of in vitro fertilization cultures for levels of bioactive lipids in order to determine these characteristics.

1 . A method for evaluating a biological characteristic in an in vitro fertilized embryo, comprising the steps of: 
 (a) obtaining a media specimen from an in vitro culture containing the embryo;    (b) measuring a concentration of one or more bioactive lipids in the media specimen; and    (c) correlating the concentration to a biological characteristic in the embryo.    
     
     
         2 . The method of  claim 1  wherein the biological characteristic is the likelihood that an in vitro fertilized embryo will develop to term.  
     
     
         3 . The method of  claim 1  wherein the biological characteristic is the likelihood that an in vitro fertilized embryo will implant upon transfer into a uterus.  
     
     
         4 . The method of  claim 1  wherein the biological characteristic is the likelihood of a multiple pregnancy.  
     
     
         5 . The method of  claim 1  wherein the correlating step is comprised of developing a profile of bioactive lipids and determining the biological characteristic from the profile.  
     
     
         6 . The method of  claim 1  wherein the bioactive lipid is selected from the group consisting of lysophospholipids, glycerophosphatidyl compounds, sphingolipids, and combinations thereof.  
     
     
         7 . The method of  claim 6  wherein the bioactive lipid is at least one lysophospholipid with the general structure:  
       
         
           
           
               
               
           
         
       
       wherein R is selected from the group consisting of hydrogen, choline, inositol, ethanolamine, serine, and glycerol; S 1  is a single fatty acid chain linked by an acyl linkage, or a hydrogen; and S 2  is a single fatty acid linked by an acyl linkage if S 1  is a hydrogen, or a hydrogen if S 1  is a single fatty acid chain.  
     
     
         8 . The method of  claim 7  wherein R is selected from the group consisting of hydrogen, choline, inositol, ethanolamine, glycerol, and serine  
     
     
         9 . The method of  claim 7  wherein the bioactive lipid is at least one lysophospholipid with the general structure:  
       
         
           
           
               
               
           
         
       
       wherein R is selected from the group consisting of hydrogen, choline, inositol, ethanolamine, serine, and glycerol; S 1  is a single long chain alkyl or akenyl, or hydrogen; and S 2  is a single long chain alkyl or alkenyl if S 1  is hydrogen, or hydrogen if S 1  is a single long chain alkyl or akenyl.  
     
     
         10 . The method of  claim 14  wherein R is selected from the group consisting of hydrogen, choline, inositol, ethanolamine, glycerol, and serine.  
     
     
         11 . The method of  claim 6  wherein the bioactive lipid is at least one glycerophosphitidyl compound selected from the group consisting of: glycerol-3-phosphate, glycerophosphitidyl inositol, glycerophosphitidyl choline, glycerophosphitidyl serine, glycerophosphitidyl glycerol, glycerophosphitidyl ethanolamine, and combinations thereof.  
     
     
         12 . The method of  claim 6  wherein the bioactive lipid is at least one sphingolipid selected from the group consisting of: sphingosine-1-phosphate, sphinganine-1-phosphate, sphingomyelin, and combinations thereof.  
     
     
         13 . The method of  claim 6  wherein the bioactive lipid is at least one sphingolipid with the general formula:  
       
         
           
           
               
               
           
         
       
       wherein S 1  is an alkyl or alkenyl chain of 15 carbons in length, and wherein R is selected from the group consisting of hydrogen, choline, inositol, ethanolamine, glycerol, and serine.  
     
     
         14 . The method of  claim 13  wherein S 1  a 15:1 alkenyl chain, and R is choline.  
     
     
         15 . The method of  claim 1  wherein the in vitro fertilized embryo is cocultured on a feeder cell line.  
     
     
         16 . The method of  claim 15  wherein the feeder cell line is of human origin.  
     
     
         17 . The method of  claim 15  wherein the feeder cell line is obtained from a patient who is to be the recipient of the in vitro fertilized embryo.  
     
     
         18 . The method of  claim 15  wherein the feeder cell line is an endometrial epithelial cell line.  
     
     
         19 . The method of  claim 1  wherein the media specimen is obtained from an in vitro culture whose embryo is between approximately one hour and approximately twenty days old.  
     
     
         20 . The method of  claim 1  wherein the embryo is between approximately one day and approximately ten days old.  
     
     
         21 . The method of  claim 1  wherein the embryo is approximately 2 days old.  
     
     
         22 . The method of  claim 1  wherein the step of measuring a concentration of one or more bioactive lipids is accomplished by mass spectroscopy.  
     
     
         23 . The method of  claim 1  wherein the step of measuring a concentration of one or more bioactive lipids is accomplished by an enzymatic chemical reaction.  
     
     
         24 . The method of  claim 23  wherein the enzymatic chemical reaction is a cycling reaction.  
     
     
         25 . The method of  claim 23  wherein the enzymatic chemical reaction comprises the use of at least one enzyme selected from the group consisting of lysophospholipase, a glycerophosphatidyl compound phosphodiesterase, glycerol-3-phosphate dehydrogenase, and glycerol-3-phosphate oxidase.  
     
     
         26 . The method of  claim 23  wherein the enzymatic chemical reaction comprises: 
 i) converting lysophospholipids in the specimen into glycero-3-phosphate by incubating with lysophospholipase and a glycerophosphatidyl compound phosphodiesterase; and  
 ii) determining the total concentration of G3P using an enzymatic cycling reaction.