Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-10
2003-05-06
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S002000, C435S007200, C435S007210, C435S007920, C435S007940, C435S287200, C435S325000, C435S375000, C435S967000, C436S063000, C436S065000, C436S172000, C436S548000, C436S808000, C436S814000, C536S024310
Reexamination Certificate
active
06558911
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to male infertility, and in particular to assays for determining fertility.
BACKGROUND OF THE INVENTION
Infertility is diagnosed as the failure to become pregnant after one year of regular, unprotected intercourse. About ten percent of couples are infertile. Male factor infertility is the sole or contributing cause in about forty percent of these cases. In 1995, approximately 60,000 cycles of ART (Advanced Reproductive Technology) were performed in the United States to treat infertility. Of these procedures, approximately 90% involved in vitro fertilization at an average cost of $7,800.00 per cycle.
Semen analysis forms the basis of the initial evaluation for assessing male-factor. infertility. In general, two to three semen analyses are performed because semen quality normally fluctuates for a given individual. Subjects are normally encouraged to refrain from intercourse for 2 to 3 days prior to evaluation. Abstinence for a shorter time can decrease ejaculate volume, while prolonged abstinence can impair sperm motility. Traditional semen analysis evaluates a number of parameters, including, ejaculate volume, sperm count, sperm motility, forward progression, sperm morphology, pH, agglutination, leukospermia, and viscosity.
Sperm morphology is recognized as an important factor in semen analysis because it is a reflection of spermatogenic development. Traditionally, sperm have been classified according to the following morphologies: oval, amorphous, tapered, duplicated, and inrnature. However, determination of the percentage of normal spermatozoa with good morphology is highly subjective, and it is difficult to identify critical sperm morphological features that are responsible for fertility potential (Szczygiel and Kurpisz, Andrologia 31:63-75 [1999]). Additionally, in some instances, infertile men have sperm with apparently normal morphology. Furthermore, the results can be biased by damage incurred during normal preparation of the sperm for analysis (e.g., pipetting, centrifugation, and washing).
It is recognized that the identification of sperm abnormalities not apparent from semen analysis may lead to more appropriate and informed treatment plans for infertility (Szczygiel and Kurpisz, supra). However, there have been few recent efforts to develop methods that provide reliable prediction of fertility or fecundity based on sperm characteristics (Amann, J. Androl. 10(2):89-98 [1989]). Accordingly, what is needed in the art are objective semen quality assays that correlate to male factor infertility in the absence of morphological data suggesting otherwise, and that are unaffected by handling of the sample. It is desirable that such assays should be indicative of fertility.
SUMMARY OF THE INVENTION
The present invention relates to male infertility, and in particular to assays for determining fertility. In some embodiments of the present invention, methods for predicting fertility are provided. Accordingly, in some embodiments of the present invention, a method is provided for assaying fertility in an animal comprising a) providing a semen sample containing sperm; and b) measuring the amount of ubiquitin in the sample, wherein the amount of ubiquitin is indicative of fertility.
The present invention is not limited to a semen sample from a particular source. Indeed, it is contemplated that a variety of semen samples may be assayed. In some embodiments, the semen sample is obtained from a variety of animals, including, but not limited to, humans, cattle, sheep, pigs, horses, buffalo, bison and other domesticated and non-domesticated animals. In other embodiments, the sample is obtained by ejaculation, electroejaculation, or from the epididymis.
In other embodiments, the method of the present invention further comprises the steps of c) providing an antibody that binds to ubiquitin; and d) combining the semen sample with the antibody under conditions wherein the antibody binds to ubiquitihated sperm.
The present invention is not limited to any particular ubiquitin antibody. In some embodiments, the ubiquitin antibody is a polyclonal antibody, while in other embodiments, the ubiquitin antibody is a monoclonal antibody. In some particularly preferred embodiments, the antibody is selected from MAB 1510, AB 1690, Ubi-1, MK-11-3, MK-12-3, UCBA798/R5H, KM691, UG 9510, and U-5504.
The present invention is not limited to any particular technique for measuring ubiquitin or the degree of ubiquitination of sperm in the semen sample. Indeed, a variety of methods of determining the degree of ubiquitination are contemplated. In some embodiments, ubiquitination is assayed by immunocytochemical techniques wherein sperm that bind a labelled ubiquitin antibody are quantified. The present invention is also not limited to any particular method of quantitation. In some embodiments, the number of sperm within a given sample that are ubiquitinated is determined microscopically by counting the number of labelled sperm in at least one subsample of the semen sample. In other embodiments, the number of labelled sperm in a given sample is determined using a videoanalysis system in conjunction with fluorescence microscopy. In other embodiments, ubiquitination is assayed by immunocytochemical techniques, wherein sperm are sequentially exposed to a ubiquitin antibody and a labelled second antibody that binds to the ubiquitin antibody. In other embodiments of the present invention, flow cytometry is used to measure ubiquitin in a semen sample. In still further embodiments, the amount of ubiquitin in a semen sample is measured by enzyme-linked immunosorbant assay (ELISA).
The present invention is not limited to any particular labelled first or second antibodies. Indeed a variety of second antibodies are contemplated, including, but not limited to those labelled with fluorescent compounds (e.g., fluorescein, rhodamine), enzymatic markers (e.g., alkaline phosphatase, horseradish peroxidase), and colloidal gold.
In other embodiments, the present invention provides methods for assaying fertility in an animal. In some embodiments, the method comprises a) providing i) a test semen sample containing sperm; and ii) an antibody that binds to ubiquitin; b) combining the semen sample with the antibody under conditions wherein the antibody binds to ubiquitinated sperm; c) measuring the amount of ubiquitin in the sample; and, d) comparing the measured amount of ubiquitin in the sample with an amount of ubiquitin in a control sample from a donor of known fertility, wherein a greater amount of ubiquitination in the test semen sample as compared to the control sample is indicative of infertility.
In still other embodiments, the present invention provides kits for assaying sperm quality. In some embodiments, the kit comprises a) a first container containing an antibody that binds to ubiquitin; and b) a second container containing a control semen sample from a donor of known fertility. In further embodiments, the kit comprises an antibody that binds to the ubiquitin binding antibody. In still further embodiments, the kit comprises a labelled second antibody that binds to the antibody that binds ubiquitin. In other embodiments, the kit further comprises instructions for assaying fertility or sperm quality in an animal.
DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and phrases are defined below:
As used herein, the term “ubiquitin” refers to a relatively small protein (approximately 76 amino acid residues) found in all cells of higher organisms (See e.g., Ciechanover, Cell 79: 3-21 [1994], incorporated herein by reference) and other ubiquitin-like proteins sharing homology with ubiquitin. In preferred embodiments, ubiquitin and ubiquitin-like molecules will be recognized by the following antibodies: MAB 1510, AB 1690, Ubi-1, MK-11-3, MK-12-3, UCBA798/R5H, KM691, UG 9510, U-5504, P4D1 (Covance, Richmond, Calif.), 221M (Biomedia, Foster City, Calif.); 1 471 732 (Boehringer Mannheim, Indianapolis, Ind.), IB3 (Calb
Gabel Gailene R.
Le Long V.
Medlen & Carroll LLP
Oregon Health & Sciences University
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