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WO1996013225A9 - Tests, dispositifs et trousses servant a determiner la fertilite masculine - Google Patents

Tests, dispositifs et trousses servant a determiner la fertilite masculine

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Publication number
WO1996013225A9
WO1996013225A9 PCT/US1995/014083 US9514083W WO9613225A9 WO 1996013225 A9 WO1996013225 A9 WO 1996013225A9 US 9514083 W US9514083 W US 9514083W WO 9613225 A9 WO9613225 A9 WO 9613225A9
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WO
WIPO (PCT)
Prior art keywords
sperm
assay
sample
pregnancy potential
antibody
Prior art date
Application number
PCT/US1995/014083
Other languages
English (en)
Other versions
WO1996013225A3 (fr
WO1996013225A2 (fr
Filing date
Publication date
Priority claimed from US08/332,825 external-priority patent/US5895749A/en
Application filed filed Critical
Priority to JP8514826A priority Critical patent/JPH11514204A/ja
Priority to EP95939003A priority patent/EP0789538A1/fr
Priority to AU40182/95A priority patent/AU4018295A/en
Publication of WO1996013225A2 publication Critical patent/WO1996013225A2/fr
Publication of WO1996013225A9 publication Critical patent/WO1996013225A9/fr
Publication of WO1996013225A3 publication Critical patent/WO1996013225A3/fr

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Definitions

  • male infertility is responsible almost 40% of the time that a couple is unable to conceive a child.
  • use of male contraceptives is on the rise. For example, according to current estimates, more than 500,000 vasectomies are performed in the U.S. each year and about 2,000,000 are performed worldwide. In addition to vasectomies, a variety of oral contraceptives for use by males are in development. Although male contraceptives decrease the probability that a fertile male will initiate a pregnancy, no male contraceptive is 100% effective. Further, most male contraceptives require a certain period of time in which to take effect.
  • a semen sample to be considered fertile must contain a sperm density of greater than 20 million spermatozoa/mL and/or a percent motility of 60% with a forward progression greater than 2 (on a 1-4 scale).
  • the semen samples should show no evidence of sperm agglutination, pyospermia or hyperviscosity (Sigman, M., et al., Evaluation of the subfertile male. In: Lipschultz, LI and SS Howards eds. Infertility in the Male, 2nd ed. Chicago: Mosby-Year Book, 1991 ; p.184).
  • male infertility is diagnosed based on low sperm motility and/or count.
  • motility analyses can produce false negatives, since viable sperm may appear non-motile due to damage sustained during processing.
  • sperm count 20 million spermatozoa/mL or greater is generally considered to be in the fertile range.
  • non-motile, non-viable sperm can be included in the count.
  • Sperm count and motility are typically assessed using commercially available instruments, such as light microscopes and computerized videoanalysis systems.
  • U.S. Patent No. 5,068,089 describes a home kit for testing fertility of human sperm based on ability of the sperm to reduce a dye. The extent of reduction (displayed colorimetrically), is said to be indicative of sperm fertilizing ability. However, this test is time consuming, requires incubation at a temperature above room temperature and does not distinguish between reduction due to sperm cells or other cells, which may be present in a semen sample.
  • U.S. Patent No. 5,219,729 describes a laboratory assay for determining the fertilizing ability of sperm based on the affinity of binding to an oocyte zona pellucida fragment. The tighter the binding, the greater the fertilizing ability of the sperm sample. However, this assay requires freshly prepared oocyte fragments and at least four hour's time during which the sperm must be kept in contact with the oocyte fragment.
  • U.S. Patent No. 5,434,057 describes assays and kits for determing male fertility based on the detection of fumarase activity.
  • fumarase is ubiquitously present in cells including cells that may be present in a sperm sample (epithelial, leukocyte and bacterial or fungal), whether fumarase activity is an accurate indicator of sperm count and motility, as claimed in the patent, is dubious.
  • a simple, rapid and accurate assay for determining the fertility of a sperm sample in a reference laboratory, doctor's office or at home is needed.
  • ARTs Assisted reproductive technologies
  • IVF in vitro fertilization
  • GIFT gamete intrafallopian transfer
  • IUI intrauterine insemination
  • ICSI sperm intracytoplasmic injection
  • IVF has an estimated success rate of about 25%, while GIFT is estimated as being successful in about 31% of attempts.
  • One factor, which may contribute to an unsuccessful ART attempt is that not all sperm samples are capable of fertilization. According to recent studies, male infertility is responsible almost 40% of the time that a couple is unable to conceive a child.
  • cryopreserved sperm in which cell viability post-thaw is lost more rapidly at later times than in fresh cells.
  • Sublethal cryodamage has been shown to be due in part to membrane embrittlement during the phase transitions involved in freezing and thawing (Alvarez, J.G. and B.T. Storey (1993) J. Androl. 14 (3): 199-209).
  • cryopreservation of sperm samples is routinely performed in ART procedures and in fact is required in order to test the donor for the presence of transmissible infectious agents (e.g., HIV) prior to insemination.
  • transmissible infectious agents e.g., HIV
  • a donor is typically tested six months after producing a particular sample and only if the test is negative will the stored sample be used for insemination.
  • the invention features devices, procedures and kits for isolating and quantitating motile sperm from a sperm-containing sample (e.g. semen) and/or for determining the pregnancy potential of a sperm sample.
  • the devices comprise a container for isolating a sperm containing sample, which is preferably adapted to separate sperm based on motility.
  • the container can optionally be conformed to facilitate collection (e.g. funnel-shaped).
  • a retainer for accomodating entry and migration of motile sperm, but not non-motile cells in the sperm containing sample is positioned inside of the container.
  • isolated motile sperm can be obtained from the retainer and separately tested.
  • the retainer is a compartment (e.g. a tube), which is in fluid communication with the sperm containing sample.
  • the retainer is a fluid, which is less dense than the sperm containing sample and thereby facilitates passage of motile sperm, but not non-motile cells.
  • the retainer includes a porous membrane, which separates the sperm containing sample from an isolation area and which thereby prevents passage of non-motile sperm or other cells that may be present in the sperm containing sample, but allows passage of motile sperm into the isolation area.
  • the invention features assays for identifying sperm samples with high pregnancy potential.
  • the assays can be used to provide an indication of the fertility status of the male donor or can be selected for use in an Artificial Reproductive Technology (ART).
  • the process comprises the steps of: i) obtaining multiple sperm samples from a donor over time, ii) obtaining an aliquot from each sperm sample, iii) testing each aliquot to determine pregnancy potential, and iv) using at least one sperm sample having high pregnancy potential in an ART to initiate a pregnancy.
  • the testing is based on quantitating an indicator of lipid peroxidation or a change in an indicator induced by a stress.
  • a particularly preferred indicator of sperm lipid peroxidation is sperm superoxide dismutase (SOD) activity, which can be quantitated, for example, using an anti-SOD antibody.
  • SOD superoxide dismutase
  • the invention features screening assays for detecting the pregnancy potential of sperm samples and thereby identifying potentially infertile males or evaluating males following vasectomy or once a particular contraceptive has been administered.
  • the screening assay is based on determining motile sperm count.
  • motile sperm are first isolated and counted. Quantitation of less than about 50,000 motile spermatozoa/mL indicates that the contraceptive has been effective and that the sperm sample has low pregnancy potential.
  • quantitation of sperm is performed directly on the sperm containing sample (without separation of motile and immotile sperm) and quantitation of less than about 20 million spermatozoa/mL indicates that the sample has low pregnancy potential; greater than about 20 million spermatozoa/mL and less than about 40 million spermatozoa/mL indicates that the sample has borderline pregnancy potential; and greater than about 40 million spermatozoa/mL indicates that the sample has high pregnancy potential.
  • the cutoff value of 20 million spermatozoa/mL includes both motile and immotile spermatozoa. Although this value is not diagnostic of infertility, it is intended to be used as part of a screening test. In this way, males that use the kit to test their fertility potential and have less than about 20 million spermatozoa/mL will be alerted to a potential infertility problem.
  • the invention features sperm diagnostic kits or systems which comprise a number of simple reagents and devices packaged in a box.
  • the kit can include a sperm isolation means and a means for identifying a high pregnancy potential sperm sample.
  • the sperm isolation means is of an appropriate conformation (e.g. funnel shaped) to facilitate collection.
  • a reagent for liquefying semen e.g. pronase or chymotrypsin ⁇ is included in the kit for use in sperm isolation.
  • the means for identifying sperm with high pregnancy potential is based on lipid peroxidation and the means for screening for pregnancy potential (e.g. infertility or effective male contraception) is based on sperm count.
  • the instant disclosed assays, devices and kits are easy to perform and can be completed in less than about 15 minutes' time.
  • the assays, devices and kits provide information which is useful, for example, in monitoring the impact of changes in such factors as diet, sleep, exercise, exposure to smoke or other carcinogens, and intake of alcohol or drugs on the fertility of sperm samples produced thereafter.
  • the devices, kits and methods can indicate whether a particular male fertility or contraceptive treatment has been effective or whether sperm subsequently obtained from the same male will initiate a pregnancy upon contact with an oocyte.
  • the assays, devices and kits are useful for determining whether a particular sperm sample is suitable for use in an ART.
  • Figure 1 is a perspective view of one embodiment of a sperm isolation device, which includes a tube for receiving and capturing motile sperm.
  • Figure 2 is a perspective view of another embodiment of a sperm isolation device, which includes a less dense fluid layer in contact with a more dense sperm-containing sample.
  • Figure 3 is a perspective view of yet another embodiment of a sperm isolation device, in which a porous membrane separates the sperm sample from a motile sperm collection area.
  • Figure 4 is a graph plotting the motility recovery after cryopreservation for sperm samples having a stress test score of greater than about 0.8 and samples having a stress test score of less than about 0.8.
  • the invention pertains to easy to use devices that can rapidly recover motile sperm from a sperm containing sample (e.g. semen) and assays and kits, which employ the devices for use in identifying a high pregnancy potential sperm sample.
  • the invention also pertains to assays and kits for screening males for infertility and for confirming whether a male contraceptive is effective or has taken effect.
  • Preferred sperm isolation devices are based on sperm motility and comprise a housing means for holding sperm, an isolation means selectively disposable within the housing means for isolating motile sperm and a means for testing the motile sperm to determine pregnancy potential.
  • a container 10 that has side walls 12, 14, 16 and 18 and a bottom portion 20 forming a sample well 21 for receiving a sample 30 containing ejaculate from a male donor. Although illustrated as cubical, the container 10 can have any selected shape or size suitable for the capture and retention of a sperm containing sample.
  • a retainer 24 can be positioned within the sample well 21 of the container, and is preferably placed in fluid communication with the sample 30.
  • the retainer 24 can be mounted to the floor portion 20 of the container 10 by a number of known methods, including the use of an adhesive, or can be supported within the container 10 by any suitable support means. Although illustrated as cylindrical, the retainer 24 can have any selected shape or size suitable for the isolation and retention of motile sperm, and can include commercially available instruments, such as tubes, capillaries, straws, pipettes, and other suitable receptacles having an internal conduit. Additionally, the container 10, although illustrated as having a substantially rectangular shape, can be configured to have any selected shape, such as a funnel-like configuration, to assist the male donor in collecting and capturing a sperm sample. The container 10 and retainer 24 can be made from any suitable biocompatible material, such as glass or plastic.
  • the illustrated sample 30 can contain both motile and non-motile sperm.
  • the retainer 24 is positioned within the well 21 or constructed to allow the motile sperm to migrate into the conduit 22 of the receptacle 24.
  • the receptacle 24 captures the motile sperm, which are then analyzed to determine the pregnancy potential of the sperm sample. For example, after a sufficient period of time has elapsed to ensure that motile sperm present in the sample 30 have migrated into the retainer 24.
  • the retainer can be removed from the container 10 and placed within a second container 31. e.g., a test tube, containing a means for testing the motile sperm to determine the pregnancy potential of the sample 34.
  • a detection mechanism 36 can be mounted within the conduit 22 of the retainer 24.
  • the motile sperm that migrate into the conduit 22 preferably contact the detection mechanism 36.
  • the detection mechanism preferably includes a suitable membrane or substrate containing a means for testing motile sperm to determine the pregnancy potential of the sperm containing sample.
  • Motile sperm can alternatively be isolated from non-motile cells in a sperm sample based on contact with a mechanical or fluid barrier or gradient.
  • a relatively dense sperm-containing sample can be contacted with a less dense fluid layer, which is preferably of a similar temperature, pH and salt concentration as the sperm-containing sample.
  • a relatively dense sperm-containing sample can be contacted with a less dense fluid layer, which is preferably of a similar temperature, pH and salt concentration as the sperm-containing sample.
  • like parts are represented throughout with the same reference numerals plus a superscript prime.
  • the illustrated container 10' includes a first fluid layer 40 and a second fluid layer 46.
  • the first fluid layer 40 includes a sperm sample from the male donor and optionally an appropriate reagent for liquefying the semen sample, e.g. pronase or chymotrypsin.
  • the layer 40 typically includes motile and/or non-motile sperm.
  • the second fluid layer 46 is preferably a fluid having a density that is less than the density of the first fluid layer 40, thus creating a density gradient along the height of the container 10'.
  • the decreasing density between layers 40 and 46 promote the migration of motile sperm from the sample fluid layer 40 into the collection fluid layer 46.
  • This axial layered construction of the fluids 40 and 46 can be accomplished by selecting appropriate fluids that are emissible or partially emissible relative to one another.
  • a sperm sample is placed within the container or is mixed with a reagent for liquefying ejaculate prior to introduction into the container 10'.
  • a second fluid layer 46 having a lower density is then introduced into the container 10' and is separated from and disposed axially above the first fluid layer 40.
  • the motile sperm present in the sample layer 40 migrate into the second fluid layer 46 in response to the diminishing density gradient along the height of the container 10'.
  • the motile sperm are isolated in the second fluid layer 46.
  • a selected period of time e.g., five minutes
  • the solution preferably contains a hypotonic solution having peroxidase-conjugated anti-SOD and/or anti-GPx IgG polyclonal antibodies.
  • a test strip containing bound sheep anti-SOD and/or anti-GPx IgG polyclonal antibodies is placed into the test solution, removed and then placed in a final solution containing a peroxidase substrate (e.g. hydrogen peroxide and
  • test strip In response to the presence of motile sperm, the test strip changes to a selected color.
  • the pipette containing motile sperm can be directly applied to a test strip, which contains a sperm reagent.
  • the second collection fluid layer 46 can be replaced with a porous membrane 60.
  • the permeable porous membrane 60 introduces or presents a mechanical resistance to the migration of non-motile sperm from the sample layer 40' into or through the membrane 60.
  • the porous membrane 60 facilitates the migration and hence the isolation of motile sperm in the isolation layer 10' by allowing motile sperm to pass between sample layer 40' and the membrane 60.
  • the porous membrane 60 can be composed of any suitable biocompatible material that facilitates the passage of motile sperm without introducing damage thereto.
  • the invention relates to preferred methods for determining the pregnancy potential of a sperm sample.
  • a sperm sample with "high pregnancy potential" as described herein has a greater than about 50% probability for initiating a pregnancy upon contact with an oocyte.
  • a sperm sample having high pregnancy potential has an increased probability of initiating a pregnancy, contact of a high pregnancy potential sperm sample with an oocyte does not guarantee a successful pregnancy.
  • Other factors such as inability of an oocyte to decondense human sperm chromatin, defective oocyte DNA (e.g. due to age), two-cell embryo block or early embryo demise, may prevent the initiation of a pregnancy even by a high pregnancy potential sperm sample.
  • Preferred sperm containing samples for use in the disclosed assays are obtained from a human or animal (e.g. a bull, stallion, ram or other domesticated animal or an endangered animal). To ensure accuracy, tests are preferably performed on freshly collected ejaculate.
  • Preferred tests for identifying sperm samples with high pregnancy potential are lipid peroxidation tests, which measure an indicator of lipid peroxidation or a change in an indicator over a period of time under defined conditions. The value obtained is then compared with a standard value for that particular indicator to determine the pregnancy potential of that sperm sample.
  • a stress test measures a change in a particular indicator of lipid peroxidation over a period of time in response to a stress (a stress test).
  • a stress test score can be obtained by dividing a measured post-stress value of an indicator of lipid peroxidation by a measured pre-stress value.
  • appropriate stresses or stressing agents for obtaining a test score include radiation (such as thermal (e.g. as described in Example 2), electromagnetic), freeze-thawing, oxidation or exposure to chemical agents (e.g. oxidizing agents such as the ferrous iron/ascorbate system described in Example 3).
  • lipid peroxidation indicators that change in response to a stress include:
  • Motility When motility is used as an indicator of lipid peroxidation and the stress is thermal (e.g. incubation for at least about 1 hour at a temperature in the range of about 27-45°C), a test score of greater than about 0.8 indicates high pregnancy potential. A test score of less than about 0.8 indicates low potential.
  • Lipid peroxidation breakdown products Samples that have a relatively high post-stress value of lipid peroxidation breakdown products (such as lipid hydroperoxides, malonaldehyde, pentane and ethane) relative to pre-stress value (test score ⁇ 0.5) have a low probability of resulting in a successful pregnancy, while test scores of about 0.5 or greater increase the probability of initiating a pregnancy.
  • the lipids can be analyzed spectrophotometrically. For example, lipid hydroperoxides can be extracted with hexane and detected at 233nm. Malonaldehyde can be measured at 532nm following reaction with the thiobarbituric acid (Tapel, A.L. et al., (1959) Arch Biochem Biophys, #0:326). Pentane and ethane can be measured by head-space gas chromatography.
  • Ratio of membrane phospholipids Samples that oxidize at high rates have phosphatidylethanolamine (PE)/ phosphatidylcholine (PC) ratios ⁇ about 0.5, while samples that oxidize at low rates (test scores > about 0.8 as indicated by loss of motility) have PE/PC ratios of greater than about 0.7. Therefore, sperm with high pregnancy potential have PE PC ratios greater than about 0.7, but less than about 1.5, sperm with some pregnancy potential have PE/PC ratios of greater than about 0.5 and less than about 0.7, and sperm with low pregnancy potential have PE/PC ratios of less than about 0.5. PE and PC can be measured, for example, by high-performance thin-layer chromatography (Alvarez, J.G. et al., (1987) J Liquid Chromatogr 10: 3557).
  • lipid peroxidation indicators that can be directly measured as an indication of pregnancy potential include:
  • Oxidation of DNA Samples that oxidize at high rates have higher levels of oxo-8-deoxyguanosine (oxo°dG), which can be measured, for example, by high-pressure liquid chromatography (HPLC) using electrochemical detection (Fraga, C.G. et al., (1991) Proc Natl Acad Sci #5:11003.).
  • oxo°dG oxo-8-deoxyguanosine
  • Superoxide dismutase fSODI activity Solid phase-bound anti Cu/Zn- SOD antibodies and enzyme labeled anti-Cu/Zn-SOD antibodies can be used to detect SOD activity as described in Example 3.
  • Sperm samples with SOD activities greater than or equal to about 9U/10 8 cells have been shown to be associated with high pregnancy rates after IVF (i.e. have high pregnancy potential), while activities less than about 7U/10 8 cells are associated with low pregnancy rates (i.e. low pregnancy potential).
  • Surface-SOD immunofluorescence can be measured, for example, by flow cytometry using sheep anti -Cu/Zn- SOD IgG polyclonal antibodies and FITC- conjugated rabbit anti-sheep secondary antibodies (Alvarez, J.G. 18th Annual Meeting American Andrology Society, Tampa, FL., 1993, abstract 170). Greater than about 300 FITC units is indicative of high pregnancy potential, while less than about 300 FITC units is indicative of low pregnancy potential.
  • Ratio of unsaturated fatty acids to saturated fattv acids Unsaturated fatty acids are prone to oxidation while saturated fatty acids are insensitive to this process. Determination of the ratio of the unsaturated fatty acid, docosahexaenoic acid (22:6) to the saturated fatty acid palmitic acid (16:0) or of other unsaturated fatty acids to saturated fatty acids can be used to predict pregnancy potential. Samples that oxidize at high rates will have low docosahexaenoic acid/palmitic acid ratios. Unsaturated/saturated fatty acids can be measured by gas chromatography following alkaline methanolysis with IN sodium methoxide at 40C for 1 hour (Alvarez, J.G. and J.C. Touchstone, Practical Manual on Lipid Analysis. Series of Monographs: I- Fatty Acids.
  • a ratio of unsaturated to saturated fatty acids of greater than or equal to about 1.0 indicates high pregnancy potential; a ratio greater than or equal to about 0.5 and less than about 1.0 indicates borderline pregnancy potential; and a ratio of less than about 0.5 indicates low pregnancy potential.
  • Creatine kinase activity The concentration of creatine kinase in sperm reflects the degree of cytoplasmic extrusion during the last phase of spermatogenesis. Samples exhibiting abnormally high levels of creatine kinase activity have also been found to have high rates of lipid peroxidation as determined by the PE/PC ratios (PE/PC ratios ⁇ 0.5 and increased malonaldehyde production). This correlation supports the detection of creatine kinase activity as a means for determining pregnancy potential of a sperm sample.
  • Creatine kinase activity in human sperm can be measured by spectrophotometric analysis at 365 nm of the NADPH generated during reaction of creatine kinase with ADP to produce creatine and ATP, followed by reaction of hexokinase with ATP and glucose to produce glucose-6- phosphate, followed by reaction of glucose-6-phosphate dehydrogenase with glucose-6-phosphate and NADP to produce NADPH.
  • the NADPH generated under these conditions is proportional to the activity of creatine kinase in human sperm (Huszar, G. (1988) Gamete Res 19:61,).
  • Creatine kinase (CK) exists in two isoforms: MM and BB. Sperm with high pregnancy potential have a CK-MM/CK-MM+CK-BB ratio of greater than or equal to about 10%. Table 2
  • Lipid peroxidation indicator values or changes in value in response to a stress can be quantitated by techniques and devices, which are well-known to one of skill in the art.
  • the particular lipid peroxidation indicator chosen may be dictated by the degree of accuracy required and the availability of instruments to detect the results.
  • the indicator of sperm lipid peroxidation is based on sperm superoxide dismutase (SOD) activity.
  • SOD superoxide dismutase
  • sperm SOD activity is tested using an SOD antibody.
  • an antibody for use in detecting a sperm lipid peroxidation indicator can be any material that binds an antigen (e.g. polyclonal, monoclonal or single chain antibody or antibody fragment, such as an Fab of Fab' 2 fragment).
  • Immunodetection of sperm SOD or another antigenic indicator of lipid peroxidation can be accomplished using any of a number of competitive or non-competitive assay procedures.
  • competitive immunoassays are performed by adding SOD to a sperm containing sample, so that the sperm and the SOD compete for a limited number of antibody binding sites resulting in the formation of sperm-antibody and labeled SOD- antibody complexes.
  • concentration of labeled SOD and SOD antibody By maintaining the concentration of labeled SOD and SOD antibody constant, the amount of labeled SOD-antibody complex formed is inversely proportional to the amount of sperm present in the sample. A quantitive determination of the sperm SOD can therefore be made based on the labeled SOD-antibody complex.
  • competitive assays can be homogeneous (i.e. not requiring separation of antibody bound tracer (e.g. labeled SOD) from free tracer, since the antigen-antibody interaction causes, directly or indirectly, a measurable change in the signal obtained from the label group of the tracer).
  • competitive assays can be heterogeneous (i.e. requiring separation of bound tracer from free tracer prior to determining the amount of ligand in the sample).
  • non-competitive assays involve incubating a sperm containing sample with an immobilized SOD antibody for a period of time sufficient to reach equilibrium with regard to the formation of antibody-sperm conjugates.
  • the SOD antibody can be directly or indirectly labeled.
  • indirect labeling can be carried out after a wash step to remove unbound sperm by contacting the immobilized antibody-sperm complexes with a second, labeled antibody that is specific for the antibody-sperm complex. Following a second wash step to remove unbound second antibody, the amount of bound second antibody can be detected and measured as an indication of bound sperm.
  • An example of this procedure is described in the following Example 5.
  • Exemplary competitive and non-competitive immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).
  • FPIA fluorescence polarization immunoassay
  • FIA fluorescence immunoassay
  • EIA enzyme immunoassay
  • NIA nephelometric inhibition immunoassay
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • radioisotopes e.g. ⁇ H, ⁇ C, ⁇ 25
  • fluorescent compounds e.g. fluorescein, rhodamine, allophycocyanin, phycoerythin, erythrosin, europian, luminol, luciferin and coumarin
  • colored or uncolored beads or particles e.g. silica gel, controlled pore glass, magnetic, Sephadex Sepharose, cellulose, metal (e.g. gold) or latex).
  • Preferred supports for immobilizing antibodies include membranes (e.g. polyethylene, polypropylene, polyamide, polyvinylidenedifluoride, glass fiber, paper), beads or particles and tubes, (e.g., glass, plastic or metal capillaries, straws or pipettes).
  • membranes e.g. polyethylene, polypropylene, polyamide, polyvinylidenedifluoride, glass fiber, paper
  • beads or particles and tubes e.g., glass, plastic or metal capillaries, straws or pipettes.
  • the invention also features processes for increasing the success rate for initiating a preganancy using an assisted reproductive technology (ART) (i.e. a procedure for contacting a sperm with an ovum to initiate a pregnancy).
  • ARTs include in vitro fertilization (IVF), gamete intrafallopian transfer (GIFT) intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI). Procedures for performing ARTs are well- known to practitioners. It is expected that additional ARTs will be developed over time.
  • a preferred process of the invention involves obtaining multiple sperm samples from a donor (e.g. a male partner from a couple undergoing an ART) over time.
  • a donor e.g. a male partner from a couple undergoing an ART
  • a donor can provide a new sperm sample once every other day.
  • An aliquot (e.g. lx l ⁇ 6 cells) of each sample can then be obtained for testing, while the remainder can be banked (e.g. cryopreserved) for potential future use in an ART.
  • the process includes a means for correlating a particular aliquot with the banked sample from which it was obtained. For example, an aliquot taken from a sample, as well as the sample itself obtained on day 1 can be labelled #1. A subsequently obtained sample from the same donor and an aliquot taken from that sample can be labelled #2, etc..
  • All aliquots provided by a particular donor can then be tested to identify sperm samples having "high pregnancy potential" (e.g. sperm having a greater than 50% probability of initiating a pregnancy upon contacting an oocyte).
  • the sperm sample indicated as having the higher pregnancy potential can then be used in an ART and the remaining, lower pregnancy potential samples can be discarded.
  • Example 2 provides the results of a blind prospective cohort study of 33 couples undergoing in vitro fertilization (IVF) and 1 1 couples undergoing gamete intrafallopian transfer (GIFT), no successful pregnancies resulted from sperm samples which exhibited a stress test score of less than about 0.8.
  • IVF in vitro fertilization
  • GIFT gamete intrafallopian transfer
  • Table 3 shows how the stress test score and therefore the pregnancy potential of sperm obtained from the same male can vary with time.
  • Figure 4 shows that sperm samples with stress test scores greater than about 0.8 have a high motility recovery, even after cryopreservation. Therefore it appears that a further benefit of using sperm samples indicated as having high pregnancy potential in an ART results from the fact that such samples are not damaged by cryopreservation to the same degree as sperm with low pregnancy potential.
  • identification of sperm samples with low pregnancy potential can also be useful, for example, as a screen for detecting potentially infertile males.
  • identification of sperm samples with low pregnancy potential can also be useful, for example, as a screen for detecting potentially infertile males.
  • identifying a sperm sample as having low pregnancy potential use of the sample in an ART can be avoided.
  • the user can determine whether a particular male contraceptive is effective or has taken effect. For example, a vasectomy typically requires a phase in period in which to take effect.
  • the use of assays to identify sperm samples with low pregnancy potential is therefore useful for confirming the efficacy of a male contraceptive.
  • Low pregnancy potential sperm samples can be identified by lipid peroxidation tests as described above.
  • low pregnancy potential sperm samples can be identified based on quantification of spermatozoa (non-motile and motile) in a sperm containing sample (e.g. ejaculate). If the number obtained is less than about 20 million spermatozoa/mL, the sperm sample is considered to have low pregnancy potential.
  • sperm targets or sperm components that can be quantitated as an indication of the number of sperm present in a sperm containing sample, include a sperm protein (e.g. a sperm flagella protein, a glycolytic enzyme, an antioxidant enzyme (e.g. glutathione peroxidase or superoxide dismutase), a nuclear protein, an acrosomal protein, ⁇ - tubulin, lactate dehydrogenase (LDH-X), protamine, acrosin or a mitochondrial protein.); or a sperm lipid (e.g.
  • a sperm protein e.g. a sperm flagella protein, a glycolytic enzyme, an antioxidant enzyme (e.g. glutathione peroxidase or superoxide dismutase), a nuclear protein, an acrosomal protein, ⁇ - tubulin, lactate dehydrogenase (LDH-X), protamine, acrosin or
  • Preferred targets for sperm quantitation are selective for and abundant on sperm cells.
  • a sperm reagent e.g. sperm antibody, ligand, lectin or substrate
  • sperm reagents include labeled (e.g. enzyme, tracer (e.g. radioactive), dye or color particle labeled) or unlabeled anti-sperm antibodies (e.g. anti- human sperm polyclonal antibody; Arnel Products Co., Inc, Cherokee Station, New York, N.Y.; Chemicon International Inc., Temecula, California) or labeled or unlabeled antibodies against a sperm component (e.g. a sperm protein or sperm lipid).
  • labeled e.g. enzyme, tracer (e.g. radioactive), dye or color particle labeled
  • unlabeled anti-sperm antibodies e.g. anti- human sperm polyclonal antibody; Arnel Products Co., Inc, Cherokee Station, New York, N.Y.; Chemicon International Inc., Temecula, California
  • sperm reagents include labeled (e.g. dye or tracer labeled) or unlabeled reagents that interact with a sperm protein (e.g. Protein Reagent (0.3% tetrabromophenol, Miles Scientific, Connecticut) or rhodamine 123, which accumulates within sperm mitochondria.
  • a sperm protein e.g. Protein Reagent (0.3% tetrabromophenol, Miles Scientific, Connecticut
  • rhodamine 123 which accumulates within sperm mitochondria.
  • the labeled reagent e.g. rhodamine
  • Preferred methods for counting sperm employ detectably labeled antibodies that specifically bind a sperm antigen (e.g. anti-human sperm polyclonal antibodies and antibodies specific to an epitope of the sperm flagellum, nuclear proteins, glycolytic enzymes, acrosome etc.).
  • One method for quantitating sperm involves incubating a sperm sample with colored particles containing anti-sperm antibodies for an appropriate period of time to allow the sperm antigens to react with the antibody bound colored particles; ii) filtering the sample of step i), so that sperm/colored particle/antibody complex is retained on the filter and unbound, colored particle/antibody and seminal plasma protein passes through the filter; and iii) visualizing the color intensity.
  • sperm colored particle/antibody complexes can be quantitated by comparing the color of the niter to a color chart, which depicts various color possibilities for various quantities of sperm. Less than about 20 million spermatozoa/mL indicates low pregnancy potential.
  • the filter membrane can be configured to indicate a "+" (i.e. fertility), if a sufficient amount of sperm/detectable particle/antibody complex is retained on the filter membrane to indicate that the sample is suitable for initiating a pregnancy (greater than about 20 million spermatozoa/mL) and a "-" (i.e. infertility) if an insufficient amount of sperm/detectable particle/antibody complex is retained on the filter membrane (less than about 20 million spermatozoa/mL).
  • sperm can first be isolated. Seminal plasma-free sperm can then be contacted with anti-sperm antibody coated colored particles. After a sufficient period of time to allow antibodies and antigens to react, unbound antibody coated colored particles can be removed from the mixture and sperm colored particle/antibody complex detected and quantitated. For example, the quantity of sperm in the sample can be quantitated using a color chart or a "+" or "-" filter as described above. Alternatively, sperm can be quantitated by detecting the appearance of agglutination in a drop of sample following addition of anti-sperm antibodies with bound latex particles.
  • low pregnancy potential can be determined by quantitating only motile sperm in a sperm sample.
  • Motile sperm can be isolated from a sperm containing sample, for example, using the devices and procedures described above. Preferred methods for quantitating sperm employ detectably labeled antibodies that specifically bind a sperm antigen. Motile sperm can be quantitated using detectably labeled, sperm specific antibodies, such as anti-glutathione peroxidase (GPx) antibody. As with sperm SOD, irnrnunodetection of sperm GPx or other sperm antigen can be accomplished using any of a number of competitive or noncompetitive assay procedures. In addition, sperm specific antibodies can be labeled (e.g. enzymatically) and if necessary immobilized by procedures that are well known in the art or as described above for SOD antibodies.
  • the invention features sperm diagnostic kits or systems comprising a number of simple reagents and devices packaged in a box.
  • the kit components can include one or more of the devices or components illustrated in Figures 1-3, and reagents for determining the pregnancy potential of sperm samples.
  • Preferable kits include reagents for liquefying semen (e.g. chymotrypsin or pronase).
  • reagents for liquefying semen e.g. chymotrypsin or pronase
  • preferable kits are based on measuring an indicator of lipid peroxidation or a change in an indicator over a period of time under defined conditions.
  • a kit can be based on determining the number of spermatozoa present in a sperm sample, wherein greater than about 40 million motile spermatozoa/mL indicates high pregnancy potential; less than about 20 million motile spermatozoa/mL indicates low pregnancy potential; and greater than about 20 million, but less than about 40 million indicates borderline pregnancy potential.
  • kits for identifying a sperm sample with low pregnancy potential are based on determining the number of spermatozoa/mL present in the sample, wherein less than about 50,000 spermatozoa/mL indicates low pregnancy potential or successful contraception.
  • Reagents and devices for use in determining sperm pregnancy potential can include dispensing devices (e.g. capillaries or pipettes) for delivering a defined volume of isolated sperm from a collection container into a container which includes a means for determining the pregnancy potential of the sample, for example by quantitating the extent of sperm lipid peroxidation or change in lipid peroxidation resulting from a stress; and/ or sperm count.
  • the means for detecting sperm lipid peroxidation is based on sperm superoxide dismutase (SOD) activity and/or the means for detecting sperm count is based on sperm protein concentration.
  • the means for detecting sperm SOD activity employs a colorimetrically labeled SOD antibody and the means for detecting sperm protein employs gold particles.
  • the kit also preferably includes a color chart against which concentrations of SOD and/or protein can be compared against known values. The kit can be optimized to develop color based on the protein levels corresponding to sperm concentrations greater than or equal to about 50,000 spermatozoa/mL .
  • a preferred kit for home determination of the pregnancy potential of a sperm sample can include: i) a collection container (optionally conformed to capture ejaculate, and/or containing a solution (e.g. enzymatic) that facilitates semen liquefaction); ii) a second container in which is contained anti-sperm antibodies covalently bound to colored particles in a hypotonic solution; iii) a membrane or filter for capturing sperm/colored particle or sperm colored particle/antibody complexes and allowing passage of unbound, colored particle or colored particle/antibody; and iv) a chart for interpreting the results obtained based on the color of the filter. Indication of less than about 20 million spermatozoa/mL indicates low pregnancy potential when using the Male Fertility approach; or less than about 50,000 spermatozoa/mL when using the Male Contraceptive approach.
  • a solution e.g. enzymatic
  • Example 1 Kit for Isolating Motile Sperm From a Semen Sample
  • tube #1 for collecting and liquefying ejaculate containing 30mg/mL of dextran and 5mg/mL of chymotrypsin in an isotonic solution;
  • tube #2 which indicates three volumes: lower (1 mL), middle (1.5 mL) and upper (2 mL).
  • the three volumes can be indicated by black and red marks (the top and the bottom mark being of the same color);
  • BSA bovine serum albumin
  • test strip containing a means for quantitating sperm e.g. protein reagent
  • the ejaculate was collected in the collection tube (tube #1) containing 5mg/mL of chymotrypsin and 30mg/mL of dextran in an isotonic solution. The mixture was allowed to stand at room temperature for about 5 minutes.
  • an appropriate volume of the isotonic solution (container #3) was used to overlay the semen/dextran/chymotrypsin mixture to the upper mark of each see-through container, resulting in the formation of a two layer density gradient: the lower layer consisting of the semen/dextran/chymotrypsin mixture and the upper layer consisting of the isotonic solution.
  • the dropper was disposed of.
  • the mixture was allowed to stand for at least 5 minutes at room temperature or optionally in a 37°C heating block.
  • a sterile capillary pipette was inserted to reach the middle mark of the container and applied to a test strip which contained a means for quantitating sperm, e.g., protein reagent, lipid reagent.
  • Example 2 Temperature Stress for Predicting Pregnancy Potential of a Sperm Sample
  • sperm samples were washed using the standard Percoll procedure and resuspended in HTF medium containing 10% plasmanate. An aliquot of the sperm suspension was utilized for artificial reproductive technologies (ART, e.g. IVF and GIFT) and a lOO ⁇ L aliquot of the same sperm suspension was incubated at 40°C for 4h (stress test). Stress test scores were expressed as the ratio of final to initial motility. Analysis was performed by stepwise multiple regression. The independent variables were: women's age; HS motility before and after the stress test; stress test score; and the number of embryos transferred per IVF cycle. The dependent variables were: pregnancy and fertilization outcome.
  • Example 4 A similar test as described in Example 4 was carried out, except that the stress was provided by adding lO ⁇ L of 0.125mM ferrous iron in PBS (phosphate based saline) and 0.6mM ascorbate also in PBS to 30 ⁇ L sperm suspensions for a total volume of 50 ⁇ L and incubating the mixture for 0.5h at 37°C with gentle shaking. Results indicate that the values obtained correlate with the stress test score at 40°C for 4h.
  • SOD activity in the sperm samples examined ranged from 1.5 to 12 U/10° cells (P ⁇ 0.001). All six sperm samples with SOD activities less than or equal to 3.3U/10 8 cells resulted in failed fertilization (P ⁇ 0.001). Between 4.7 and 7.1U/10 8 cells, fifteen out of sixteen oocytes were fertilized but no ongoing pregnancies resulted.
  • the ejaculate was collected in the collection tube (tube #1) containing 5mg/mL of chymotrypsin. After about three to five minutes (at which time the semen should be liquefied), an aliquot (about 50 ⁇ L) of the liquefied semen was added to another test tube (tube #2), which contained horseradish peroxidase-conjugated sheep anti-human superoxide dismutase (SOD) IgG polyclonal antibodies (The Binding Site, Inc., 5889 Oberlin Drive, San Diego, CA 92121) and horseradish peroxidase-conjugated sheep anti-human glutathione peroxidase (GPx) IgG polyclonal antibodies (The Binding Site, Inc., 5889 Oberlin Drive, San Diego, CA 92121) dissolved in a hypotonic solution.
  • SOD horseradish peroxidase-conjugated sheep anti-human superoxide dismutase
  • GPx horse
  • test strip After five minutes, a dip test strip with bound sheep anti-human SOD and anti-human GPx Ig G polyclonal antibodies (The Binding Site, Inc., 5889 Oberlin Drive, San Diego, CA 92121) was inserted into test tube#2. After five minutes, the test strip was removed from the tube and rinsed with tap water.
  • test strip was then dipped into another test tube (tube #3), which contained the peroxidase substrate, hydrogen peroxide (H2O2) and diaminobenzidine (DAB); (Sigma Chemical Co., St. Louis, MO 63178).
  • tube #3 which contained the peroxidase substrate, hydrogen peroxide (H2O2) and diaminobenzidine (DAB); (Sigma Chemical Co., St. Louis, MO 63178).
  • the color ratio of SOD/GPx provides an indication of pregnancy potential by comparison with a chart provided with the kit. Color in the GPx pad indicates equal or greater than about 20 million spermatozoa/mL. If the color in the SOD pad is higher than the color in the GPx pad, a high pregnancy potential of the sperm is indicated. If the color in the SOD pad is equal to or lower than the color in the GPx pad, a low pregnancy potential is indicated.
  • a male contraceptive that suppresses spermatogenesis e.g. a GnRH antagonist
  • a vasectomy was collected in the collection tube (tube #1) containing 5mg/mL of chymotrypsin in an isotonic solution.
  • test tube #2 was transferred to a filter membrane, so that sperm/colored particle/antibody complex was retained and unbound, colored particle/antibody passed through the filter into a reservoir.
  • the color of the filter can then compared to various color possibilities depicted on a chart that is included with the kit to determine the number of sperm present in the sample and thereby determine the efficacy of the male contraceptive treatment.
  • This approach allows one to detect in semen a concentration of at least 50,000 spermatozoa/mL. This sensitivity is comparable to that obtained with the regular light microscope commonly used in andrology laboratories.
  • Example 7 Assay for Monitoring the Efficacy of Male Contraception (Plus (+) or Minus (-) Approach)
  • the ejaculate was collected in the collection tube (tube #1) containing 5mg/mL of chymotrypsin in an isotonic solution. After about 3 to 5 minutes (at which time the semen should be liquefied) a drop (50 ⁇ l) (Male Fertility Kit) or 0.5 mL (Male Contraceptive Kit) of the liquefied semen was added to another test tube (tube #2) containing a hypotonic solution.
  • window #1 was used to apply the sample where after the sample was applied, the soluble sperm antigens reacted with anti-human sperm antibodies conjugated to gold or colored latex particles included in a reservoir in the membrane; window #2 had a horizontal colored line.
  • a second anti-human sperm antibody bound in the vertical position to the membrane underlying window #2 reacted with the sperm antigen/antibody/particle complex producing a colored vertical line when the sperm concentration in semen was above 50,000 spermatozoa/mL in which case a plus (+) sign appeared. If the concentration of sperm in semen was below 50,000 spermatozoa/mL, a minus (-) sign appeared.
  • test tube containing the isotonic or hypotonic solution 3) (1) test tube containing the isotonic or hypotonic solution; 4) (2) droppers;
  • chymotrypsin solution (dextran-free) was added to the semen collection tube in a volume equal to the semen volume. The mixture was allowed to stand at room temperature for about 5 minutes in order for the semen to liquefy.
  • test tube #2 containing an isotonic solution (e.g. phosphate buffered saline or Earle's Medium) or a hypotonic solution (e.g. distilled water) .
  • an isotonic solution e.g. phosphate buffered saline or Earle's Medium
  • hypotonic solution e.g. distilled water
  • test tube #2 One drop (50 ⁇ L) (Male Fertility Kit) or 0.5 mL (Male Contraceptive Kit) of test tube #2 was added to a filter (e.g. blocked nitrocellulose, 5 ⁇ m pore size or microfiber glass filter, 2.7 ⁇ m pore size) mounted on a liquid reservoir.
  • a filter e.g. blocked nitrocellulose, 5 ⁇ m pore size or microfiber glass filter, 2.7 ⁇ m pore size
  • Seminal plasma proteins which were about 10-20 nm in size passed through the filter, while sperm cells which were about 1000 times bigger (50 ⁇ m in size) were retained.
  • anti-sperm antibody-coated colored particles e.g. gold particles that have an intrinsic pink color; or a colored latex particle
  • the filter procedure the antibody used to coat the latex particle did not have to be highly specific for the sperm cell (i.e. it could exhibit some cross-reactivity with seminal plasma proteins).
  • Example 9 Male Fertility & Contraceptive Screening Kits (Filter Approach with Preincubation of the Antibody and the Sperm Antigens)
  • test tube containing the isotonic or hypotonic solution 3) (1) test tube containing the isotonic or hypotonic solution; 4) (2) droppers;
  • chymotrypsin solution (dextran-free) was added to the semen collection tube in a volume equal to the semen volume. The mixture was allowed to stand at room temperature for about 5 minutes in order for the semen to liquefy.
  • one drop (about 50 ⁇ L) (Male Fertility Kit) or 0.5 mL (Male Contraceptive Kit) of the semen/chymotrypsin mixture in the semen collection tube was added to another test tube (test tube #2) containing anti-sperm antibody-coated colored particles (e.g. gold particles that have an intrinsic pink color; or a colored latex particle) in an isotonic solution (e.g. phosphate buffered saline or Earle's Medium) or hypotonic solution (e.g. distilled water) .
  • isotonic solution e.g. phosphate buffered saline or Earle's Medium
  • hypotonic solution e.g. distilled water
  • test tube containing an isotonic or hypotonic solution and sperm-specific antibody- coated particles (uncolored or colored); 4) (2) droppers;
  • chymotrypsin solution (dextran-free) was added to the semen collection tube (tube #1 ) in a volume equal to the semen volume. The mixture was allowed to stand at room temperature for about 5 minutes in order for the semen to liquefy.
  • Contraceptive Kit of the semen/chymotrypsin mixture was added to another test tube (test tube #2) containing an isotonic solution (e.g. phosphate buffered saline or Earle's Medium) or hypotonic solution (e.g. distilled water) and sperm-specific antibody-coated particles (uncolored or colored).
  • an isotonic solution e.g. phosphate buffered saline or Earle's Medium
  • hypotonic solution e.g. distilled water
  • sperm-specific antibody-coated particles uncolored or colored
  • test tube #2 was deposited on a slide, which was of a color that contrasted with the sperm specific antibody coated latex particles, so that agglutination, indicating the potential presence of fertile sperm could be discerned on the slide.
  • the absence of agglutination when using the Male Fertility approach was indicative of less than about 20 million spermatozoa/mL; borderline agglutination was indicative of greater than about 20 million spermatozoa/mL and less than about 40 million spermatozoa/mL; and the presence of high agglutination was indicative of greater than about 40 million spermatozoa/mL.
  • the absence of agglutination when using the Male Contraceptive approach was indicative of less than about 50,000 spermatozoa/mL.
  • Example 11 Male Fertility & Contraceptive Screening Kits (Non Immunoreactive/Filter Approach)
  • test tube containing the isotonic or hypotonic solution 3) (1) test tube containing the isotonic or hypotonic solution; 4) (2) droppers;
  • chymotrypsin solution (dextran-free) was added to the semen collection tube (tube #1 ) in a volume equal to the semen volume. The mixture was allowed to stand at room temperature for about 5 minutes in order for the semen to liquefy.
  • one drop (50 ⁇ L) (Male Fertility Kit) or 0.5 mL (Male Contraceptive Kit) of the liquefied semen was added to another test tube (tube #2) containing gold particles (that have an intrinsic pink color) in a hypotonic or isotonic solution.
  • One drop (Male Fertility Kit) or 0.5 mL (Male Contraceptive Kit) of the contents of tube #2 was added onto the filter (nitrocellulose with 5 ⁇ m pore size or microfiber glass filter with a pore size of 2.7 ⁇ m).
  • Gold particles have an intrinsic pink color and high affinity for biomolecules (e.g. proteins or lipids) and therefore can bind to both spermatozoa and seminal plasma proteins.
  • the ejaculate was collected in the collection tube (tube #1) containing 5mg/mL of chymotrypsin in an isotonic solution. After about 3 to 5 minutes (at which time the semen should have been liquefied) a drop (about 50 ⁇ L) of the liquefied semen was added to a test tube (tube #2) containing anti-glutathione peroxidase (GPx) antibodies bound to yellow colored particles and anti-superoxide dismutase (SOD) antibodies bound to blue colored particles in a hypotonic or isotonic solution. The mixture was incubated for up to about 10 minutes to allow the sperm antigens exposed after the hypotonic treatment to react with the antibodies.
  • GPx anti-glutathione peroxidase
  • SOD anti-superoxide dismutase
  • test tube #2 was transferred to a filter membrane, so that sperm/colored particle/antibody complex was retained and unbound, colored particle/antibody passed through the filter into a reservoir. If the filter appeared green, the SOD/GPx ratio was about 1 : 1 and the sample had borderline pregnancy potential; if the filter appeared bluish, high SOD content was indicated and the sample had high pregnancy potential; and if the filter appeared yellowish, low SOD content was indicated and the sample had low pregnancy potential.
  • squeeze bottle # 1 (1) squeeze bottle containing the liquefying enzyme (e.g. chymotrypsin or pronase at 5 mg/ml . ) (squeeze bottle # 1 ) ;
  • the liquefying enzyme e.g. chymotrypsin or pronase at 5 mg/ml .
  • squeeze bottle #1 two drops (per milliliter of semen) of a solution of pronase solution (5mg/mL) (Sigma Chemical Corp.. St. Louis, MO) in Earle's medium was added to the ejaculate in the collection tube. The mixture was allowed to stand at room temperature for about 5 minutes in order for the semen to liquefy.
  • pronase solution 5mg/mL
  • one drop (Male Fertility Kit) or four drops (Male Contraceptive Kit) of the liquefied semen was added to another squeeze bottle (squeeze bottle #2) which had the top removed and contained 150 ⁇ L (Male Fertility Kit) or 250 ⁇ L (Male Contraceptive Kit) of a hypotonic solution. Then, one drop (Male Fertility Kit) or 4 drops (Male Contraceptive Kit) of the Rhodamine- 123 (Molecular Probes, Inc. Eugene, OR) solution in squeeze bottle #3 was added to squeeze bottle #2 and the top placed back on. The contents were then mixed by tapping with a finger.

Abstract

Tests, dispositifs et trousses servant à identifier les échantillons de sperme à fort potentiel de fécondation (par exemple dans les techniques de reproduction assistée) et les échantillons de sperme à faible potentiel de fécondation (par exemple pour identifier les mâles potentiellement infertiles ou pour évaluer l'efficacité des moyens de contraception masculins).
PCT/US1995/014083 1994-10-31 1995-10-31 Tests, dispositifs et trousses servant a determiner la fertilite masculine WO1996013225A2 (fr)

Priority Applications (3)

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JP8514826A JPH11514204A (ja) 1994-10-31 1995-10-31 男性の授精能を判定するための検定、装置及び器具
EP95939003A EP0789538A1 (fr) 1994-10-31 1995-10-31 Tests, dispositifs et trousses servant a determiner la fertilite masculine
AU40182/95A AU4018295A (en) 1994-10-31 1995-10-31 Assays, devices and kits for determining male fertility

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US33282694A 1994-10-31 1994-10-31
US08/332,825 1994-10-31
US08/332,825 US5895749A (en) 1994-10-31 1994-10-31 Male fertility and contraception home test kits
US08/332,826 1994-10-31

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US5935800A (en) * 1994-10-31 1999-08-10 Beth Israel Deaconess Medical Center Assays and kits for determining male fertility
US5866354A (en) * 1996-12-16 1999-02-02 The State Of Oregon Acting By And Through The State Of Board Of Higher Education On Behalf Of Oregon State University Method for measuring mobility of sperm
WO1999066331A1 (fr) * 1998-06-17 1999-12-23 Genosis, Inc. Technique et trousse permettant de detecter l'infertilite masculine
GB9817795D0 (en) * 1998-08-14 1998-10-14 Genosis Inc A method and device for separation and detection of spermatozoa in a sample
PT1159617E (pt) * 1999-03-09 2005-08-31 Leopold Flohe Metodo para detectar problemas de infertilidade masculina
JP2002537853A (ja) * 1999-03-09 2002-11-12 フローエ レオポルド ＰＨＧＰｘ活性測定に基づく男性用避妊薬の探索方法
DE102007045935B4 (de) 2007-09-25 2015-02-12 Philipps-Universität Marburg Fertilitätstest
FI20096013A (fi) 2009-10-02 2011-04-03 Finnzymes Oy Menetelmä reaktioseoksen valmistelemiseksi ja tähän liittyvät tuotteet
US12065698B2 (en) 2009-10-02 2024-08-20 Thermo Fisher Scientific Baltics Uab Sample processing apparatus and method
EP2388588B1 (fr) * 2010-05-18 2013-10-30 Justus-Liebig-Universität Gießen Procédé et test rapide destinés à la détermination de la fertilité de spermatozoïdes
CN107205808B (zh) 2014-11-25 2020-01-17 艾图生物科学股份有限公司 从生物样品的氧化还原电位测定生育力潜力
KR101879496B1 (ko) * 2016-07-13 2018-07-17 경북대학교 산학협력단 Acrbp 항체를 유효성분으로 포함하는 포유동물의 정소세포 동정용 조성물
CN107290547A (zh) * 2017-08-07 2017-10-24 广州市微米生物科技有限公司 一种不孕不育快速检测试剂盒及其制备方法

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US4009260A (en) * 1973-04-19 1977-02-22 Schering Aktiengesellschaft Fractionation of sperm
US4007087A (en) * 1975-10-17 1977-02-08 Gametrics Limited Sperm fractionation and storage
FR2539628B1 (fr) * 1983-01-25 1988-11-10 Fondation Rech Hormonologie Procede de separation de cellules mobiles, notamment de spermatozoides mobiles, ainsi que dispositif pour la mise en oeuvre de ce procede
SE449003B (sv) * 1985-10-18 1987-03-30 Pharmacia Ab Sett att in vitro avskilja spermier med egen rorelseformaga medelst penetrationsmedium i vilket ingar hyaluronsyrasalt
FR2709250B1 (fr) * 1993-08-23 1995-10-13 Ccd Laboratoire Dispositif pour sélectionner des spermatozoïdes.

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