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WO2004057019A1 - Sperm fertility assay - Google Patents

Sperm fertility assay Download PDF

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Publication number
WO2004057019A1
WO2004057019A1 PCT/CA2003/002014 CA0302014W WO2004057019A1 WO 2004057019 A1 WO2004057019 A1 WO 2004057019A1 CA 0302014 W CA0302014 W CA 0302014W WO 2004057019 A1 WO2004057019 A1 WO 2004057019A1
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WO
WIPO (PCT)
Prior art keywords
sperm
carrier matrix
hyaluronic acid
indicator substance
sperm sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CA2003/002014
Other languages
French (fr)
Inventor
Robert Casper
Stephen L. Corson
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Individual
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Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to AU2003289792A priority Critical patent/AU2003289792A1/en
Publication of WO2004057019A1 publication Critical patent/WO2004057019A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides

Definitions

  • This invention relates to a device for assessing the fertilizing ability of a sperm sample and in particular to a device that contains hyaluronic acid for assessing the fertilizing ability of a sperm sample.
  • Fertility testing has become a more prevalent and desired test as a result of an increase in couples seeking advice and assistance on infertility because of their inability to conceive. In such situations both females and males must undergo a fertility test to ascertain if they are infertile.
  • U.S. Patent No. 5,434,057 discloses a test kit that assesses the amount of motile sperm in a sample
  • U.S. Patent No. 5,605,803 discloses a test that determines the total sperm concentration in a sample.
  • kits that are available can only be utilized in a laboratory or doctor's office and are not easily accessible or easy to use by the average person. However, some kits have been developed that do not require extensive training in order to use them.
  • Hyaluronidase is an enzyme that is present in the sperm acrosome. It has been demonstrated previously that in the presence of detergent this acrosomal enzyme is released from the sperm head. Once released hyaluronidase is free to act, for example, by hydrolizing hyaluronic acid into its metabolites. The hyaluronidase activity correlates to the sperm count and also to sperm motility and morphology, as discussed in Abdul-Aziz M, MacLusky NJ, Bhavnani B, Casper RF., 1995, Hyaluronidase activity in human semen: correlation with fertilization in INF. Fertil Steril, 64:1147-53.
  • CA 2,154819 discloses a method of measuring the amount of hyaluronidase enzyme present in the sperm sample. The method included the use of a substrate that can be enzymatically acted upon by the hyaluronidase, the substrate being suspended in a semi solid substance.
  • the sperm sample to be assessed is pretreated with a non-ionic detergent to ensure the maximum release of the hyaluronidase is achieved from the sperm in the sample.
  • the aliquot of the treated sample is then applied to the semi solid substance containing the substrate and any hyaluronidase enzyme present in the aliquot will react with the substrate. Over a fixed period of time the amount of substrate being modified by the enzyme will be proportional to the amount of enzyme that was present in the aliquot. Therefore, in order to measure the amount of enzyme in the aliquot the substrate modified by the enzyme can be measured and the measurement used to determine the amount of enzyme present in the aliquot and subsequently in the sample.
  • the present invention provides a device for assessing the fertilizing ability of a sperm sample utilising hyaluronic acid.
  • a device for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to a marker dye and a non-ionic detergent.
  • a device for assessing the fertilizing ability of a sperm sample, the sperm sample having been contacted with a non-ionic detergent, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to a marker dye.
  • the present invention further provides a method, for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a solid support having an insoluble carrier matrix containing a non-ionic detergent, hyaluronic acid and means for generating a visual signal operably linked to the hyaluronic acid, contacting the carrier matrix with a sperm sample and detecting the visual signal.
  • the means for generating a visual signal is a marker dye.
  • the present invention further provides a method, for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a solid support having a carrier matrix containing hyaluronic acid and means for generating a visual signal operably linked to the hyaluronic acid, mixing a sperm sample with a non-ionic detergent, contacting the carrier matrix with the mixture and detecting the visual signal.
  • the means for generating a visual signal is a marker dye.
  • the present invention further provides a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance.
  • the present invention further provides a method for assessing the fertilizing ability of a sperm sample comprising the steps of providing a device comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance, contacting the carrier matrix with the sperm sample and assessing the means for detecting the indicator substance.
  • the present invention further provides a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a marker dye operable to detect the presence of the indicator substance.
  • the present invention further provides a device, for assessing the fertilizing ability of .a sperm sample, the sperm sample having been contacted with a non-ionic detergent, the device comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, and a marker dye operable to detect the presence of the indicator substance
  • the present invention further provides a device, for assessing the fertilizing ability of a sperm sample, the sperm sample having been contacted with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities in, the seminal plasma or sperm, the device comprising a solid support having a carrier matrix containing a non-ionic detergent and a marker dye operable to detect the presence of the indicator substance.
  • the present invention further provides a device, for assessing the fertilizing ability of a sperm sample, the sperm sample having been contacted with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities in, the seminal plasma or sperm and a non-ionic detergent, the device comprising a solid support having a carrier matrix containing a marker dye operable to detect the presence of the indicator substance.
  • the present invention further provides a method for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance, contacting the carrier matrix with the sperm sample and detecting the amount of the means for detecting the indicator substance.
  • the means for detecting the indicator substance is a marker dye
  • the step of detecting the amount of the means is the generation of a visual signal.
  • the present invention further provides a method for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, and a means for generating a visual signal operable to detect the presence of the indicator substance, contacting the sperm sample with a non-ionic detergent, contacting the carrier matrix with the mixture of the sperm sample and the non-ionic detergent and detecting the visual signal.
  • the means for generating a visual signal is a marker dye.
  • the present invention further provides a method for assessing the fertilizing ability of a sperm sample, comprising the steps of contacting the sperm sample with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, providing a solid support having a carrier matrix containing a means for generating a visual signal operable to detect the presence of the indicator substance and a non- ionic detergent, contacting the carrier matrix with the mixture of the sperm sample, and the hyaluronic acid and detecting the visual signal.
  • the means for generating a visual signal is a marker dye
  • the present invention further provides a kit comprising the solid support of any one of the above devices and an instruction leaflet which outlines the method as set out above for assessing the fertilizing ability of a sperm sample.
  • the present invention further provides a kit for assessing the fertilizing ability of a sperm sample comprising a device having a solid support containing a carrier matrix which contains hyaluronic acid operably linked to a marker dye and a non-ionic detergent, and an instruction leaflet that outlines the method comprising the steps of contacting the carrier matrix with a sperm sample and detecting the visual signal.
  • the present invention further provides a kit for assessing the fertilizing ability of a sperm sample comprising a device having a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance and an instruction leaflet that outlines the method comprising the steps of contacting the carrier matrix with a sperm sample and assessing the means for detecting the indicator substance.
  • the present invention provides a device for assessing the fertilizing ability of a sperm sample.
  • the device comprises a solid support having a carrier matrix that contains hyaluronic acid operably linked to a marker dye and a non-ionic detergent.
  • the non-ionic detergent releases hyaluronidase from the sperm head.
  • the hyaluronidase is then free to hydrolyse the hyaluronic acid present.
  • the marker dye is released from the hyaluronic acid.
  • the marker dye is operable to change colour and when linked to the hyaluronic acid emits a first colour or is colourless, and when released from the hyaluronic acid will emit a second colour. This change in colour will therefore indicate the presence of hyaluronidase in the sperm sample.
  • the device comprises a solid support having a carrier matrix that contains hyaluronic acid operably linked to a marker dye.
  • a sperm sample is pre-treated with a non-ionic detergent.
  • the detergent releases hyalorunidase from the head of the sperm.
  • the carrier matrix is then contacted with the pre-treated sperm sample, and the hyaluronidase in the sample is then free to hydrolyse the hyaluronic acid present in the carrier matrix.
  • the marker dye is released from the hyaluronic acid.
  • the support used in the above embodiments may vary depending on the type of carrier matrix to be used.
  • the carrier matrix of the present invention may be a solid or liquid or gel.
  • the carrier matrix may be the whole support, or a part of the support or a structure or substance that is attached to or contained within the support.
  • the carrier matrix of the embodiments of the present invention will contain chemical compositions that will participate in the chemical reactions of the device of the present invention.
  • the chemical compounds of the present invention may be applied to the carrier matrix and dried. Alternatively the chemical compounds may be present in a liquid or gel form and be held in that state within the carrier matrix.
  • the carrier matrix may be an insoluble bibulous or non-bibulous material that maintains its structural integrity when exposed to physiological fluids such as semen.
  • Suitable bibulous materials include, but are not limited to, paper, cellulose, synthetic resin fleeces, glass fibers, and woven and non-woven fabrics.
  • suitable non-bibulous materials include, but are not limited to organo-plastic materials, such as polypropylene.
  • the device may be a take home test kit comprised of a solid support formed from a suitable plastic material, and the carrier matrix may be formed from a synthetic resin.
  • the carrier matrix may be attached to the solid support using a suitable adhesive such as an epoxy resin.
  • suitable adhesive such as an epoxy resin.
  • the device may be a take home test kit comprised of a solid support formed from a suitable plastic material, for example a capsule or test tube like structure, and the carrier matrix may be formed from a gel, for example agar.
  • Suitable marker dyes include any colorigenic substance that can be bound to or encapsulated by hyaluronic acid in such a way that hydrolysis of hyaluronic acid by hyaluronidase results in a color change.
  • Such possible dyes include, but are not limited to, fluorescent dyes such as Fura-2 and Fluo-3, which could be used to detect hyaluronidase release of ions such as calcium bound to hyaluronic acid; 2',7'-bis-(carboxyethyl)- 5-(and-6)-carboxyfluorescein, 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate and other fluorescent dyes that can reflect pH changes as a result of hyaluronic acid hydrolysis; biotin and its derivatives, alkaline phosphatase substrates, indolyl type substrates, coumarin- or fluorescein- based fluorogenic substrates, redox enzyme substrates such as dihydrorhodamine 123, bioluminescent substrates such as coelenterazine, ⁇ -galactosidase conjugated to streptavidin, avidin or antibodies as secondary detection reagents could be used to detect chro
  • Non-ionic detergents are non-denaturing and permit the solubilization of membranes without interfering with biological activity. They are suitable for the study of protein conformations and for the separation of hydrophilic proteins from membrane spanning hydrophobic proteins. The vast majority of all non-ionic detergents are- condensation products of ethylene oxide with a hydrophobe.
  • non-ionic detergents include, but are not limited to, alkyl aryl polyether sulfates, alcohol sulfonates, alkyl phenol polyglycol ethers, and polyethylene glycol alkyl aryl ethers, Triton-X 100, Nonidet p-40, Tween 20, Tween 80, tetramethyl ammonium chloride, sodium or lithium dodecyl sulphate, octyl-B-D-glucopyranoside, hexadecyltrimethyl ammonium bromide, ' and Brij-35.
  • a device for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance.
  • the indicator substance that is absent from or found in trace quantities may be, but is not limited to, silver, gold, platinum, iron, copper, zinc, calcium, chromium, titanium, nickel, manganese, magnesium and other metals, ions or elements.
  • trace quantities is understood to mean not present in amounts sufficient to biologically or chemically interact with the marker dye to a sufficient degree to cause a color change above an acceptable baseline level.
  • the means for detecting the indicator substance may vary depending on the indicator substance that is used in the device.
  • the means for detecting the indicator substance may be a marker dye that is operable to detect the presence of the indicator substance, and when detection occurs emits a colour or undergoes a colour change.
  • the means for detecting the indicator substance may be a chemical process that will detect the release of the indicator substance from the hyaluronic acid after hydrolysis.
  • a chemical process similar to that used in the exposure of photographic film may be used to detect silver particles that were bound to hyaluronic acid and are released upon hydrolysis of the hyaluronic acid by hyaluronidase.
  • a device for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, and a means for detecting the indicator substance.
  • a sperm sample that has been pre-treated with a non-ionic detergent is contacted with the carrier matrix.
  • a device for assessing the fertilizing ability of a sperm sample, the sperm sample being pre-treated with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, the device comprising a solid support having a carrier matrix containing a non- ionic detergent and a means for detecting the indicator substance.
  • a method for assessing the fertilizing ability of a sperm sample comprising the steps of providing a device comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance, contacting the support with a sperm sample, and assessing the means for detecting the amount the indicator substance.
  • a method for assessing the fertilizing ability of a sperm sample comprising the steps of pre-treating a sperm sample with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, providing a device comprising a solid support having a carrier matrix containing a non-ionic detergent and a means for detecting the indicator substance, and assessing the means for detecting the indicator substance.
  • An example of one embodiment of the invention discussed above includes providing a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably " linked to Calcium, a non-ionic detergent and Fura- 2TM.
  • a sperm sample is applied to the carrier matrix the non-ionic detergent will release any hyaluronidase present in the sperm head. Any released hyaluronidase will then hydrolyse the hyaluronic acid in the carrier matrix causing the release of the Calcium from the acid.
  • the fluorescent dye, Fura-2TM will then detect the presence of the Calcium and can then be detected.
  • a further example of an embodiment of the invention includes providing a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to silver particles and a non-ionic detergent.
  • a sperm sample is applied to the carrier matrix the non-ionic detergent will release any hyaluronidase present in the sperm head. Any released hyaluronidase will then hydrolyse the hyaluronic acid in the carrier matrix causing the release of the silver particles from the acid.
  • the carrier matrix may then be processed in a manner similar to that of the exposure of a photographic film or plate and therefore upon exposure will indicate the presence of the silver particles.

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Abstract

The present invention provides a device, for assessing the fertilizing ability of a sperm sample, having a solid support including a carrier matrix containing hyaluronic acid operably linked to a marker dye and a non-ionic detergent.

Description

SPERM FERTILITY ASSAY
FIELD OF THE INVENTION
This invention relates to a device for assessing the fertilizing ability of a sperm sample and in particular to a device that contains hyaluronic acid for assessing the fertilizing ability of a sperm sample.
BACKGROUND OF THE INVENTION
Fertility testing has become a more prevalent and desired test as a result of an increase in couples seeking advice and assistance on infertility because of their inability to conceive. In such situations both females and males must undergo a fertility test to ascertain if they are infertile.
Male fertility tests can actually test for different parameters. For example, U.S. Patent No. 5,434,057 (Dorian) discloses a test kit that assesses the amount of motile sperm in a sample, whereas U.S. Patent No. 5,605,803 (Herr et al.) discloses a test that determines the total sperm concentration in a sample.
Many male fertility test kits that are available can only be utilized in a laboratory or doctor's office and are not easily accessible or easy to use by the average person. However, some kits have been developed that do not require extensive training in order to use them.
Hyaluronidase is an enzyme that is present in the sperm acrosome. It has been demonstrated previously that in the presence of detergent this acrosomal enzyme is released from the sperm head. Once released hyaluronidase is free to act, for example, by hydrolizing hyaluronic acid into its metabolites. The hyaluronidase activity correlates to the sperm count and also to sperm motility and morphology, as discussed in Abdul-Aziz M, MacLusky NJ, Bhavnani B, Casper RF., 1995, Hyaluronidase activity in human semen: correlation with fertilization in INF. Fertil Steril, 64:1147-53.
It has been previously demonstrated that hyaluronidase activity correlates with sperm concentration motility and moφhology, as discussed in CA Patent Application No. 2,154,819 published March 3, 1996. This application disclosed that by measuring hyaluromdase activity an index of the potential fertilizing ability of the sperm is provided. CA 2,154819 discloses a method of measuring the amount of hyaluronidase enzyme present in the sperm sample. The method included the use of a substrate that can be enzymatically acted upon by the hyaluronidase, the substrate being suspended in a semi solid substance. The sperm sample to be assessed is pretreated with a non-ionic detergent to ensure the maximum release of the hyaluronidase is achieved from the sperm in the sample. The aliquot of the treated sample is then applied to the semi solid substance containing the substrate and any hyaluronidase enzyme present in the aliquot will react with the substrate. Over a fixed period of time the amount of substrate being modified by the enzyme will be proportional to the amount of enzyme that was present in the aliquot. Therefore, in order to measure the amount of enzyme in the aliquot the substrate modified by the enzyme can be measured and the measurement used to determine the amount of enzyme present in the aliquot and subsequently in the sample.
In view of the prior art it is therefore desirable to provide a device and method for assessing sperm fertility that is easy to use and provides an accurate measurement of the functional fertilizing capacity of the sperm.
It is further desired to provide a device and method for assessing sperm fertility that measures the enzymatic activity of hyaluronidase.
SUMMARY OF THE INVENTION
The present invention provides a device for assessing the fertilizing ability of a sperm sample utilising hyaluronic acid.
In one embodiment of the present invention there is provided a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to a marker dye and a non-ionic detergent.
In another embodiment of the present invention there is provided a device, for assessing the fertilizing ability of a sperm sample, the sperm sample having been contacted with a non-ionic detergent, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to a marker dye.
The present invention further provides a method, for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a solid support having an insoluble carrier matrix containing a non-ionic detergent, hyaluronic acid and means for generating a visual signal operably linked to the hyaluronic acid, contacting the carrier matrix with a sperm sample and detecting the visual signal. In a preferred embodiment the means for generating a visual signal is a marker dye.
The present invention further provides a method, for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a solid support having a carrier matrix containing hyaluronic acid and means for generating a visual signal operably linked to the hyaluronic acid, mixing a sperm sample with a non-ionic detergent, contacting the carrier matrix with the mixture and detecting the visual signal. In a preferred embodiment the means for generating a visual signal is a marker dye.
The present invention further provides a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance.
The present invention further provides a method for assessing the fertilizing ability of a sperm sample comprising the steps of providing a device comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance, contacting the carrier matrix with the sperm sample and assessing the means for detecting the indicator substance.
The present invention further provides a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a marker dye operable to detect the presence of the indicator substance.
The present invention further provides a device, for assessing the fertilizing ability of .a sperm sample, the sperm sample having been contacted with a non-ionic detergent, the device comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, and a marker dye operable to detect the presence of the indicator substance The present invention further provides a device, for assessing the fertilizing ability of a sperm sample, the sperm sample having been contacted with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities in, the seminal plasma or sperm, the device comprising a solid support having a carrier matrix containing a non-ionic detergent and a marker dye operable to detect the presence of the indicator substance.
The present invention further provides a device, for assessing the fertilizing ability of a sperm sample, the sperm sample having been contacted with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities in, the seminal plasma or sperm and a non-ionic detergent, the device comprising a solid support having a carrier matrix containing a marker dye operable to detect the presence of the indicator substance.
The present invention further provides a method for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance, contacting the carrier matrix with the sperm sample and detecting the amount of the means for detecting the indicator substance. In a preferred embodiment the means for detecting the indicator substance is a marker dye, and the step of detecting the amount of the means is the generation of a visual signal.
The present invention further provides a method for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, and a means for generating a visual signal operable to detect the presence of the indicator substance, contacting the sperm sample with a non-ionic detergent, contacting the carrier matrix with the mixture of the sperm sample and the non-ionic detergent and detecting the visual signal. In a preferred embodiment the means for generating a visual signal is a marker dye.
The present invention further provides a method for assessing the fertilizing ability of a sperm sample, comprising the steps of contacting the sperm sample with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, providing a solid support having a carrier matrix containing a means for generating a visual signal operable to detect the presence of the indicator substance and a non- ionic detergent, contacting the carrier matrix with the mixture of the sperm sample, and the hyaluronic acid and detecting the visual signal. In a preferred embodiment the means for generating a visual signal is a marker dye
The present invention further provides a kit comprising the solid support of any one of the above devices and an instruction leaflet which outlines the method as set out above for assessing the fertilizing ability of a sperm sample.
The present invention further provides a kit for assessing the fertilizing ability of a sperm sample comprising a device having a solid support containing a carrier matrix which contains hyaluronic acid operably linked to a marker dye and a non-ionic detergent, and an instruction leaflet that outlines the method comprising the steps of contacting the carrier matrix with a sperm sample and detecting the visual signal.
The present invention further provides a kit for assessing the fertilizing ability of a sperm sample comprising a device having a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance and an instruction leaflet that outlines the method comprising the steps of contacting the carrier matrix with a sperm sample and assessing the means for detecting the indicator substance.
DETAILED DESCRIPTION OF THE INVENTION
In a preferred embodiment, the present invention provides a device for assessing the fertilizing ability of a sperm sample. In one embodiment of the present invention the device comprises a solid support having a carrier matrix that contains hyaluronic acid operably linked to a marker dye and a non-ionic detergent.
When a sperm sample comes into contact with the solid support the non-ionic detergent releases hyaluronidase from the sperm head. The hyaluronidase is then free to hydrolyse the hyaluronic acid present. Upon hydrolysis of the hyaluronic acid by hyaluronidase the marker dye is released from the hyaluronic acid. In one embodiment of the present invention the marker dye is operable to change colour and when linked to the hyaluronic acid emits a first colour or is colourless, and when released from the hyaluronic acid will emit a second colour. This change in colour will therefore indicate the presence of hyaluronidase in the sperm sample. In another embodiment of the present invention the device comprises a solid support having a carrier matrix that contains hyaluronic acid operably linked to a marker dye. In use, a sperm sample is pre-treated with a non-ionic detergent. The detergent releases hyalorunidase from the head of the sperm. The carrier matrix is then contacted with the pre-treated sperm sample, and the hyaluronidase in the sample is then free to hydrolyse the hyaluronic acid present in the carrier matrix. Upon hydrolysis of the hyaluronic acid by hyaluronidase, the marker dye is released from the hyaluronic acid.
As will be understood by a person skilled in the art the support used in the above embodiments may vary depending on the type of carrier matrix to be used. The carrier matrix of the present invention may be a solid or liquid or gel. As will also be understood the carrier matrix may be the whole support, or a part of the support or a structure or substance that is attached to or contained within the support.
The carrier matrix of the embodiments of the present invention will contain chemical compositions that will participate in the chemical reactions of the device of the present invention. The chemical compounds of the present invention may be applied to the carrier matrix and dried. Alternatively the chemical compounds may be present in a liquid or gel form and be held in that state within the carrier matrix.
The carrier matrix may be an insoluble bibulous or non-bibulous material that maintains its structural integrity when exposed to physiological fluids such as semen. Suitable bibulous materials include, but are not limited to, paper, cellulose, synthetic resin fleeces, glass fibers, and woven and non-woven fabrics. Examples of suitable non-bibulous materials include, but are not limited to organo-plastic materials, such as polypropylene.
Variations in the support and the carrier matrix may be used in the device of the present invention. For example the device may be a take home test kit comprised of a solid support formed from a suitable plastic material, and the carrier matrix may be formed from a synthetic resin. The carrier matrix may be attached to the solid support using a suitable adhesive such as an epoxy resin. Other ways of attaching the carrier matrix to the solid support will be understood by a person skilled in the art. Alternatively the device may be a take home test kit comprised of a solid support formed from a suitable plastic material, for example a capsule or test tube like structure, and the carrier matrix may be formed from a gel, for example agar.
Suitable marker dyes, that may be used in the above embodiments, include any colorigenic substance that can be bound to or encapsulated by hyaluronic acid in such a way that hydrolysis of hyaluronic acid by hyaluronidase results in a color change. Such possible dyes include, but are not limited to, fluorescent dyes such as Fura-2 and Fluo-3, which could be used to detect hyaluronidase release of ions such as calcium bound to hyaluronic acid; 2',7'-bis-(carboxyethyl)- 5-(and-6)-carboxyfluorescein, 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate and other fluorescent dyes that can reflect pH changes as a result of hyaluronic acid hydrolysis; biotin and its derivatives, alkaline phosphatase substrates, indolyl type substrates, coumarin- or fluorescein- based fluorogenic substrates, redox enzyme substrates such as dihydrorhodamine 123, bioluminescent substrates such as coelenterazine, β-galactosidase conjugated to streptavidin, avidin or antibodies as secondary detection reagents could be used to detect chromogenic β- galactosidase substrates such as X-gal, which produces a dark blue precipitate on enzymatic hydrolysis or others yielding red-, pink-, puφle- and green-colored precipitate on enzyme hydrolysis.
Non-ionic detergents are non-denaturing and permit the solubilization of membranes without interfering with biological activity. They are suitable for the study of protein conformations and for the separation of hydrophilic proteins from membrane spanning hydrophobic proteins. The vast majority of all non-ionic detergents are- condensation products of ethylene oxide with a hydrophobe. Examples of suitable non-ionic detergents include, but are not limited to, alkyl aryl polyether sulfates, alcohol sulfonates, alkyl phenol polyglycol ethers, and polyethylene glycol alkyl aryl ethers, Triton-X 100, Nonidet p-40, Tween 20, Tween 80, tetramethyl ammonium chloride, sodium or lithium dodecyl sulphate, octyl-B-D-glucopyranoside, hexadecyltrimethyl ammonium bromide, ' and Brij-35.
In a further embodiment of the invention there is provided a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance. The indicator substance that is absent from or found in trace quantities may be, but is not limited to, silver, gold, platinum, iron, copper, zinc, calcium, chromium, titanium, nickel, manganese, magnesium and other metals, ions or elements. The use of the term "trace quantities" is understood to mean not present in amounts sufficient to biologically or chemically interact with the marker dye to a sufficient degree to cause a color change above an acceptable baseline level.
It will be understood by a person skilled in the art that the means for detecting the indicator substance may vary depending on the indicator substance that is used in the device. For example, the means for detecting the indicator substance may be a marker dye that is operable to detect the presence of the indicator substance, and when detection occurs emits a colour or undergoes a colour change.
Alternatively the means for detecting the indicator substance may be a chemical process that will detect the release of the indicator substance from the hyaluronic acid after hydrolysis. For example, a chemical process similar to that used in the exposure of photographic film may be used to detect silver particles that were bound to hyaluronic acid and are released upon hydrolysis of the hyaluronic acid by hyaluronidase.
In another embodiment of the present invention there is provided a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, and a means for detecting the indicator substance. In use, a sperm sample that has been pre-treated with a non-ionic detergent is contacted with the carrier matrix.
In a further embodiment of the invention there is provided a device, for assessing the fertilizing ability of a sperm sample, the sperm sample being pre-treated with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, the device comprising a solid support having a carrier matrix containing a non- ionic detergent and a means for detecting the indicator substance.
In a further embodiment there is provided a method for assessing the fertilizing ability of a sperm sample, comprising the steps of providing a device comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance, contacting the support with a sperm sample, and assessing the means for detecting the amount the indicator substance.
In a further embodiment there is provided a method for assessing the fertilizing ability of a sperm sample, comprising the steps of pre-treating a sperm sample with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, providing a device comprising a solid support having a carrier matrix containing a non-ionic detergent and a means for detecting the indicator substance, and assessing the means for detecting the indicator substance.
Further details of the preferred embodiments of the present invention are illustrated in the following examples, which are provided for illustration only and are understood to be non- limiting with respect to the appended claims.
An example of one embodiment of the invention discussed above includes providing a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably "linked to Calcium, a non-ionic detergent and Fura- 2™. When a sperm sample is applied to the carrier matrix the non-ionic detergent will release any hyaluronidase present in the sperm head. Any released hyaluronidase will then hydrolyse the hyaluronic acid in the carrier matrix causing the release of the Calcium from the acid. The fluorescent dye, Fura-2™, will then detect the presence of the Calcium and can then be detected.
A further example of an embodiment of the invention includes providing a device, for assessing the fertilizing ability of a sperm sample, comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to silver particles and a non-ionic detergent. When a sperm sample is applied to the carrier matrix the non-ionic detergent will release any hyaluronidase present in the sperm head. Any released hyaluronidase will then hydrolyse the hyaluronic acid in the carrier matrix causing the release of the silver particles from the acid. The carrier matrix may then be processed in a manner similar to that of the exposure of a photographic film or plate and therefore upon exposure will indicate the presence of the silver particles.
The prefened embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto with departing from the spirit of the invention on scope of the appended claims.

Claims

CLAIMS:
1. A device, for assessing the fertilizing ability of a sperm sample, comprising: a solid support having a carrier matrix containing a non-ionic detergent and hyaluronic acid operably linked to or encapsulating a marker dye.
2. A device for assessing the fertilizing ability of a sperm sample, comprising: a solid support having a carrier matrix containing a non-ionic detergent, hyaluronic acid and means for generating a visual signal operably linked to the hyaluronic acid wherein the detergent is operable to release hyaluronidase from the sperm sample which will hydrolize the hyaluronic acid and the means for generating the visual signal responds to the hydrolysis to indicate the presence of hyaluronidase.
3. A method, for assessing the fertilizing ability of a sperm sample, comprising the steps of: providing a solid support having an insoluble carrier matrix containing a non- ionic detergent, hyaluronic acid and means for generating a visual signal operably linked to the hyaluronic acid; contacting the carrier matrix with a sperm sample; and detecting the visual signal.
4. A kit for assessing the fertilizing ability of a sperm sample comprising: a solid support having a carrier matrix containing hyaluronic acid operably linked to a marker dye and a non-ionic detergent; and an instruction leaflet.
5. A device, for assessing the fertilizing ability of a sperm sample, comprising:
a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance.
6. A method, for assessing the fertilizing ability of a sperm sample, comprising the steps of: providing a device comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance; contacting the carrier matrix with a sperm sample; and assessing the means for detecting the indicator substance.
7. A device, for assessing the fertilizing ability of a sperm sample, the sperm sample being pre-treated with a non-ionic detergent, the device comprising: a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, and a means for detecting the indicator substance.
8. A method, for assessing the fertilizing ability of a sperm sample, comprising the steps of: pre-treating a sperm sample with a non-ionic detergent; providing a device comprising a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, and a means for detecting the indicator substance; contacting the carrier matrix with the pre-treated sperm sample; assessing the means for detecting the indicator substance.
9. A device, for assessing the fertilizing ability of a sperm sample, the sperm sample being pre-treated with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, the device comprising: a solid support having a carrier matrix containing a non-ionic detergent and a means for detecting the indicator substance.
10. A method for assessing the fertilizing ability of a sperm sample, comprising the steps of: pre-treating a sperm sample with hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm; providing a device comprising a solid support having a carrier matrix containing a non-ionic detergent and a means for detecting the indicator substance; contacting the carrier matrix with the pre-treated sperm sample; assessing the means for detecting the indicator substance.
11. A kit for assessing the fertilizing ability of a sperm sample, comprising: a solid support having a carrier matrix containing hyaluronic acid operably linked to an indicator substance that is absent from, or found in trace quantities, in the seminal plasma or sperm, a non-ionic detergent and a means for detecting the indicator substance; and an instruction leaflet.
PCT/CA2003/002014 2002-12-23 2003-12-23 Sperm fertility assay Ceased WO2004057019A1 (en)

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Citations (2)

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US5719031A (en) * 1996-08-14 1998-02-17 Molecular Probes, Inc. Dye labeled polymers as reagents for measuring polymer degradation

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