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(12) Patent Application Publication (10) Pub. No.: US 2006/01992.04 A1 Dix Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2006/01992.04 A1 Dix Et Al US 2006O1992.04A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/01992.04 A1 Dix et al. (43) Pub. Date: Sep. 7, 2006 (54) GENETIC TESTING FOR MALE FACTOR (60) Provisional application No. 60/327.525, filed on Oct. INFERTILITY 5, 2001. (75) Inventors: David Jacob Dix, Raleigh, NC (US); Publication Classification Stephen A. Krawetz, Detroit, MI (US); David Miller, Belmont Grove (GB) (51) Int. Cl. CI2O I/68 (2006.01) Correspondence Address: (52) U.S. Cl. .................................................................. 435/6 BROWDY AND NEIMARK, P.L.L.C. 624 NINTH STREET, NW SUTE 3OO WASHINGTON, DC 20001-5303 (US) (57) ABSTRACT (73) Assignees: G EPA, Washington, DC (US); Wayne RNA in sperm can be used as a diagnostic to distinguish ate University, Detroit, MI (US); Uni- - versity of Leeds, Belmont Grove (GB) betweendiagnostic normal transcripts and affectedis provided individuals. which is A compared list of specific with (21) Appl. No.: 11/357,423 transcripts obtained from the sperm of a subject. Correlation between the two transcripts is used to identify normal sperm (22) Filed: Feb. 21, 2006 or affected sperm. Addition, genetic testing for male infer tility or damage to spermatozoa is accomplished by provid Related U.S. Application Data ing a microarray of DNA probes with a sample of sperma tozoa to determine the mRNA fingerprints of the sample, and (63) Continuation-in-part of application No. 10/264,321, comparing the mRNA fingerprints of the sample with the filed on Oct. 4, 2002, now abandoned. mRNA fingerprints of normal fertile male spermatozoa. Patent Application Publication Sep. 7, 2006 Sheet 1 of 11 US 2006/01992.04 A1 200 204 203t 203 Patent Application Publication Sep. 7, 2006 Sheet 2 of 11 US 2006/01992.04 A1 910'zz/18z'e?ood(d) 'LIdL 9L0'zz/ZGL’,si?saL.(Í) Mae****: Patent Application Publication Sep. 7, 2006 Sheet 3 of 11 US 2006/0199204 A1 A / G. 3 CELLULAR COMPONENT BIOCHEMICAL FUNCTION A. % E integral possiveprotein membrone I DNA-binding protein ID Nucleus Transferase Cytoplasm Inhibitor or Repressor Membrane fraction :::: Receptor (signaling) Plasma membrane RNA-binding protein integral membrane protein Activator Extracellular space Oxidoreductose Soluble fraction Protease (other than proteosomol) Mitochondrion Regulatory subunit Endoplastic reticulum E. Active transporter, secondary ES Cytosol Ribosomol SubUnit Action Cytoskeleton Lysine Peripheralprotein plasmo membrone Cho?nel (possive transprotein) Extracellular & 1somerase E Protein conjugation factor Adhesin/agglutinin O Cytoskeleton GFP binding protein/GFP/ese Nucleolus Cho per ones Extrocellular ?notrix Other S Other Patent Application Publication Sep. 7, 2006 Sheet 4 of 11 US 2006/01992.04 A1 Patent Application Publication Sep. 7, 2006 Sheet 6 of 11 US 2006/01992.04 A1 ·UHO diuojoºldu. ex= Patent Application Publication Sep. 7, 2006 Sheet 7 of 11 US 2006/01992.04 A1 s Patent Application Publication Sep. 7, 2006 Sheet 8 of 11 US 2006/01992.04 A1 Affected (8293Normal transcripts) (7227 transcripts) Fig. 8. The partitioning of transcripts present in the normal individuals data set (left), both datasets (middle) and affected individuals data set (right) above the threshold value. Patent Application Publication Sep. 7, 2006 Sheet 9 of 11 US 2006/01992.04 A1 g Ngue - Affected Normal 'Fig. 9. Analysis of RNA transcripts present in sperm from thirteen normal and eight affected samples. The heat map shows that the levels of transcripts present in normal and affected individuals that can be distinguished. Six strikingly different regions from the Affymetrix analysis are shown that can be used to classify and diagnose individuals. Patent Application Publication Sep. 7, 2006 Sheet 10 of 11 US 2006/01992.04 A1 Fig. 10. Clustering of sperm RNA samples based on Illumina BeadChip array transcript data. Sperm transcripts from normal individuals are clearly distinguished from affected individuals. Patent Application Publication Sep. 7, 2006 Sheet 11 of 11 US 2006/0199204 A1 Fig.11. Clustering of the 661 transcripts consistently detected across all three array platforms, using data from Affymetrix arrays. Left panel, an example of Cluster 3.0 and TreeView consistently clustering normal individuals separate from affected individuals. Right panel, a segment of the corresponding 661 transcript heat map generated visually demonstrating the diagnostic capacity of the arrays. US 2006/01992.04 A1 Sep. 7, 2006 GENETIC TESTING FOR MALE FACTOR 0007 With the exception of obvious defects such as INFERTILITY aZoospermia, globoZoospermia, and immotile ciliary Syn drome, the extreme heterogeneity of normal fertile human CROSS-REFERENCE TO RELATED semen suggests that most idiopathic male factor infertility is APPLICATIONS not a result of monogenic disorders. Moreover, all known 0001. The present invention is a continuation-in-part of monogenic disorders that affect the testes affect other tissues application Ser. No. 10/264,321, filed Oct. 5, 2002, which to an equal or greater extent. Accordingly, it is reasonable to application claims priority from provisional application Ser. assume that the majority of idiopathic male factor infertility No. 60/327,525, filed Oct. 5, 2001, the entire contents of that has testes-restricted phenotypes is not monogenic, but both of which are hereby incorporated by reference. oligo- or poly-genic in origin. 0008 Two recent developments offer considerable prom FIELD OF THE INVENTION ise towards identifying oligo- and/or poly-genic factors that 0002 The present invention relates to methods, kits, and influence male fertility. First, the discovery of mRNAs in tools for distinguishing between sperm of normal and ejaculate spermatozoa makes it possible to obtain transcrip affected individuals. tional information from male germ cells using non-invasive procedures. It is expected that these mRNAs provide a BACKGROUND OF THE INVENTION window to past events of spermatogenesis, echoing tests for gene expression. Interestingly, data mining and functional 0003 Predicting the fertility of a male is very useful in a assessment suggests that in addition to delivering the hap variety of contexts. For example, the artificial insemination loid male genome, spermatozoa also deliver a critical industry is interested in knowing the likelihood that fertili complement of mRNAs to the oocyte. Secondly, microas zation will occur if a female is artificially inseminated with says make it possible to construct detailed gene expression a particular male's semen. Alternatively, human fertility profiles. clinics are concerned with achieving impregnation, and evaluating the sperm count of a male is one step in this 0009 Most laboratory investigations of semen quality are procedure. Thus, whether in the context of animal breeding, relatively poor indicators of fertility because they are sub the artificial insemination industry, or human fertility clinics, jective and predominantly rely on physiological and mor determination of the fertility of the male is very important. phological criteria. This is exemplified in the recent study that compared the predictive results from over 250 indepen 0004 Ten percent of the male population have abnor dent studies (Gynecol Obstet Invest. 2005:59(2) 86-91). mally low sperm counts, and approximately one in six Only 4 could be could be directly compared as the variation couples experiences difficulty in conceiving a child. Male among the others was too great. The consequences of using factor infertility accounts for 40-50% of the cases in which immature spermatids or other compromised germ cells in assisted reproductive techniques are recommended. The intra-cytoplasmic spermatozoa induction (CISI) procedures great majority (>98%) of infertile men actually produce need careful reappraisal, considering that spermatozoal sperm, but, for some reason, those sperm are often unable to mRNA is required for the production of normal offspring. fertilize an egg. Chromosomal anomalies are associated with approximately one third of non-obstructive male factor 0010 Thus, there is a need for an effective, efficient and infertility affecting some 2% of the infertile male population. accurate method and/or device for determining male fertil Half of these men (15%) present with abnormal karyotypes, ity. More specifically, there is a need for a method for which the other half (12-15%) present with microdeletions determining if a male is fertile using microarrays in analyZ in the Azoospermic Factor (AZF) region of Yq (i.e., DAZY/ ing mRNAS of spermatozoa. RBM). SUMMARY OF THE INVENTION 0005 Couples having difficulty starting a family must 0011. It is an object of the present invention to overcome undergo an extensive battery of tests, including a testicular the aforementioned difficulties of the prior art. biopsy. However, it has not yet been possible to identify which couples will never conceive, so that these couples can 0012. It is another object of the present invention to forgo the lengthy, expensive, and ultimately futile infertility provide a method for detecting if a male is a normal fertile therapy and begin considering other options, such as sperm male using microarrays in analyzing the mRNA of sperma donors. tOZOa. 0006 Testes-specific defects have only been demon 0013. It is a further object of the present invention to strated in men with sub-microscopic microdeletions of the Y provide a kit for detecting normal fertile males using chromosome encompassing one or more genes. It is reason microarrays in analyzing the mRNA of
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