Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-19
2003-04-01
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S002000, C435S007210, C435S007800, C435S007940, C435S007950, C435S332000, C435S334000, C435S806000, C435S975000, C436S503000, C436S518000, C436S519000, C436S063000, C436S172000, C530S388200, C530S388220, C530S852000
Reexamination Certificate
active
06541206
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a biochemical method for determining the fertilization potential in sperm and, in particular, to a method which utilizes the measurement of particular proteins in spermatozoa which reflect sperm development, maturity, DNA integrity and fertility.
2. Description of Related Art
The testicle serves as site for spermatogenesis, sperm production and sperm maturation. Diagnosis of possible problems in spermatogenesis is normally carried out with the determination of sperm concentrations in the ejaculate. The sperm count fluctuates in all men, but the fluctuations are more detectable in oligospermic (OS) men with sperm counts below the 20×10
6
sperm/ml semen of normal or “normospermic” (NS) level. Low sperm concentrations may be a consequence of pituitary problems, deficient hormone levels, testicular disorders and diminished sperm production, retrograde ejaculation, partial obstruction of the ejaculatory tract, age, environmental factors, fever or excess heat, exposure to organic solvents in the work place, etc.
It has been shown that male factor is a contributory cause of infertility in about 40% to 50% of the couples. The overwhelming majority of these infertile and subfertile men are oligospermic and/or asthenospermic (sperm motility is lower than 50%). Management problems exist with respect to these infertile men, as these men may try to father children for years without success. With intrauterine placement of the sperm the pregnancy rates for couples with male factor infertility are only about 15-20% per cycle. This is in spite of the fact that there is no detectable difference in sperm concentrations and motility among the groups of husbands who do or do not cause pregnancy following intrauterine insemination treatment. The pregnancy rates with in vitro fertilization treatment are higher, but adequate sperm concentration or sperm motility does not assure the occurrence of pregnancies. There are also men who suffer from unexplained male infertility, i.e. sperm with diminished fertility in spite of normal sperm concentrations.
Beyond the classical semen analysis parameters, i.e., sperm concentration, motility and velocity, present approaches to evaluate selected sperm functions include the assessment of motile sperm yield following migration or “swim-up”; measurements of acrosine activity, i.e., the enzyme which facilitates sperm penetration; the hypoosmotic sperm swelling procedure which probes the integrity of the sperm membrane, the sperm-hemizona assay which tests the ability of sperm to bind to segments of human oocytes, the sperm chromatin structure assay; the human sperm zona-free hamster oocyte penetration test, which is more consistent on the negative side (e.g. penetration rates below 15-20% and diminished success in human in vitro fertilization) than it is a measure of fertility. None of these tests, with the exception of the hemi-zona assay, address the overall physiological soundness of the spermatozoa or showed a high correlation with fertilizing potential or occurrence of pregnancies.
It became increasingly apparent that a new approach was necessary for the assessment of sperm fertility/infertility. This new approach was based on measurement of objective sperm biochemical parameters which were shown to have an essential role in the management of male infertility. Such a method for testing sperm quality and fertilizing potential was developed and disclosed in U.S. Pat. No. 4,945,044, “Objective Biochemnical Method for Determining Fertilization Potential in Oligospermic Men” by the inventor of the present application. This method comprised obtaining a sperm sample; detecting the CK enzyme isoforms from the sperm sample, measuring a first CK enzyme concentration for CK-X isoform of the CK enzyme; and determining the sperm quality parameter based upon the first CK enzyme concentration. A second CK enzyme isoform, the CK-B was also measured as a basis for determining the sperm quality parameter. Measurements of the levels of CK-X and CK-B isoforms by electrophoresis and fluorescence visualization technique were used to establish the first and second enzyme concentrations. The sperm quality parameter, as expressed (% CK-X/(CK-X+CK-B)) was proportional to the ratio of the CK-X isoform level to the sum of CK-X and CK-B isoform levels. Sperm fractions which met a predetermined minimum sperm quality level were deemed to be of adequate fertility and selected for use in vivo or in vitro fertilization attempts on oocytes. The validity and predictive value of CK-X/CK-M ratio has been demonstrated in several clinical studies.
Despite the increase in accuracy of the sperm quality parameter described in the '044 patent, the method of testing for the CK-X isoform of the CK enzyme in sperm requires an electrophoretic analysis which is not readily available outside of laboratories. Cost pressures in health care, including reproductive health care, have encouraged the development of tests which do not require relatively expensive laboratory analysis. Sperm quality tests which could be performed in physicians' offices, at relatively lower cost and with more readily available equipment, would represent a great advance in fertility treatment.
Bearing in mind the problems and deficiencies of the prior art, it is therefore an object of the present invention to provide an improved method for testing of sperm quality, which also shows high correlation with fertilizing potential and occurrence of pregnancies.
It is another object of the present invention to provide a method and kit for testing sperm quality which may be readily performed outside of a laboratory environment.
A further object of the invention is to provide a sperm quality test which may be performed at lower cost and in physicians' offices.
Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specification.
SUMMARY OF THE INVENTION
The above and other objects, which will be apparent to those skilled in the art, are achieved in the present invention which provides, in one aspect, a method of testing sperm quality comprising obtaining a sample of sperm to be tested; detecting and measuring amount of testis-specific chaperone protein in the sperm; and determining a sperm quality parameter based upon the amount of chaperone protein, wherein an increased amount of the chaperone protein indicates a higher sperm quality. Preferably, the chaperone protein is detected and measured either by binding one or more antibodies specific to the sperm chaperone protein to the sperm and measuring the antibody content or measuring ATP bound to the sperm chaperone protein. In the case of the latter method, the chaperone protein may be detected and easured by measuring ATP bound to the sperm chaperone protein, and such easuring is by chaperone protein-bound and CK-B generated ATP measurement, or by haperone protein ATP bioluminescence. Preferably, the chaperone protein is detected and measured by a chaperone protein-specific immuno-assay to determine ATP bound to the sperm chaperone protein.
The preferred method further includes detecting and measuring the amount of ATP generated by the CK-B enzyme in the sperm, and comparing the amount of testis-specific chaperone protein detected and measured in the sperm to the amount of ATP bound to CK-B enzyme detected and measured in the sperm. This provides a ratio of sperm containing the testis-specific chaperone protein to sperm CK-B enzyme, which is used to determine the sperm quality parameter. The chaperone protein may be detected and measured by enzymatic determination of the sperm ATP, with and without CK substrate, in order to measure the chaperone protein-bound ATP versus ATP generated by the sperm CK-B, or by chemical measurement of the chaperone protein-bound ATP in sperm.
Any of these detection methods preferably further include measuring the total amount of sperm in the sample and comparing the amount of testis-specific chaperone
DeLio & Peterson LLC
Grun James L.
Huszar Gabor B.
Nguyen Bao-Thuy L.
LandOfFree
Objective biochemical method for assessment of sperm quality does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Objective biochemical method for assessment of sperm quality, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Objective biochemical method for assessment of sperm quality will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3064960