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Sdad assay and uses thereof

Sdad assay and uses thereof description/claims

This application is a non-provisional of, claims priority to and the benefit of U.S. Provisional Patent Application Ser. No. 60/957,117, filed on Aug. 21, 2007, the entire contents of which are incorporated herein by reference.

The process of fertilization involves a series of events including: a) sperm binding to, and then penetration of the oocyte zona pellucida via the acrosome reaction, b) sperm fusion with the oocyte plasma membrane, c) oocyte activation (release of the cortical granules to prevent multiple sperm from fertilizing the egg), d) sperm activation and pronucleous formation (Abou-Haila A. et al. 2000: Furlong, L. et al. 2005).

Sperm activation initiates post-fertilization upon entry into the ooplasm where the compacted sperm DNA undergoes many alterations as it develops into a male pronucleus. During the first phase of sperm activation or decondensation, the chromatin condensation acquired during spermiogenesis is reversed. Protamines are exchanged with histones reformatting the sperm chromatin allowing for pronucleus formation (Ballhom et al., Wasserman et al.). Once the sperm decondensation is complete, the DNA synthesis phase occurs, that is followed by recondensation of the sperm chromatin in preparation for the cell division that will result in the 2-cell stage embryo (Longo et. al., (1991)).

A need exists in the medical arts for a clinical sperm screening for a protocol that provides assessment of sperm activation events that are not screened. These sperm activation events include: sperm DNA accelerated decondensation (SDAD) and/or sperm earlyDNA synthesis (SEDS). A need continues to exist in the clinical arts for a panel of tests of sperm activation events useful in prescribing the assisted reproductive technology (ART) having the highest probablility for success for a particular patient and/or reproductively challenged couple, and that has the least emotional and financial impact. A need also exists for a test that can identify males determined to have a varicocele(s) who can benefit from a varicocelectomy. Finally, a need exists for protocol(s) that provide a more rapid processing and assessment of sperm analyzed in sperm activation tests that include sperm DNA decondensation.

In a general sense, a rapid sperm screening assay that is predictive for human sperm reproductive and/or fertilizing capacity is provided in the present disclosure. In some embodiments the assay provides for a highly predictive method for identifying males who produce sperm with low fertilizing capacity, or in the cases where sperm results in a pregnancy, a high probability that the pregnancy will not progress beyond the first trimester, i.e. a low probability of a take home baby. In one aspect, the present invention identifies such males when their sperm is analyzed in a 5 minute to 10 minute in vitro assay that demonstrates a quantifiable difference between a patient's sperm, and the sperm from a known fertile male. In yet other embodiments, this in vitro assay is the sperm DNA accelerated decondensation (SDAD) Test. In other embodiments, the in vitro assay is the sperm early DNA synthesis (SEDS) Test. In yet other embodiments, the in vitro assays include the SDAD Test, SDD Test, and SEDS Test.

In another aspect, a sperm screening method is provided that is more highly predictive of a take-home baby likelihood when using an assisted reproductive technique (ART). By way of example, such as assisted reproductive technique is the intracytoplasmic sperm injection (ICSI). The presently disclosed method provides a predictive value that is highly statistically significant (p<0.01).

In some embodiments, a screening method is provided that is useful in identifying male sperm donors who will have a low probability of successful ART attempts at pregnancy (success being defined as live birth), such as when using ICSI. Other sperm screening assays, such as the sperm DNA decondensation (SDD) Test, the sperm penetration assay (SPA), and the sperm chromatin structure assay (SCSA) have all been found to not be predictive of ICSI live birth outcome. Thus, in some aspect, the present disclosure describes a screening method that can predict ICSI outcome. In some embodiments, this screening method comprises obtaining a semen sample from a potential sperm donor that has been determined to provide a normal sperm assay result in a SPA and SDD Test, and assessing the sample for sperm DNA accelerated decondensation (SDAD) in a frog egg extract as described herein. In some embodiments, the sperm DNA accelerated decondensation (SDAD) is a measure of chromatin DNA decondensation evidenced after about a 5 (five) minute incubation interval in a frog egg extract. In other embodiments, a sperm sample with essentially complete sperm chromatin DNA decondesation at the five (5) minute time interval identifies an unsatisfactory sperm donor for an ICSI pregnancy attempt.

In some embodiments, a screening method is provided for identifying a male sperm donor having a low probability of an successful ART attempts at pregnancy (e.g., IUI and IVF). The screening method has relatively low predictive capacity for determining a couple's success, success being defined as live birth, when sperm from the male partner is used in an ICSI attempt at pregnancy. In some embodiments, the screening method comprises obtaining a semen sample from a potential sperm donor that has been determined to have an abnormal SDD Test score a (delay in DNA decondensation when incubated in frog egg extract). In other embodiments, the delay in sperm DNA decondensation is a measure of chromatin DNA decondensation evidenced after about a 15 (fifteen) minute incubation interval in a frog egg extract. In other embodiments, a sperm sample with an abnormal response in the SDD Test identifies an unsatisfactory sperm donor for an IUI and/or IVF pregnancy attempt. However, because the SDD Test has no predictive capacity for determining if a patient will succeed in an ICSI attempt at pregnancy, such patients can go directly to ICSI attempts at pregnancy.

It is also an object of the invention to provide an automated sperm screening assay having predictive value for assuring pregnancy outcome and/or a successful pregnancy attempt, where natural, or ART attempts at pregnancy have failed, and that this automated approach provides the same predictive value determined by the presently used manual approach that is not amiable to processing and the scoring of large numbers of samples. In other embodiments, the particular sperm screening assay is the SDAD or SDD Tests, or both. In other embodiments, the automated assay is a high throughput sperm screening method that employs a 96-microtiter well plate, each plate comprising a volume of a frog egg extract. Optionally, and in some embodiments, each microtiter well plate may include frog egg extract containing a DNA labeling agent, such as tritiated thymidine triphosphate (when autoradiography is used to analyze DNA synthesis) or 5-Bromo-2′-deoxyuridine (BUdR), when sperm will be stained with a fluorescent tagged anti-BUdR antibody, and the DNA synthesis analyzed using an automated system/image analysis system described herein. Once the extract sperm mixture is incubating, aliquots will be removed at 5 minutes (SDAD Test), and 15 minutes (SDD Test) and the sperm in these aliquots fixed and stained for subsequent analysis as described herein, and scored using an image analysis system. In another embodiment sperm, in an additional aliquot will be analyzed manually using phase contrast microscopy at a 5 minute time point (SDAD Test) and a 15 minute time point (SDD Test), as described herein.

In another embodiment, a method is described for using the SDD Test to identify an unsatisfactory sperm donor whose abnormal score is determined to be related to the patient also having a varicocele(s). When identified, such individuals have been found to benefit from a varicocelectomy.

The following definitions are used throughout the description of the present invention:

As used in the description of the present invention, the term, “hyperdecondensed sperm” is defined as sperm with a 2-fold increase in size over that observed in the fully decondensed sperm. As used in the present description, a “successful” pregnancy is defined as a live birth resulting from a pregnancy achieved using an assisted reproductive technique (ART).

As used in the description of the present invention, the term, “reproductively challenged couple” is defined as a human male and a human female that have been involuntarily infertile for 1 or more years. Approximately forty percent (40%) of these couples are infertile due to male factor(s), forty percent (40%) are infertile due to female factor(s), and 20% are infertile due to combined male and female factors.

As used in the description of the present inventor, the term, “take-home baby” is defined as a live human birth.

The following abbreviations are used throughout the description of the invention:

LPO=lipid peroxidation

SEDS=sperm early DNA synthesis;

HSAA=human sperm activation assay;

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