Real time multipoint assay for optimizing performance

Abstract

Described herein are methods of optimizing the performance of a mammalian semen sample with respect to outcomes such as fertility, using a multipoint assay to determine an optimum time point for preparing the semen sample for use in insemination. The multipoint assay is based upon a kinetic model of biomarker expression during sperm capacitation relative to the outcome of interest.

1 . A method of optimizing sperm performance of a semen sample upon insemination of said semen sample, comprising:
 i) selecting a marker, wherein expression of the marker in the semen sample changes with time during capacitation;   ii) determining the level or location of expression of the marker in the semen sample at a plurality of time points during incubation of said semen sample before insemination of said semen sample;   iii) determining a timepoint for preparing said semen sample for use in insemination, wherein said timepoint is based upon a calibration of the marker expression displayed by said semen sample in step (ii) to a kinetic model of biomarker expression during sperm capacitation relative to said performance;   iv) preparing at about said timepoint of step (iii) said semen sample for use in insemination;   thereby optimizing said performance of said semen sample upon insemination of said semen sample.   
     
     
         2 . A method of optimizing sperm performance of a semen sample upon insemination of said semen sample, wherein said semen sample has been exposed to a treatment which modulates the rate of capacitation, comprising:
 i) selecting a marker, wherein expression of the marker in the semen sample changes with time during capacitation;   ii) determining the level or location of expression of the marker in the semen sample at a plurality of time points after exposure to said treatment and during incubation of said semen sample before insemination of said semen sample,   iii) determining a timepoint for preparing said semen sample for use in insemination, wherein said timepoint is based upon a calibration of the marker expression displayed by said semen sample in step (ii) to a kinetic model of biomarker expression during sperm capacitation relative to said performance;   iv) preparing at about said timepoint of step (iii) said semen sample for use in insemination;   thereby optimizing said performance of said semen sample upon insemination of said semen sample.   
     
     
         3 . The method of  claim 1  or  2 , wherein the semen sample is mammalian. 
     
     
         4 . The method of  claim 1  or  2 , wherein the semen sample is human. 
     
     
         5 . The method of  claim 1  or  2 , wherein the sperm performance comprises enhanced fertility. 
     
     
         6 . The method of  claim 1  or  2 , wherein the sperm performance comprises gender bias. 
     
     
         7 . The method of  claim 1  or  2 , wherein the sperm performance comprises female gender bias. 
     
     
         8 . The method of  claim 1  or  2 , wherein the sperm performance comprises male gender bias. 
     
     
         9 . The method of  claim 1  or  2 , wherein preparing said semen sample for use in insemination comprises freezing said semen sample or vitrification of said semen sample. 
     
     
         10 . The method of  claim 9 , wherein the sperm performance comprises resistance to freezing when preparing said semen sample for use in insemination. 
     
     
         11 . The method of  claim 9 , wherein the sperm performance comprises resistance to vitrification when preparing said semen sample for use in insemination. 
     
     
         12 . The method of  claim 1  or  2 , wherein the marker and the biomarker are identical. 
     
     
         13 . The method of  claim 1  or  2 , wherein the marker and/or the biomarker is an Fc receptor. 
     
     
         14 . The method of  claim 1  or  2 , further comprising more than one marker. 
     
     
         15 . The method of  claim 1  or  2 , wherein the kinetic model of biomarker expression during sperm capacitation comprises more than one biomarker. 
     
     
         16 . The method of  claim 1  or  2 , wherein the kinetic model of biomarker expression during sperm capacitation comprises a biomarker which binds to the constant region of an antibody. 
     
     
         17 . The method of  claim 1  or  2 , wherein the semen sample is not treated by fixation. 
     
     
         18 . The method of  claim 1  or  2 , wherein the semen sample is not exposed to treatment to increase permeability of the membrane. 
     
     
         19 . The method of  claim 1  or  2 , wherein the first of said plurality of time points is obtained immediately after collection of said semen sample. 
     
     
         20 . The method of  claim 1  or  2 , wherein the marker and the biomarker are identical. 
     
     
         21 . The method of  claim 1  or  2 , wherein the marker and the biomarker is an Fc receptor. 
     
     
         22 . The method of  claim 1  or  2 , further comprising more than one marker. 
     
     
         23 . The method of  claim 1  or  2 , wherein the kinetic model of biomarker expression during sperm capacitation comprises more than one biomarker. 
     
     
         24 . The method of  claim 1  or  2 , wherein the kinetic model of biomarker expression during sperm capacitation comprises the biomarker Fc receptor. 
     
     
         25 . The method of  claim 1  or  2 , wherein the kinetic model of biomarker expression during sperm capacitation is developed using ejaculates from the same species of said semen sample. 
     
     
         26 . The method of  claim 1  or  2 , wherein said incubation of said semen sample comprises incubation at a temperature ranging from approximately 40 C to 4° C. 
     
     
         27 . The method of  claim 26 , wherein said incubation of said semen sample occurs at a temperature gradient ranging from approximately 40 C to 12° C. 
     
     
         28 . The method of  claim 2 , wherein said treatment decelerates the rate of capacitation. 
     
     
         29 . The method of  claim 2 , wherein said treatment accelerates the rate of capacitation. 
     
     
         30 . The method of  claim 2 , wherein said treatment arrests capacitation at a specific stage. 
     
     
         31 . The method of  claim 2 , wherein said treatment comprises exposing said semen sample to a chemical agent which modulates the rate of capacitation. 
     
     
         32 . The method of  claim 31 , wherein said agent is selected from the group consisting of endocannibinoids, bicarbonate and 8-butyryl cAMP. 
     
     
         33 . The method of  claim 2 , wherein said treatment comprises exposing said semen sample to an environmental stimulus which modulates the rate of capacitation. 
     
     
         34 . The method of  claim 33 , wherein said environmental stimulus is selected from the group consisting of barometric pressure, atmospheric pressure, temperature change and agitation. 
     
     
         35 . The method of  claim 33 , wherein said treatment comprises exposing said semen sample to an atmospheric condition which modulates the rate of capacitation. 
     
     
         36 . The method of  claim 35 , wherein said atmospheric condition comprises a gas which is selected from the group consisting of a nitric oxide, ozone, argon, cyanide and CO 2 . 
     
     
         37 . The method of  claim 36 , wherein said the CO 2  concentration is greater than 5%. 
     
     
         38 . The method of  claim 1  or  2 , wherein the marker or biomarker is expressed in the extracellular portion of said semen sample. 
     
     
         39 . The method of  claim 1  or  2 , wherein the marker or biomarker is expressed by a sperm cell. 
     
     
         40 . The method of  claim 39 , wherein the marker or biomarker is expressed on the cell surface of said sperm cell. 
     
     
         41 . The method of  claim 39 , wherein the marker or biomarker is expressed in a membrane of the sperm cell. 
     
     
         42 . The method of  claim 41 , wherein the marker or biomarker is a change selected from the group consisting of membrane isotrophy, membrane fluidity, membrane charge, membrane permeability, membrane budding, membrane hydrophobicity, lipid raft structure, a lipid subdomain of the membrane, ion channel permeability, tyrosine phosphorylation, kinase activation, protease activation, kinase inactivation, protease inactivation, change in presence or amount of a ligand-binding molecule(s), and a calcium gradient. 
     
     
         43 . The method of  claim 39 , wherein the marker or biomarker is a lipid selected from the group of phosphatidyl serine, phosphatidlycholine, phosphatidyl ethanolamine and sphingomyelin. 
     
     
         44 . The method of  claim 39 , wherein the marker or biomarker further comprises its cellular location of the sperm. 
     
     
         45 . The method of  claim 39 , wherein the marker or biomarker is intracellularly expressed with respect to said sperm cell. 
     
     
         46 . The method of  claim 45 , wherein the intracellular marker or biomarker is selected from the group consisting of: intracellular pH, intracellular concentration of HCO 3 , intracellular concentration of fragmented DNA, mitochondrial Calcium and intracellular concentration of Calcium. 
     
     
         47 . The method of  claim 39  where the marker or biomarker binds a glycoprotein or an anionic polysaccharide. 
     
     
         48 . The method of  claim 39 , where the marker or biomarker binds a glycosaminoglycan, a sulfated glycosaminoglycan, a sulfated glycan and a sulfated polylactosaminoglycans. 
     
     
         49 . The method of  claim 39 , where the marker or biomarker is selected from the group consisting of heparin, fucoidan, ZP3, a glycoprotein or fragment thereof derived from the egg vestments, and a Lewis antigen. 
     
     
         50 . The method of  claim 39 , where the marker or biomarker binds a dye. 
     
     
         51 . The method of  claim 39 , wherein the marker or biomarker is a physiologic activity selected from the group consisting of percent of sperm displaying motility, motility grade, frequency of beating of flagella of said sperm cell, ability of said sperm cell to penetrate mucus, loss of adhesion of said sperm cell, hypoosmotic swelling of said sperm cell, chemotaxis, thermotaxis and metabolic status of said sperm cell. 
     
     
         52 . The method of  claim 39 , where the marker or biomarker comprises a molecule secreted or expelled by said sperm cell. 
     
     
         53 . The method of  claim 38 , wherein the marker or biomarker comprises the appearance of exocytic vesicles or hybrid vesicles in solution. 
     
     
         54 . The method of  claim 52 , where in the secreted or expelled molecule is selected from the group consisting of an enzyme, a proenzyme, an agent, a reactive oxygen species, an exosomal vesicle, a dye and DNA fragments. 
     
     
         55 . The method of  claim 52 , where in the secreted or expelled agent is selected from the group consisting of an antibody bound fluorophore, a fluorophore and an enzyme. 
     
     
         56 . The method of  claim 1  or  2 , wherein said biomarker is selected from the group consisting of Fertility-associated antigen, soybean trypsin inhibitor, an Fc receptor and CD46. 
     
     
         57 . The method of  claim 1  or  2 , wherein said marker is selected from the group consisting of Fertility-associated antigen, soybean trypsin inhibitor, an Fc receptor and CD46. 
     
     
         58 . The method according to  claim 9 , wherein freezing said semen sample comprises adding a cryoprotectant selected from the group consisting of trehalose, glycerol, propylene glycol, dimethylsulfoxide, sucrose and egg yolk. 
     
     
         59 . The method of  claim 39 , where the marker or biomarker is permeability of said sperm to a dye. 
     
     
         60 . The method of  claim 59 , where the marker or biomarker is oscillatory behavior of said sperm in response to dye uptake and/or release. 
     
     
         61 . The method of  claim 49 , wherein the egg vestments of fragment thereof, is from the zona pellicida or fragments thereof.