Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
1999-01-15
2001-01-09
Jones, Dwayne C. (Department: 1614)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C514S054000
Reexamination Certificate
active
06171778
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention is generally in the area of improved solutions containing arabinogalactan for washing and separating sperm.
Sperm must be washed prior to use in most diagnostic or research protocols, such as infertility testing, in vitro fertilization and freezing. Washing is done to limit damage to the sperm cell by components of seminal plasma, such as antibodies, white blood cells, red blood cells and bacteria. Washing also removes dying or dead sperm which release enzymes and other products which are toxic to healthy sperm.
Generally, sperm are separated by allowing the motile sperm to swim away from the debris (sperm swim-up), by centrifuging the sperm through a gradient and collecting a pellet of live sperm (washing), or by passing the sperm through a column that binds the dead or unhealthy sperm. Each of these current techniques has its own disadvantages. Swim-up only recovers a small number of the normal sperm. Column methods have poor selectivity for the normal sperm in the ejaculate. Centrifugation through a density gradient results in high sperm recovery but also can produce products which are toxic to sperm, so that an additional wash step is required to remove the products. Centrifugation also can produce sperm with reduced motility and fertility during in vitro fertilization. Parrish et al.,
Theriogenology
, 44:859-869 (1995); and Tanphaichitr et al.,
Gamete Research
, 20:67-81 (1988).
Methods and compositions for washing and separating sperm have been the subject of many studies. Trounson and Gardner,
Handbook of In Vitro Fertilization
, CRC Press, Boca Raton, 1994, pp. 46-50. Bongso et al.,
Fertility and Sterility
, 51:850-854 (1989) discloses a study of the improvement of concentration and motility of sperm following separation in Ficoll, a synthetic polymer of sucrose. U.S. Pat. No. 4,007,087 to Ericsson discloses fractionating sperm in a medium including soluble materials such as proteins, peptides and dextran. U.S. Pat. No. 4,327,177 to Shrimpton discloses the separation of sperm by density in a nutrient media derived from mammalian milk. U.S. Pat. No. 4,605,558 to Shrimpton discloses a method of separating X and Y sperm in a density gradient and an osmolality gradient in a medium derived from milk. Platov et al.,
Ovtsevodstvo
, 10:38-39 (1980) (Abstract) discloses the use of gum arabic, cherry resin and apricot resin in a sheep semen freezing medium.
Arabinogalactan is a water-soluble polysaccliaride which can be isolated from trees of the genus Larix, particularly
Larix occidentalis
Nuttall (western larch). Methods for the preparation of ultrarefined arabinogalactan are disclosed in U.S. Pat. No. 5,116,969. Hill et al.,
J. Lab. Clin. Med
., 111:73-83 (1988) discloses the use of arabinogalactan to obtain washed murine platelets by centrifugation. The preparation of arabinogalactan derivatives and fragments is described in Prescott, et al.,
Carbohydrate Research
, 278:113-128 (1995); and U.S. Pat. No. 5,478,576 to Jung, et al., the disclosures of which are incorporated herein by reference.
There is a need for the development of improved products for washing and separating sperm for protocols such as infertility testing, in vitro fertilization, and freezing.
It is therefore an object of the invention to provide solutions for washing and separating sperm to produce sperm samples with optimal motility and viability, and which can be used in a variety of diagnostic and research applications, such as fertility testing, and in vitro fertilization.
SUMMARY OF THE INVENTION
Compositions including arabinogalactan, and methods for their use in washing and separating sperm are provided. Use of solutions including arabinogalactan produces motile sperm samples which are suitable for use in a variety of diagnostic and research applications. In one embodiment, the wash is provided with an effective amount of arabinogalactan to permit washing or separation of the sperm, while maintaining sperm viability properties during the washing or separation procedure. Using tie improved sperm wash solution, non-sperm substances that are capable of having a detrimental effect on sperm viability, such as seminal plasma, white blood cells, red blood cells, freezing extender agents, sperm debris, and media components, are removed from the sperm sample. The inclusion of arabinogalactan in the wash allows the motile sperm to be separated without damage to these sperm. In a preferred embodiment, the arabinogalactan is ultrarefined.
The improved sperm wash product is compatible with sperm samples obtained from variety of different mammals, including human, bovine and equine sperm samples. The sperm wash including arabinogalactan can be used to separate motile sperm, while optimizing a wide range of viability properties of the sperm, including sperm motility, sperm numbers, percentage of living sperm recovered, sperm membrane function, sperm penetration rate, sperm fertilization rate and/or oocyte development following fertilization. The washed sperm may be used in a variety of diagnostic or research protocols including infertility testing, sperm toxicology testing, and in vitro fertilization.
DETAILED DESCRIPTION OF THE INVENTION
A sperm wash solution including arabinogalactan, which is highly compatible with sperm is provided, as well as methods for using the wash to wash or separate sperm, to produce sperm which can be used in a variety of diagnostic or research applications. The presence of arabinogalactan in the solution has a protective effect on sperm in a sample during processing. In a preferred embodiment, the arabinogalactan is ultrarefined. The concentration of arabinogalactan in the solution can be optimized for a particular sperm to sample. Sperm washed in a solution comprising arabinogalactan have improved properties in comparison to sperm processed in existing sperm wash products, including improved sperm motility, sperm numbers, percenage of living sperm, sperm membrane function, sperm penetration rate, sperm fertilization rate and oocyte development following fertilization.
Arabinogalactan
Arabinogalactan (“AG”) is a water-soluble polysaccharide which can be isolated from trees of the genus Larix, particularly
Larix occidetitalis
Nuttall (western larch). Arabinogalactan may constitute up to 35% of the total heartwood of some species. Stout, “Larch Arabinogalactan” in
Industrial Gums
, R. L. Whistle Ed., Academic Press, New York, pp. 307-310, 1959. It is highly water soluble and can be purified from larch chips.
As used herein, the term “arabinogalactan,” unless otherwise specified, includes naturally occurring or synthetic arabinogalactan, portions of arabinogalactan, such as degradation products, and chemically or 25 biochemically modified arabinogalactan or portions thereof which have been modified using methods available in the art, which are effective in a sperm solution during sperm washing or separation to protect sperm motility.
In one preferred embodiment, ultrarefined arabinogalactan is used in the sperm wash. Methods for the preparation of ultrarefined arabinogalactan are disclosed in U.S. Pat. No. 5,116,969, the disclosure of which is incorporated herein by reference. Ultrarefined arabinogalactan of greater than 95%, or optionally, greater than 99.9% purity (Larex UF™) is available from Larex, International, St. Paul, Minn. As defined herein “ultrarefined arabinogalactan” refers to arabinogalactan, isolated from a plant source such as trees of the genus Larix, with a purity greater than 95%. In a preferred embodiment, the molecular weight of the ultrarefined arabinogalactan is between about 10,000 and 30,000 daltons (by size exclusion chromatography with pullulan reference).
Arabinogalactan provides a useful low cost alternative to the use of other compositions commonly used in sperm washing products, such as Percoll™, colloidal polyvinylpyrrolidone coated silica, available, for example, from Sigma Chemical Company, St. Louis, Mo. The use of Percoll gradients in centrifugation for washing sperm is described, for exampl
Ellington Joanna E.
Oliver Sylvia Adams
Advanced Reproduction Technologies Inc.
Jones Dwayne C.
Seed Intellectual Property Law Group PLLC
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