CA2693546A1 - Detection of human cysteine-rich secretory protein (crisp1) in semen and medical applications related thereto - Google Patents
Detection of human cysteine-rich secretory protein (crisp1) in semen and medical applications related thereto Download PDFInfo
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Abstract
The invention relates to the detection of human cysteine-rich secretory protein (CRISP1) as a marker for the presence or absence of epididymal secretions in an ejaculate.
Described herein are methods for assessing the presence of epididymal secretions in human semen, methods for evaluating successfulness of vasectomy in men, methods for evaluating successfulness of vasovasostomy in men, and methods for discriminating between obstructive and non-obstructive azoospermia in men. Generally, the methods comprise detecting in the semen sample presence or absence of CRISP1. Also described are kits useful for diagnostic applications in humans according to the described evaluation methods.
Described herein are methods for assessing the presence of epididymal secretions in human semen, methods for evaluating successfulness of vasectomy in men, methods for evaluating successfulness of vasovasostomy in men, and methods for discriminating between obstructive and non-obstructive azoospermia in men. Generally, the methods comprise detecting in the semen sample presence or absence of CRISP1. Also described are kits useful for diagnostic applications in humans according to the described evaluation methods.
Description
Our ref.: 273859.90 DETECTION OF HUMAN CYSTEINE-RICH SECRETORY PROTEIN (CRISPI) IN SEMEN
AND MEDICAL APPLICATIONS RELATED THERETO
FIELD OF THE INVENTION
The invention relates to the field of biology. More particularly, it concerns an epididymal specific protein which is detectable in human semen.
BACKGROUND OF THE INVENTION
In the USA, more than 525 000 vasectomies are performed yearly on men aged between 29 and 45 years. Worldwide, more than 100 million men rely on vasectomy for contraception purposes. However, due to changes in their personal life, an increasing number of men undergo surgical vasectomy reversal (vasovasostomy). In the USA only, this surgical procedure is performed on 250-300 000 men yearly. Therefore, there is a need for efficient, sensitive and non-invasive methods and kits for evaluating successfulness of vasectomy and of vasovasostomy in men.
Azoospermia is a medical condition of a male not having any measurable level of sperm in his semen. Azoospermia has two forms: (i) non-obstructive azoospermia, where there is a problem with spermatogenesis, and; (ii) obstructive azoospermia, where spermatozoa are created, but cannot be mixed with the rest of the ejaculatory fluid due to a physical obstruction, generally an obstruction of the epididymis or of the deferent duct (vas deferens).
Unfortunately, it is currently very difficult to discriminate between both forms: it requires either a biopsy of the testicle which is invasive and painful; or an ultrasonography of the scrotum which is a complex medical procedure that can only be performed by doctors in hospitals or clinics. There is thus an urgent need for a quick and non-invasive method for discriminating between obstructive and non-obstructive azoospermia.
Human cysteine-rich secretory protein (CRISP1) is one of the major epididymal secreted protein. It plays important functions in sperm physiology (Gibbs et ai., Endocr Rev 2008;29:865-97; Cohen et al., Asian J Androl 2007;9:528-32; Roberts et al., Mol Cell Endocrinol 2006;250:122-7) and expression of this protein in the epididymis is greatly affected by vasectomy (Thimon et al., Biol Reprod 2008;79:262-73). However, there is contradictive evidence as to the specificity of Crispl gene expression.
Although it has been Our ref.: 273859.90 reported that the human Crispl gene is expressed only in the epididymis (Kratzschmar et al., Eur. J. Biochem. 1996; 236, 827-836; Hayashi et al., Genomics 1996;32:367-74), some authors have shown expression of human Crisp1 not only in the epididymis, but also in the vas deferens and seminal vesicles by using more sensitive detection methods (Nolan et al., Biol. Reprod. 2006; 74, 984-991). Accordingly, it was uncertain before the present invention that the absence of the CRISPI protein in human semen could be an indicator of obstructive azoospermia. Moreover, it was unknown that the absence or presence of human CRISP1 in the semen could serve as a reliable marker for evaluating in men successfulness of vasectomy or vasovasostomy.
Many mammalian members of the CRISP family of genes are known, including the rat and mouse members which have been the subject of a U.S. patent application published under No. US 2006/027803.
As it will be apparent from a review of the disclosure, drawings and description of the features of the invention below, the present invention addresses these needs and other needs by providing methods and kits which are based on the detection of CRISPI.
BRIEF SUMMARY OF THE INVENTION
The invention relates to the detection of CRISPI as a marker for the presence or absence of epididymal secretions in a mammalian ejaculate.
According to one particular aspect, the invention relates to a method for assessing the presence of epididymal secretions in a human semen sample. The method comprises detecting in the semen sample presence or absence of human cysteine-rich secretory protein (CRISPI). Absence of CRISPI in the seminal plasma is indicative of an absence of epididymal secretions in the ejaculate.
The invention also relates to a method for evaluating successfulness of vasectomy in a man.
The method comprises obtaining a semen sample from a vasectomized man and detecting in the semen sample the presence or absence of human cysteine-rich secretory protein (CRISPI). Absence of CRISP1 is indicative of a successful vasectomy while presence of CRISPI is indicative of an unsuccessful vasectomy.
AND MEDICAL APPLICATIONS RELATED THERETO
FIELD OF THE INVENTION
The invention relates to the field of biology. More particularly, it concerns an epididymal specific protein which is detectable in human semen.
BACKGROUND OF THE INVENTION
In the USA, more than 525 000 vasectomies are performed yearly on men aged between 29 and 45 years. Worldwide, more than 100 million men rely on vasectomy for contraception purposes. However, due to changes in their personal life, an increasing number of men undergo surgical vasectomy reversal (vasovasostomy). In the USA only, this surgical procedure is performed on 250-300 000 men yearly. Therefore, there is a need for efficient, sensitive and non-invasive methods and kits for evaluating successfulness of vasectomy and of vasovasostomy in men.
Azoospermia is a medical condition of a male not having any measurable level of sperm in his semen. Azoospermia has two forms: (i) non-obstructive azoospermia, where there is a problem with spermatogenesis, and; (ii) obstructive azoospermia, where spermatozoa are created, but cannot be mixed with the rest of the ejaculatory fluid due to a physical obstruction, generally an obstruction of the epididymis or of the deferent duct (vas deferens).
Unfortunately, it is currently very difficult to discriminate between both forms: it requires either a biopsy of the testicle which is invasive and painful; or an ultrasonography of the scrotum which is a complex medical procedure that can only be performed by doctors in hospitals or clinics. There is thus an urgent need for a quick and non-invasive method for discriminating between obstructive and non-obstructive azoospermia.
Human cysteine-rich secretory protein (CRISP1) is one of the major epididymal secreted protein. It plays important functions in sperm physiology (Gibbs et ai., Endocr Rev 2008;29:865-97; Cohen et al., Asian J Androl 2007;9:528-32; Roberts et al., Mol Cell Endocrinol 2006;250:122-7) and expression of this protein in the epididymis is greatly affected by vasectomy (Thimon et al., Biol Reprod 2008;79:262-73). However, there is contradictive evidence as to the specificity of Crispl gene expression.
Although it has been Our ref.: 273859.90 reported that the human Crispl gene is expressed only in the epididymis (Kratzschmar et al., Eur. J. Biochem. 1996; 236, 827-836; Hayashi et al., Genomics 1996;32:367-74), some authors have shown expression of human Crisp1 not only in the epididymis, but also in the vas deferens and seminal vesicles by using more sensitive detection methods (Nolan et al., Biol. Reprod. 2006; 74, 984-991). Accordingly, it was uncertain before the present invention that the absence of the CRISPI protein in human semen could be an indicator of obstructive azoospermia. Moreover, it was unknown that the absence or presence of human CRISP1 in the semen could serve as a reliable marker for evaluating in men successfulness of vasectomy or vasovasostomy.
Many mammalian members of the CRISP family of genes are known, including the rat and mouse members which have been the subject of a U.S. patent application published under No. US 2006/027803.
As it will be apparent from a review of the disclosure, drawings and description of the features of the invention below, the present invention addresses these needs and other needs by providing methods and kits which are based on the detection of CRISPI.
BRIEF SUMMARY OF THE INVENTION
The invention relates to the detection of CRISPI as a marker for the presence or absence of epididymal secretions in a mammalian ejaculate.
According to one particular aspect, the invention relates to a method for assessing the presence of epididymal secretions in a human semen sample. The method comprises detecting in the semen sample presence or absence of human cysteine-rich secretory protein (CRISPI). Absence of CRISPI in the seminal plasma is indicative of an absence of epididymal secretions in the ejaculate.
The invention also relates to a method for evaluating successfulness of vasectomy in a man.
The method comprises obtaining a semen sample from a vasectomized man and detecting in the semen sample the presence or absence of human cysteine-rich secretory protein (CRISPI). Absence of CRISP1 is indicative of a successful vasectomy while presence of CRISPI is indicative of an unsuccessful vasectomy.
Our ref.: 273859.90 The invention also relates to a method for evaluating successfulness of vasovasostomy in a man. The method comprises obtaining a semen sample from a vasovasostomized man and detecting in the sample the presence or absence of human cysteine-rich secretory protein (CRISPI). Presence of CRISP1 is indicative of a successful vasovasostomy while absence of CRISPI is indicative of an unsuccessful vasovasostomy.
The invention further relates to a method for discriminating between obstructive and non-obstructive azoospermia in men. The method comprises obtaining a sample from an ejaculate of an azoospermic man and detecting in the sample the presence or absence of human cysteine-rich secretory protein (CRISP1). Presence of CRISPI is indicative of a non-obstructive azoospermia condition while absence of CRISPI is indicative of an obstructive azoospermia condition.
The invention further relates to a kit for evaluating successfulness of vasectomy and vasovasostomy in men. The kit comprises a user manual or instructions and at least one detection component for detecting in a human ejaculate the presence or absence of human cysteine-rich secretory protein (CRISPI ).
An advantage of the present invention is that it provides efficient, sensitive and non-invasive means for evaluating successfulness of vasectomy and of vasovasostomy in men.
The invention further provides quick and non-invasive methods for discriminating between obstructive and non-obstructive azoospermia.
Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 comprises pictures showing immunohistological localization of CRISP1 in the caput (A, D), corpus (B, E), and cauda (C, F) epididymides from normal (A, B, C) and vasectomized men (D, E, F). C* and F* are negative controls. Arrows show lumen positively stained for CRISPI.
Figure 2 comprises pictures showing in situ localization of the CrispI mRNA
along normal (A, B, C) and vasectomized (D, E, F) human epididymides. Histological sections of human Our ref.: 273859.90 Caput (A, D), corpus (B, E), and cauda (C, F) segments of the epididymis were probed with DIG-labeled antisense or sense (negative control) (B*, E*) CRISP1 RNA probes..
Arrows indicate principal cells positively stained for CRISPI mRNA.
Figure 3A is a dot graph representing densitometric determination of the quantity of CRISPI associated with 107 spermatozoa of fertile and vasovasostomized men.
CRISP1 is expressed as a ratio of TUBA used as a ubiquitous protein.
Figure 3B is a panel showing examples of Western blots of CRISPI detection performed on protein extracted from 107 spermatozoa of fertile and vasovasostomized men.
Figures 4A and 4B are pictures showing immunolocalization of CRISP1 on ejaculated spermatozoa of fertile (Fig. 4A) and vasovasostomized (Fig. 4B) men. A' and B' are negative control using goat IgGs. Immune complexes appear as a dark staining. Arrows indicate the acrosomal region.
Figure 5 is a dot graph showing relative amount of CRISPI in seminal plasma from normal, vasectomized and vasovasostomized men. CRISP1 amounts are expressed as a ratio of an internal standard (acid phosphatase). Bars indicate average and "n" indicates the number of samples.
Figure 6 is a panel showing Western blot detection of CRISPI in tissues homogenates from testis (1), epididymis (2), vas deferens (3), prostate (4), and seminal vesicles (5).
Figure 7 displays the amino acid sequence of CRISP1.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention is based on the identification of CRISP1 in semen.
Surprisingly, the present inventors have found that CRISP1 is highly specific to the epididymis and that absence of CRISP1 in human semen correlates with a complete absence of any epididymal secretions in an ejaculate. This epididymal specificity makes CRISP1 a reliable marker for evaluating in men successfulness of vasectomy or vasovasostomy and/or for discriminating between obstructive and non-obstructive azoospermia.
The invention further relates to a method for discriminating between obstructive and non-obstructive azoospermia in men. The method comprises obtaining a sample from an ejaculate of an azoospermic man and detecting in the sample the presence or absence of human cysteine-rich secretory protein (CRISP1). Presence of CRISPI is indicative of a non-obstructive azoospermia condition while absence of CRISPI is indicative of an obstructive azoospermia condition.
The invention further relates to a kit for evaluating successfulness of vasectomy and vasovasostomy in men. The kit comprises a user manual or instructions and at least one detection component for detecting in a human ejaculate the presence or absence of human cysteine-rich secretory protein (CRISPI ).
An advantage of the present invention is that it provides efficient, sensitive and non-invasive means for evaluating successfulness of vasectomy and of vasovasostomy in men.
The invention further provides quick and non-invasive methods for discriminating between obstructive and non-obstructive azoospermia.
Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 comprises pictures showing immunohistological localization of CRISP1 in the caput (A, D), corpus (B, E), and cauda (C, F) epididymides from normal (A, B, C) and vasectomized men (D, E, F). C* and F* are negative controls. Arrows show lumen positively stained for CRISPI.
Figure 2 comprises pictures showing in situ localization of the CrispI mRNA
along normal (A, B, C) and vasectomized (D, E, F) human epididymides. Histological sections of human Our ref.: 273859.90 Caput (A, D), corpus (B, E), and cauda (C, F) segments of the epididymis were probed with DIG-labeled antisense or sense (negative control) (B*, E*) CRISP1 RNA probes..
Arrows indicate principal cells positively stained for CRISPI mRNA.
Figure 3A is a dot graph representing densitometric determination of the quantity of CRISPI associated with 107 spermatozoa of fertile and vasovasostomized men.
CRISP1 is expressed as a ratio of TUBA used as a ubiquitous protein.
Figure 3B is a panel showing examples of Western blots of CRISPI detection performed on protein extracted from 107 spermatozoa of fertile and vasovasostomized men.
Figures 4A and 4B are pictures showing immunolocalization of CRISP1 on ejaculated spermatozoa of fertile (Fig. 4A) and vasovasostomized (Fig. 4B) men. A' and B' are negative control using goat IgGs. Immune complexes appear as a dark staining. Arrows indicate the acrosomal region.
Figure 5 is a dot graph showing relative amount of CRISPI in seminal plasma from normal, vasectomized and vasovasostomized men. CRISP1 amounts are expressed as a ratio of an internal standard (acid phosphatase). Bars indicate average and "n" indicates the number of samples.
Figure 6 is a panel showing Western blot detection of CRISPI in tissues homogenates from testis (1), epididymis (2), vas deferens (3), prostate (4), and seminal vesicles (5).
Figure 7 displays the amino acid sequence of CRISP1.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention is based on the identification of CRISP1 in semen.
Surprisingly, the present inventors have found that CRISP1 is highly specific to the epididymis and that absence of CRISP1 in human semen correlates with a complete absence of any epididymal secretions in an ejaculate. This epididymal specificity makes CRISP1 a reliable marker for evaluating in men successfulness of vasectomy or vasovasostomy and/or for discriminating between obstructive and non-obstructive azoospermia.
Our ref.: 273859.90 As used herein, the term "ejaculate" or "semen" generally refers to the organic fluid which is ejected from the penis following an ejaculation by a subject. Depending of the subject's condition, the semen may or may not comprise spermatozoa.
As used herein, the term "seminal plasma" or "seminal fluid" generally refers to the liquid supernatant, after sedimentation or centrifugation of semen, which has been contributed by the testis and the various sex glands (e.g. the seminal vesicles, the prostate and the bulbourethral glands).
As used herein, the term "epididymal secretions" generally refers to the biological materials which have been contributed by the epididymis.
As used herein, the term "CRISPI" generally refers to the human cysteine-rich secretory protein. CRISP1 is an epididymal protein (248 amino acids) which is known to those skilled in the art since it has been the subject of many scientific publications. The GeneBankTM
(NCBI) accession number of human CRISPI is D38451, the UniProtTM accession number is P54107 and the UniGeneTM accession number is Hs.109620. Figure 7 provides the complete amino acid sequence of CRISPI in humans (SEQ ID NO: 1). The methods of the invention encompass detection of the full-length CRISPI protein, and also the detection of fragments thereof comprising at least 10, 25, 50, 100, 150, 200, 225, 240, 245, or 247 contiguous amino acids of SEQ ID NO: 1.
Medical applications As demonstrated in the Examplification section, CRISPI is a protein highly specific to the epididymis such that absence of CRISPI in semen is indicative of a complete absence of epididymal secretions in the ejaculate. This surprising finding opens different avenues for research, diagnostic and medical applications.
In one embodiment, detecting absence or presence of epididymal secretions in the ejaculate is especially useful for discriminating between obstructive and non-obstructive azoospermia.
Therefore, the methods described herein may be useful to replace and/or complement various infertility assessment procedures, including but not limited to, physical examination, sperm count, biopsy of the testicle, ultrasonography. Detecting in semen the presence or absence of CRISPI may also be useful to the medical profession when investigating conditions such as azoospermia, sexual dysfunction, congenital defects of the sperm, and hypogonadism. Therefore, the methods described herein may also be carried out in the Our ref.: 273859.90 course of various assisted reproduction procedures, including but not limited to in vitro fertilization (IFV), artificial insemination (AI), gamete intrafallopian transfer (GIFT), embryo transfer, intracytoplasmic sperm injection (ICSI), etc. The invention further encompasses diagnosis and management of human male fertility (e.g. fertility clinics) using any of the methods, techniques and kits described herein.
As described hereinbefore, detecting the presence or absence of CRISPI in a semen sample is useful for assessing successfulness of vasectomy or successfulness of vasovasostomy. According to a particular embodiment, a semen sample from a vasectomized man is obtained and the sample is assessed for detecting therein the presence or absence of CRISP1. Since in vasectomized men the vasa deferentia have been severed, and then tied or sealed, epididymal proteins and sperm should not be present in the ejaculate. Therefore, absence of CRISP1 in the ejaculate would be indicative of a successful vasectomy while presence of CRISPI would be indicative of an unsuccessful vasectomy. Similarly, according to another embodiment, if the semen sample is obtained from a vasovasostomized man (i.e. the vasa deferentia have been reconnected for reversing the vasectomy), epididymal proteins and sperm should be present in the ejaculate.
Accordingly, presence of CRISPI would be indicative of a successful vasovasostomy while absence of CRISPI would be indicative of an unsuccessful vasovasostomy.
Detection techniques Different methods and tools can be used for measuring the presence or absence of CRISP1 in semen. A preferred agent for detecting CRISP1 is an antibody capable of binding to CRISP1 (and/or to a fragment thereof). More preferably, the antibody is highly specific to CRISPI. Antibodies can be polyclonal, or more preferably monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. According to some embodiments, the antibody is labeled. The term "labeled", with regard to an antibody, is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include, but are not limited to, detection of a primary antibody using a fluorescently labeled secondary antibody.
Specific antibodies directed against the human CRISP1 are available and include those commercialized by Santa Cruz Biotechnology (CRISP-1 (H-75) antibody (rabbit IgG, Catalog # sc-33802; CRISP-1 (N-15) antibody, goat IgG, Catalog # sc-21276), Abnova (mouse Our ref.: 273859.90 polyclonal antibody raised against a full-length human CRISP1 protein; Catalog #
H00000167-B01); Sigma (anti-CRISPI antibody produced in rabbit, Catalog #
AV53580 ), and Abcam (CRISPI rabbit polyclonal antibody, Catalog #ab74785).
Therefore, the present invention encompasses various CRISPI immunodetection and/or CRISP1 quantification methods, including but not limited to ELISA, flow cytometry and densitometric analysis of Western blots.
Those skilled in the art know how to use specific antibodies to make different tools useful in the immunodetection and quantification, including but not limited to antibody coated colored beads, antibody coated magnetic beads, strips and dip sticks coated with antibodies, etc.
It is also conceivable according to the invention, that the biological activity of CRISP1 be measured in a semen sample. Therefore, in some embodiments, it is an activity of CRISP1 which is assessed using methods and techniques known to those skilled in the art. For instance, published patent application US 2006/275803 suggests that CRISPI may have a protease activity.
Kits Another aspect of the invention pertains to commercial packages or kits for carrying out the methods and assays of the invention. Kits according to the invention may be used for discriminating between obstructive and non-obstructive azoospermia and/or for evaluating successfulness of vasectomy and successfulness of vasovasostomy.
Kits according to the invention will employ, unless otherwise indicated, conventional techniques for protein detection. A kit of the invention may comprise one or more of the following elements: a buffer for the homogenization of a sperm sample;
purified sperm fertility proteins (and/or a fragment thereof), including but not limited to CRISP1 to be used as controls; buffers (e.g. incubation buffer(s), substrate and assay buffer(s), modulator buffer(s)); detection materials (e.g. antibodies, fluorescein-labelled derivatives, luminogenic substrates, detection solutions, scintillation counting fluid, antibody coated beads, etc.);
laboratory supplies (e.g. reaction tubes or microplates (e.g. 96- or 384-well plates)); a user manual or instructions, etc. The kit and methods of the invention may be configured such as to permit a quantitative detection or measurement of the protein(s) of interest (e.g.
cytofluorimetry, LuminexTM, ECL PlexTM Western Blotting Detection System, etc.).
As used herein, the term "seminal plasma" or "seminal fluid" generally refers to the liquid supernatant, after sedimentation or centrifugation of semen, which has been contributed by the testis and the various sex glands (e.g. the seminal vesicles, the prostate and the bulbourethral glands).
As used herein, the term "epididymal secretions" generally refers to the biological materials which have been contributed by the epididymis.
As used herein, the term "CRISPI" generally refers to the human cysteine-rich secretory protein. CRISP1 is an epididymal protein (248 amino acids) which is known to those skilled in the art since it has been the subject of many scientific publications. The GeneBankTM
(NCBI) accession number of human CRISPI is D38451, the UniProtTM accession number is P54107 and the UniGeneTM accession number is Hs.109620. Figure 7 provides the complete amino acid sequence of CRISPI in humans (SEQ ID NO: 1). The methods of the invention encompass detection of the full-length CRISPI protein, and also the detection of fragments thereof comprising at least 10, 25, 50, 100, 150, 200, 225, 240, 245, or 247 contiguous amino acids of SEQ ID NO: 1.
Medical applications As demonstrated in the Examplification section, CRISPI is a protein highly specific to the epididymis such that absence of CRISPI in semen is indicative of a complete absence of epididymal secretions in the ejaculate. This surprising finding opens different avenues for research, diagnostic and medical applications.
In one embodiment, detecting absence or presence of epididymal secretions in the ejaculate is especially useful for discriminating between obstructive and non-obstructive azoospermia.
Therefore, the methods described herein may be useful to replace and/or complement various infertility assessment procedures, including but not limited to, physical examination, sperm count, biopsy of the testicle, ultrasonography. Detecting in semen the presence or absence of CRISPI may also be useful to the medical profession when investigating conditions such as azoospermia, sexual dysfunction, congenital defects of the sperm, and hypogonadism. Therefore, the methods described herein may also be carried out in the Our ref.: 273859.90 course of various assisted reproduction procedures, including but not limited to in vitro fertilization (IFV), artificial insemination (AI), gamete intrafallopian transfer (GIFT), embryo transfer, intracytoplasmic sperm injection (ICSI), etc. The invention further encompasses diagnosis and management of human male fertility (e.g. fertility clinics) using any of the methods, techniques and kits described herein.
As described hereinbefore, detecting the presence or absence of CRISPI in a semen sample is useful for assessing successfulness of vasectomy or successfulness of vasovasostomy. According to a particular embodiment, a semen sample from a vasectomized man is obtained and the sample is assessed for detecting therein the presence or absence of CRISP1. Since in vasectomized men the vasa deferentia have been severed, and then tied or sealed, epididymal proteins and sperm should not be present in the ejaculate. Therefore, absence of CRISP1 in the ejaculate would be indicative of a successful vasectomy while presence of CRISPI would be indicative of an unsuccessful vasectomy. Similarly, according to another embodiment, if the semen sample is obtained from a vasovasostomized man (i.e. the vasa deferentia have been reconnected for reversing the vasectomy), epididymal proteins and sperm should be present in the ejaculate.
Accordingly, presence of CRISPI would be indicative of a successful vasovasostomy while absence of CRISPI would be indicative of an unsuccessful vasovasostomy.
Detection techniques Different methods and tools can be used for measuring the presence or absence of CRISP1 in semen. A preferred agent for detecting CRISP1 is an antibody capable of binding to CRISP1 (and/or to a fragment thereof). More preferably, the antibody is highly specific to CRISPI. Antibodies can be polyclonal, or more preferably monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. According to some embodiments, the antibody is labeled. The term "labeled", with regard to an antibody, is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include, but are not limited to, detection of a primary antibody using a fluorescently labeled secondary antibody.
Specific antibodies directed against the human CRISP1 are available and include those commercialized by Santa Cruz Biotechnology (CRISP-1 (H-75) antibody (rabbit IgG, Catalog # sc-33802; CRISP-1 (N-15) antibody, goat IgG, Catalog # sc-21276), Abnova (mouse Our ref.: 273859.90 polyclonal antibody raised against a full-length human CRISP1 protein; Catalog #
H00000167-B01); Sigma (anti-CRISPI antibody produced in rabbit, Catalog #
AV53580 ), and Abcam (CRISPI rabbit polyclonal antibody, Catalog #ab74785).
Therefore, the present invention encompasses various CRISPI immunodetection and/or CRISP1 quantification methods, including but not limited to ELISA, flow cytometry and densitometric analysis of Western blots.
Those skilled in the art know how to use specific antibodies to make different tools useful in the immunodetection and quantification, including but not limited to antibody coated colored beads, antibody coated magnetic beads, strips and dip sticks coated with antibodies, etc.
It is also conceivable according to the invention, that the biological activity of CRISP1 be measured in a semen sample. Therefore, in some embodiments, it is an activity of CRISP1 which is assessed using methods and techniques known to those skilled in the art. For instance, published patent application US 2006/275803 suggests that CRISPI may have a protease activity.
Kits Another aspect of the invention pertains to commercial packages or kits for carrying out the methods and assays of the invention. Kits according to the invention may be used for discriminating between obstructive and non-obstructive azoospermia and/or for evaluating successfulness of vasectomy and successfulness of vasovasostomy.
Kits according to the invention will employ, unless otherwise indicated, conventional techniques for protein detection. A kit of the invention may comprise one or more of the following elements: a buffer for the homogenization of a sperm sample;
purified sperm fertility proteins (and/or a fragment thereof), including but not limited to CRISP1 to be used as controls; buffers (e.g. incubation buffer(s), substrate and assay buffer(s), modulator buffer(s)); detection materials (e.g. antibodies, fluorescein-labelled derivatives, luminogenic substrates, detection solutions, scintillation counting fluid, antibody coated beads, etc.);
laboratory supplies (e.g. reaction tubes or microplates (e.g. 96- or 384-well plates)); a user manual or instructions, etc. The kit and methods of the invention may be configured such as to permit a quantitative detection or measurement of the protein(s) of interest (e.g.
cytofluorimetry, LuminexTM, ECL PlexTM Western Blotting Detection System, etc.).
Our ref.: 273859.90 In one preferred embodiment, the kit is adapted for home-based uses. Indeed, home assessment of the presence or absence of sperm in an ejaculate is desirable, for instance, for confirming absence of sperm in the semen after a vasectomy procedure. Home-based diagnostic provides the benefits of an easier and faster diagnostic than an evaluation of the semen under a microscope or than obtaining a sperm count at a hospital or a clinic.
In a particular embodiment, the kit comprises a dip stick, or a strip comprising (e.g. coated or impregnated with) one or more antibodies (e.g. labeled or non-labeled antibodies) specific for CRISPI. For instance, in one particular embodiment, a user would contact a semen sample or ejaculate with the dip stick or strip and a positive signal (e.g. a coloration) would appear if CRISPI is detected. The dip stick or strip could also comprise control antibodies for detecting control proteins (e.g. seminal plasma proteins).
The kits of the invention may also be particularly useful for diagnostic applications in humans according to the evaluation methods described hereinbefore. More particularly, the kits disclosed herein may be helpful for laboratory and diagnostic purposes in humans during infertility diagnostic procedures (e.g. for discriminating between obstructive and non-obstructive azoospermia). The kits and methods of the invention may be used in combination with previously described assay kits.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the invention and covered by the claims appended hereto. The invention is further illustrated by the following example, which should not be construed as further limiting.
EXAMPLE
EXAMPLE 1: CRISPI expression in context to vasectomy surgical reversal MATERIAL AND METHODS
Ethic consent These studies were conducted with the approval of the ethic committee for research on human subjects of the Universite Laval.
In a particular embodiment, the kit comprises a dip stick, or a strip comprising (e.g. coated or impregnated with) one or more antibodies (e.g. labeled or non-labeled antibodies) specific for CRISPI. For instance, in one particular embodiment, a user would contact a semen sample or ejaculate with the dip stick or strip and a positive signal (e.g. a coloration) would appear if CRISPI is detected. The dip stick or strip could also comprise control antibodies for detecting control proteins (e.g. seminal plasma proteins).
The kits of the invention may also be particularly useful for diagnostic applications in humans according to the evaluation methods described hereinbefore. More particularly, the kits disclosed herein may be helpful for laboratory and diagnostic purposes in humans during infertility diagnostic procedures (e.g. for discriminating between obstructive and non-obstructive azoospermia). The kits and methods of the invention may be used in combination with previously described assay kits.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of the invention and covered by the claims appended hereto. The invention is further illustrated by the following example, which should not be construed as further limiting.
EXAMPLE
EXAMPLE 1: CRISPI expression in context to vasectomy surgical reversal MATERIAL AND METHODS
Ethic consent These studies were conducted with the approval of the ethic committee for research on human subjects of the Universite Laval.
Our ref.: 273859.90 Biological material Human epididymides were obtained from a local organ transplantation program.
After obtaining family consent, testicles were removed while artificial circulation was maintained to preserve tissues assigned for transplantation. Donors were 26-50 years of age with no medical pathologies that could affect reproductive function, except vasectomy.
Organs were brought to the lab on ice and processed within 3 hours after orchidectomy.
Vasectomy was determined while dissecting the scrotal segment of the vas deferens.
Epididymal tissues were dissected in caput, corpus and cauda and minced in small tissue pieces.
Tissues were immediately snap frozen in liquid nitrogen for subsequent RNA extraction or preparation of protein extracts. For in situ hybridization, pieces of tissue were fixed in freshly prepared 4%
paraformaldehyde in phosphate buffered saline (PBS), embedded in OCT and kept at -80 C
until used. Other pieces of tissue were fixed by immersion in 4%
paraformaldehyde solution, dehydrated and embedded in paraffin for immunohistological localization of CRISP1.
Other tissues of the reproductive tract were used for Western blot determination of CRISPI
expression. Testicular tissues were obtained when epididymides were dissected from organ donors. Segments of the vasa deferentia were obtained from healthy patients of proven fertility who were undergoing vasectomy or vasovasostomy. Prostate and seminal vesicle tissues were obtained from patients who were undergoing prostatectomy. This surgical procedure was performed by laparoscopy under general anesthesia.
Semen samples were obtained by masturbation from healthy control donors or from vasovasostomized men undergoing post-surgical spermogram follow-up performed at the clinical andrology laboratory of our institution. The time period elapsed between vasectomy and vasovasostomy varied from 3 to 9 years. Azoospermic and oligoasthenospermic samples from vasovasostomized men were not included in this study. After liquefaction, spermatozoa were pelleted and washed twice by centrifugation at 800g in Dulbecco's phosphate-buffered saline. Seminal plasma and sperm pellets were frozen at -80 C until used.
In situ hybridization In situ hybridization and tissue section preparations were performed as previously described (Legare et at., J Androl 2004;25:30-43). Briefly, CrispI complementary DNA
(cDNA) was generated by RT-PCR using polyA RNA from normal human epididymis. The oligonucleotides used as primers were 5'-GAA-GCC-TGC-CCA-AGT-AAC-TG-3' (SEQ ID
After obtaining family consent, testicles were removed while artificial circulation was maintained to preserve tissues assigned for transplantation. Donors were 26-50 years of age with no medical pathologies that could affect reproductive function, except vasectomy.
Organs were brought to the lab on ice and processed within 3 hours after orchidectomy.
Vasectomy was determined while dissecting the scrotal segment of the vas deferens.
Epididymal tissues were dissected in caput, corpus and cauda and minced in small tissue pieces.
Tissues were immediately snap frozen in liquid nitrogen for subsequent RNA extraction or preparation of protein extracts. For in situ hybridization, pieces of tissue were fixed in freshly prepared 4%
paraformaldehyde in phosphate buffered saline (PBS), embedded in OCT and kept at -80 C
until used. Other pieces of tissue were fixed by immersion in 4%
paraformaldehyde solution, dehydrated and embedded in paraffin for immunohistological localization of CRISP1.
Other tissues of the reproductive tract were used for Western blot determination of CRISPI
expression. Testicular tissues were obtained when epididymides were dissected from organ donors. Segments of the vasa deferentia were obtained from healthy patients of proven fertility who were undergoing vasectomy or vasovasostomy. Prostate and seminal vesicle tissues were obtained from patients who were undergoing prostatectomy. This surgical procedure was performed by laparoscopy under general anesthesia.
Semen samples were obtained by masturbation from healthy control donors or from vasovasostomized men undergoing post-surgical spermogram follow-up performed at the clinical andrology laboratory of our institution. The time period elapsed between vasectomy and vasovasostomy varied from 3 to 9 years. Azoospermic and oligoasthenospermic samples from vasovasostomized men were not included in this study. After liquefaction, spermatozoa were pelleted and washed twice by centrifugation at 800g in Dulbecco's phosphate-buffered saline. Seminal plasma and sperm pellets were frozen at -80 C until used.
In situ hybridization In situ hybridization and tissue section preparations were performed as previously described (Legare et at., J Androl 2004;25:30-43). Briefly, CrispI complementary DNA
(cDNA) was generated by RT-PCR using polyA RNA from normal human epididymis. The oligonucleotides used as primers were 5'-GAA-GCC-TGC-CCA-AGT-AAC-TG-3' (SEQ ID
Our ref.: 273859.90 NO:2) and 5'-GGG-AGT-TAA-GGT-CTC-CAG-CA-3' (SEQ ID NO:3) and the PCR product was subcloned into pGEM-T (Promega, Madison, Wis, USA). The plasmid was digested and the mRNA was transcribed using SP6 and T7 RNA polymerase (Roche) in the presence of Digoxigenin-1 1-uridin-triphosphate (DIG)-UTP.
Epididymis cryosections were fixed with 4% paraformaldehyde for 5 min, incubated for 10 min in 95% ethanol/5% acetic acid at -20 C, and rehydrated. Target mRNAs were unmasked by enzymatic digestion with 10 g/mL proteinase K in PBS for 10 min at 37 C.
Sections were incubated in 0.2% glycine, post-fixed for 5 min with 4%
paraformaldehyde, acetylated with 0.25% acetic anhydride, OA M triethanolamine, pH 8 for 10 min, and washed in PBS.
Tissue sections were prehybridized for 2 hours at 42 C with 250 g/mL of salmon sperm DNA preheated in a hybridization buffer and incubated overnight at 42 C, under cover slips, with 25 L of 5 gg/ml heat-denatured antisense of sense cRNA probed with DIG.
Nonspecific staining was blocked by incubation for 1 hour with 5% heat-inactivated sheep serum in Tris buffer. Hybridization reactions were detected with an alkaline phosphatase-conjugated anti-DIG antibody diluted 1:1000 in a blocking buffer. The hybridization signal was visualized after incubation with the phosphatase substrate, nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (GIBCO-BRL, Gaithersburg, Mo, USA). Epididymis sections from normal and vasectomized men were processed in parallel to allow comparison.
Protein preparation Spermatozoa Sperm pellets (107) were resuspended in SDS-PAGE sample buffer (Laemmli UK.
Nature 1970;227:680-5) prepared without reducing reagent. After 10 min, spermatozoa were pelletted by centrifugation, the protein supernatant recovered and reduced by addition of 2%
13-mercaptoethanol followed by 5 min heating in a boiling water bath.
Seminal plasma Seminal plasma samples were centrifuged two times at 3000g for 10 min to eliminate spermatozoa and other cellular constituents. Supernatants were precipated with MEOH/CHCI3, proteins were resuspended in sample buffer and submitted to SDS-PAGE
Epididymis cryosections were fixed with 4% paraformaldehyde for 5 min, incubated for 10 min in 95% ethanol/5% acetic acid at -20 C, and rehydrated. Target mRNAs were unmasked by enzymatic digestion with 10 g/mL proteinase K in PBS for 10 min at 37 C.
Sections were incubated in 0.2% glycine, post-fixed for 5 min with 4%
paraformaldehyde, acetylated with 0.25% acetic anhydride, OA M triethanolamine, pH 8 for 10 min, and washed in PBS.
Tissue sections were prehybridized for 2 hours at 42 C with 250 g/mL of salmon sperm DNA preheated in a hybridization buffer and incubated overnight at 42 C, under cover slips, with 25 L of 5 gg/ml heat-denatured antisense of sense cRNA probed with DIG.
Nonspecific staining was blocked by incubation for 1 hour with 5% heat-inactivated sheep serum in Tris buffer. Hybridization reactions were detected with an alkaline phosphatase-conjugated anti-DIG antibody diluted 1:1000 in a blocking buffer. The hybridization signal was visualized after incubation with the phosphatase substrate, nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (GIBCO-BRL, Gaithersburg, Mo, USA). Epididymis sections from normal and vasectomized men were processed in parallel to allow comparison.
Protein preparation Spermatozoa Sperm pellets (107) were resuspended in SDS-PAGE sample buffer (Laemmli UK.
Nature 1970;227:680-5) prepared without reducing reagent. After 10 min, spermatozoa were pelletted by centrifugation, the protein supernatant recovered and reduced by addition of 2%
13-mercaptoethanol followed by 5 min heating in a boiling water bath.
Seminal plasma Seminal plasma samples were centrifuged two times at 3000g for 10 min to eliminate spermatozoa and other cellular constituents. Supernatants were precipated with MEOH/CHCI3, proteins were resuspended in sample buffer and submitted to SDS-PAGE
Our ref.: 273859.90 (Laemmli., 1970). Protein concentrations were determined by amido black staining of dot blots (Chapdelaine et al., 2001, Biotechniques 31: 478-480).
Tissues Tissues were homogenized with a PolytronTM (InterSciences, Markham, ON) in a homogenization buffer (0.01M Tris, 0.1mM EDTA, 1%SDS, and 1mM PMSF). Extracts were then centrifuged at 3000g for 15min at 4 C. Supernatants were precipitated with MEOH/CHCI3, proteins were resuspended in sample buffer and submitted to SDS-PAGE
(Laemmli, 1970). Protein concentrations were determined by amido black staining of dot blots (Chapdelaine et al., 2001).
Antibodies Goat polyclonal antibody against CRISPI was purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA, USA) and used at 1 g/ml for Western blot analysis, 5 g/ml for immunohistochemistry on epididymis, and 15 g/ml on sperm smears. Rabbit polyclonal antibody against prostatic phosphatase acid (PAP) purified from human seminal plasma was used at 1/1000 (vol/vol). This antiserum was a generous gift of Dr RR Tremblay and was used as a marker of prostatic protein secretions. Mouse monoclonal antibody against a-tubulin (Sigma, ON, Canada) was used at 1/50000 (vol/vol). Mouse monoclonal antibody against Actin-0 (Sigma, ON, Canada) was used at 1/20 000 (v/v). Monoclonal anti-phosphotyrosine antibody (clone 4G10, Upstate Biotechnology) was used at 1/10000.
Rabbit anti-goat IgG, goat anti-rabbit and a goat anti-mouse conjugated to horseradish peroxidase were purchased from BIO/CAN Scientific (Mississauga, ON, Canada) and used respectively at 1/5000 (vol/vol). A biotinylated rabbit-anti-goat secondary antibody was obtained from Dako Diagnostics (Mississauga, ON, Canada) and used at 1/400 (vol/vol).
Immunohistochemical staining Paraffin sections were prepared from fixed epididymal tissues. Ejaculated spermatozoa from normal and vasovasostomized men were washed, smear on microscopic slides, fixed with cold ethanol, and processed for CRISPI localization. Endogenous peroxidase activity was quenched with 3% H202 (v/v) in PBS for 30 min. Non-specific binding sites were then blocked with 10% goat serum in PBS for 1 h. The CRISPI-specific antibodies were diluted in PBS and applied overnight at 4 C. In control sections, the primary antibodies were replaced by the corresponding non-specific IgG and processed in parallel. Sections were subsequently incubated with biotinylated rabbit anti-goat antibody for 30 min, and with Our ref.: 273859.90 ABCTM reagent for 30 min. Immunostaining was revealed using 3-amino-9-ethylcarbazole (AEC). Harris hematoxylin was used for counterstaining, and mounted under cover slip using an aqueous mounting medium (Sigma). Slides were observed under a Zeiss Axioskop2 PIUSTM microscope (Toronto, ON, Canada) linked to a digital camera from Diagnostics Instruments (Sterling Heights, MI). Images were captured using the SpotT""
software (Diagnostics Instruments).
Western blotting Proteins were separated on a 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Towbin et al., Proc Nati Acad Sci USA 1979;76:4350-4.). After saturation with 5% milk in PBS-0.1% Tween-20TH, membranes were incubated with an anti-CRISP1, an anti-PAP, an anti-tubulin or anti-phosphotyrosine. Rabbit anti-goat, goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase was used for chemiluminescent detection of proteins (ECLTM reagent: Amersham, Baie d'Urfe, QC, Canada). CRISPI, PAP, a-tubulin and phosphotyrosine-containing proteins signals were quantitated by densitometry in the linear range of film exposure and expressed as arbitrary units.
Statistical analysis Statistical analysis was performed by analysis of variance using Super ANOVATM
software (ABACUS Concepts, Berkeley, CA). Results were compared by Student-Newman-Keuls test. Differences were considered to be significant at P <0.05.
RESULTS
Cellular localization of CRISPI mRNA and protein along the epididymis from normal and vasectomized men The epididymal origin of CRISP1 was investigated by immunohistochemistry.
CRISP1 was undetectable in the caput epididymidis, and an increasing amount of this protein was revealed in the corpus and cauda of normal tissues. As already published (Legare et at., Endocrinology 1999;140:3318-27), vasectomy has major consequences on the height of the epididymal epithelium and the diameter of the lumen (Figure 1). As deduced from Western blots, a huge amount of CRISPI was accumulating in the lumen of cauda epididymidis of vasectomized men. In both normal and vasectomized tissues, CRISP1 was undetectable in the interstitial tissues of the epididymis.
Tissues Tissues were homogenized with a PolytronTM (InterSciences, Markham, ON) in a homogenization buffer (0.01M Tris, 0.1mM EDTA, 1%SDS, and 1mM PMSF). Extracts were then centrifuged at 3000g for 15min at 4 C. Supernatants were precipitated with MEOH/CHCI3, proteins were resuspended in sample buffer and submitted to SDS-PAGE
(Laemmli, 1970). Protein concentrations were determined by amido black staining of dot blots (Chapdelaine et al., 2001).
Antibodies Goat polyclonal antibody against CRISPI was purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA, USA) and used at 1 g/ml for Western blot analysis, 5 g/ml for immunohistochemistry on epididymis, and 15 g/ml on sperm smears. Rabbit polyclonal antibody against prostatic phosphatase acid (PAP) purified from human seminal plasma was used at 1/1000 (vol/vol). This antiserum was a generous gift of Dr RR Tremblay and was used as a marker of prostatic protein secretions. Mouse monoclonal antibody against a-tubulin (Sigma, ON, Canada) was used at 1/50000 (vol/vol). Mouse monoclonal antibody against Actin-0 (Sigma, ON, Canada) was used at 1/20 000 (v/v). Monoclonal anti-phosphotyrosine antibody (clone 4G10, Upstate Biotechnology) was used at 1/10000.
Rabbit anti-goat IgG, goat anti-rabbit and a goat anti-mouse conjugated to horseradish peroxidase were purchased from BIO/CAN Scientific (Mississauga, ON, Canada) and used respectively at 1/5000 (vol/vol). A biotinylated rabbit-anti-goat secondary antibody was obtained from Dako Diagnostics (Mississauga, ON, Canada) and used at 1/400 (vol/vol).
Immunohistochemical staining Paraffin sections were prepared from fixed epididymal tissues. Ejaculated spermatozoa from normal and vasovasostomized men were washed, smear on microscopic slides, fixed with cold ethanol, and processed for CRISPI localization. Endogenous peroxidase activity was quenched with 3% H202 (v/v) in PBS for 30 min. Non-specific binding sites were then blocked with 10% goat serum in PBS for 1 h. The CRISPI-specific antibodies were diluted in PBS and applied overnight at 4 C. In control sections, the primary antibodies were replaced by the corresponding non-specific IgG and processed in parallel. Sections were subsequently incubated with biotinylated rabbit anti-goat antibody for 30 min, and with Our ref.: 273859.90 ABCTM reagent for 30 min. Immunostaining was revealed using 3-amino-9-ethylcarbazole (AEC). Harris hematoxylin was used for counterstaining, and mounted under cover slip using an aqueous mounting medium (Sigma). Slides were observed under a Zeiss Axioskop2 PIUSTM microscope (Toronto, ON, Canada) linked to a digital camera from Diagnostics Instruments (Sterling Heights, MI). Images were captured using the SpotT""
software (Diagnostics Instruments).
Western blotting Proteins were separated on a 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Towbin et al., Proc Nati Acad Sci USA 1979;76:4350-4.). After saturation with 5% milk in PBS-0.1% Tween-20TH, membranes were incubated with an anti-CRISP1, an anti-PAP, an anti-tubulin or anti-phosphotyrosine. Rabbit anti-goat, goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase was used for chemiluminescent detection of proteins (ECLTM reagent: Amersham, Baie d'Urfe, QC, Canada). CRISPI, PAP, a-tubulin and phosphotyrosine-containing proteins signals were quantitated by densitometry in the linear range of film exposure and expressed as arbitrary units.
Statistical analysis Statistical analysis was performed by analysis of variance using Super ANOVATM
software (ABACUS Concepts, Berkeley, CA). Results were compared by Student-Newman-Keuls test. Differences were considered to be significant at P <0.05.
RESULTS
Cellular localization of CRISPI mRNA and protein along the epididymis from normal and vasectomized men The epididymal origin of CRISP1 was investigated by immunohistochemistry.
CRISP1 was undetectable in the caput epididymidis, and an increasing amount of this protein was revealed in the corpus and cauda of normal tissues. As already published (Legare et at., Endocrinology 1999;140:3318-27), vasectomy has major consequences on the height of the epididymal epithelium and the diameter of the lumen (Figure 1). As deduced from Western blots, a huge amount of CRISPI was accumulating in the lumen of cauda epididymidis of vasectomized men. In both normal and vasectomized tissues, CRISP1 was undetectable in the interstitial tissues of the epididymis.
Our ref.: 273859.90 In situ hybridization localization of CrispI mRNA was in agreement with the immunohistological localization of the translational product (Figure 2). In both normal and vasectomized tissues, the CrispI mRNA was expressed by the epithelium in the corpus and cauda segments. mRNA was undetectable in the caput. The staining was restricted to the epithelium as no staining was detected in interstitial tissues.
Effect of vasectomy on CRISPI associated with spermatozoa Western blot analyses were performed on protein extracted from a constant number of spermatozoa from different men. CRISPI was detected at -32kDa. The commercially available antibody was specific to CRISP1 as shown by the absence of immunodetectable band revealed when Western blots were probed with a control serum (data not shown).
When expressed as a ratio of a constitutive protein TUBA, the quantity of CRISPI
associated with a constant number of ejaculated spermatozoa was significantly higher in vasovasostomized men when compared to normal donors (Figure 3) with a P>0.05.
CRISPI was associated with the acrosomal region of ejaculated spermatozoa from both normal and vasovasostomized men. The immune detection signal varied significantly from one sperm cell to the other in both normal and vasovasostomy samples. By opposition to Western blot analyses; it was not possible to quantitate the amount of CRISP1 immunodetected on smears of spermatozoa (Figure 4). The preimmune serum did not stain the acrosomal region but the equatorial segment was unspecifically stained for an unknown reason (Figure 4).
Vasectomy effect on CRISPI in seminal plasma CRISPI was also found as a soluble form in the seminal plasma. When Western blots were performed on 20 g of seminal plasma proteins, CRISPI was detectable in semen samples of both normal and vasovasostomized men. As for CRISPI associated to spermatozoa, CRISPI was significantly (P>0.05) more concentrated in seminal plasma of vasovasostomized men when compared to samples from normal men. Interestingly, was undetectable in seminal plasma of vasectomized men (Figure 5). This correlated well with the fact that CRISPI was undetectable on Western blots of 20 g of protein extracted from human seminal vesicles and prostate tissues (Figure 6).
Conclusions As shown herein, in human semen, CRISPI is found both associated to sperm and in the soluble fraction. Both forms are in higher concentrations in vasovasostomized men if the vas Our ref.: 273859.90 deferens were repermeabilized. CRISPI remains localized to the acrosomal region of spermatozoa in both normal and vasovasostomized. This suggests that the association of this epididymal protein to spermatozoa is not a simple coating phenomenon and that the specificity of the interaction remains even though CRISP1 is secreted in excess in some vasovasostomized men.
The present study demonstrates for the first time that CRISPI is not detectable in vasectomized men, that CRISPI expression is restricted to the epididymis only and that it is not synthesized by accessory glands of the male reproductive. The presence of CRISPI can thus be considered as a good marker of excurrent duct permeability and can be used in the clinic to discriminate between secretory and obstructive azoospermy.
Headings are included herein for reference purposes and to aid in locating certain sections These headings are not intended to limit the scope of the concepts described thereinunder, and these concepts may have applicability in other sections throughout the entire specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.
Effect of vasectomy on CRISPI associated with spermatozoa Western blot analyses were performed on protein extracted from a constant number of spermatozoa from different men. CRISPI was detected at -32kDa. The commercially available antibody was specific to CRISP1 as shown by the absence of immunodetectable band revealed when Western blots were probed with a control serum (data not shown).
When expressed as a ratio of a constitutive protein TUBA, the quantity of CRISPI
associated with a constant number of ejaculated spermatozoa was significantly higher in vasovasostomized men when compared to normal donors (Figure 3) with a P>0.05.
CRISPI was associated with the acrosomal region of ejaculated spermatozoa from both normal and vasovasostomized men. The immune detection signal varied significantly from one sperm cell to the other in both normal and vasovasostomy samples. By opposition to Western blot analyses; it was not possible to quantitate the amount of CRISP1 immunodetected on smears of spermatozoa (Figure 4). The preimmune serum did not stain the acrosomal region but the equatorial segment was unspecifically stained for an unknown reason (Figure 4).
Vasectomy effect on CRISPI in seminal plasma CRISPI was also found as a soluble form in the seminal plasma. When Western blots were performed on 20 g of seminal plasma proteins, CRISPI was detectable in semen samples of both normal and vasovasostomized men. As for CRISPI associated to spermatozoa, CRISPI was significantly (P>0.05) more concentrated in seminal plasma of vasovasostomized men when compared to samples from normal men. Interestingly, was undetectable in seminal plasma of vasectomized men (Figure 5). This correlated well with the fact that CRISPI was undetectable on Western blots of 20 g of protein extracted from human seminal vesicles and prostate tissues (Figure 6).
Conclusions As shown herein, in human semen, CRISPI is found both associated to sperm and in the soluble fraction. Both forms are in higher concentrations in vasovasostomized men if the vas Our ref.: 273859.90 deferens were repermeabilized. CRISPI remains localized to the acrosomal region of spermatozoa in both normal and vasovasostomized. This suggests that the association of this epididymal protein to spermatozoa is not a simple coating phenomenon and that the specificity of the interaction remains even though CRISP1 is secreted in excess in some vasovasostomized men.
The present study demonstrates for the first time that CRISPI is not detectable in vasectomized men, that CRISPI expression is restricted to the epididymis only and that it is not synthesized by accessory glands of the male reproductive. The presence of CRISPI can thus be considered as a good marker of excurrent duct permeability and can be used in the clinic to discriminate between secretory and obstructive azoospermy.
Headings are included herein for reference purposes and to aid in locating certain sections These headings are not intended to limit the scope of the concepts described thereinunder, and these concepts may have applicability in other sections throughout the entire specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.
Claims (18)
1. A method for assessing the presence of epididymal secretions in a human semen sample, comprising: detecting in said semen sample presence or absence of human cysteine-rich secretory protein (CRISP1); whereby absence of CRISP1 is indicative of an absence of epididymal secretions in the semen sample.
2. The method of claim 1, wherein the semen sample is from a patient who has been subjected to vasectomy or to vasovasostomy.
3. The method of claim 2, wherein absence of CRISP1 is indicative of a successful vasectomy.
4. The method of claim 2, wherein presence of CRISP1 is indicative of a successful vasovasostomy.
5. The method of claim 1, wherein said semen sample has no spermatozoa.
6. The method of claim 5, wherein said semen sample is from an azoospermic man.
7. The method of claim 6, wherein absence of CRISP1 is indicative of an obstructive azoospermia in said man.
8. The method of any one of claims 1 to 7, wherein CRISP1 comprises an amino acid sequence as set forth in SEQ ID NO: 1.
9. The method of any one of claims 1 to 8, wherein said detection comprises using an antibody binding specifically to human CRISP1.
10. A method for evaluating successfulness of vasectomy in a man, comprising:
- obtaining a semen sample from a vasectomized man; and - detecting in the semen sample the presence or absence of human cysteine-rich secretory protein (CRISP1);
whereby absence of CRISP1 is indicative of a successful vasectomy while presence of CRISP1 is indicative of an unsuccessful vasectomy.
- obtaining a semen sample from a vasectomized man; and - detecting in the semen sample the presence or absence of human cysteine-rich secretory protein (CRISP1);
whereby absence of CRISP1 is indicative of a successful vasectomy while presence of CRISP1 is indicative of an unsuccessful vasectomy.
11. A method for evaluating successfulness of vasovasostomy in a man, comprising:
- obtaining a semen sample from a vasovasostomized man; and - detecting in the semen sample the presence or absence of human cysteine-rich secretory protein (CRISP1);
whereby presence of CRISP1 is indicative of a successful vasovasostomy while absence of CRISP1 is indicative of an unsuccessful vasovasostomy.
- obtaining a semen sample from a vasovasostomized man; and - detecting in the semen sample the presence or absence of human cysteine-rich secretory protein (CRISP1);
whereby presence of CRISP1 is indicative of a successful vasovasostomy while absence of CRISP1 is indicative of an unsuccessful vasovasostomy.
12. A method for discriminating between obstructive and non-obstructive azoospermia in man, comprising:
- obtaining an ejaculate of an azoospermic man; and - detecting in the ejaculate the presence or absence of human cysteine-rich secretory protein (CRISP1);
whereby presence of CRISP1 is indicative of a non-obstructive azoospermia condition while absence of CRISP1 is indicative of an obstructive azoospermia condition.
- obtaining an ejaculate of an azoospermic man; and - detecting in the ejaculate the presence or absence of human cysteine-rich secretory protein (CRISP1);
whereby presence of CRISP1 is indicative of a non-obstructive azoospermia condition while absence of CRISP1 is indicative of an obstructive azoospermia condition.
13 The method of any one of claims 10 to 12, wherein CRISP1 comprises an amino acid sequence as set forth in SEQ ID NO: 1.
14. The method of any one of claims 10 to 13, wherein said detection comprises using an antibody specific for human CRISP1.
15. A kit for evaluating successfulness of vasectomy and/or for evaluating successfulness of vasovasostomy in men, comprising: a user manual or instructions and at least one detection component for detecting in a human ejaculate the presence or absence of human cysteine-rich secretory protein (CRISP1).
16. A kit for discriminating between obstructive and non-obstructive azoospermia in men comprising: a user manual or instructions and at least one detection component for detecting in a human ejaculate the presence or absence of human cysteine-rich secretory protein (CRISP1).
17. The kit of claim 15 or 16, wherein said at least one detection component comprises an antibody binding specifically to CRISP1.
18. The kit of any one of claims 15 to 17, wherein said at least one detection component is found on a strip or a dip stick.
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| US25763909P | 2009-11-03 | 2009-11-03 | |
| US61/257,639 | 2009-11-03 |
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| CA (1) | CA2693546A1 (en) |
Cited By (27)
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| CN103257242A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | Application of CRISP (cysteine-rich secretory protein)1 kit for diagnosing carcinoma of buccal mucosa |
| CN103257238A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | Application of CRISP (cysteine-rich secretory protein)1 test paper for diagnosing carcinoma of maxillary sinus |
| CN103257228A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | Novel application of CRISP1 (cysteine-rich secretory protein 1) kit for diagnosis on carcinoma of palate |
| CN103257241A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | New application of CRISP (cysteine-rich secretory protein)1 to medical instrument |
| CN103257240A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | Application of CRISP (cysteine-rich secretory protein)1 kit for diagnosing carcinoma of tongue |
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| CN103267860A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing kit for diagnosing jawbone carcinoma |
| CN103267856A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of kit for diagnosing jaw cancer |
| CN103267862A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing gold label kit for diagnosing maxillary sinus carcinoma |
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| CN103267858A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing gold label test paper for diagnosing salivary gland carcinoma |
| CN103267863A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | CRISP1 (cystein-rich secretory protein 1) test paper for quickly diagnosing carcinoma of maxillary sinus |
| CN103278629A (en) * | 2013-05-22 | 2013-09-04 | 苏州市马尔泰新材料有限公司 | Gold-labelled kit for quickly diagnosing oropharynx carcinoma |
| CN103278645A (en) * | 2013-05-22 | 2013-09-04 | 苏州市马尔泰新材料有限公司 | Application of gold-labelled test strip for quickly diagnosing carcinoma of lip |
| CN103278644A (en) * | 2013-05-22 | 2013-09-04 | 苏州市马尔泰新材料有限公司 | Gold-labelled CRISP (cysteine-rich secretory protein)1 kit for quickly diagnosing carcinoma of palate |
| CN103278646A (en) * | 2013-05-22 | 2013-09-04 | 苏州市马尔泰新材料有限公司 | Application of gold-labelled cysteine-rich secretory protein (CRISP)1 kit for diagnosing mouth floor carcinoma |
| CN103293319A (en) * | 2013-05-22 | 2013-09-11 | 苏州市马尔泰新材料有限公司 | Gold-labeled test paper for rapidly diagnosing carcinoma jaw |
| CN103293320A (en) * | 2013-05-22 | 2013-09-11 | 苏州市马尔泰新材料有限公司 | Application of cysteine-rich secretory protein 1 (CRISP 1) in preparation of gingival cancer diagnosis products |
| CN103293296A (en) * | 2013-05-22 | 2013-09-11 | 苏州市马尔泰新材料有限公司 | Application of cysteine-rich secretory protein 1 (CRISP 1) in preparation of kit for diagnosing carcinoma of floor of mouth |
| CN103308682A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Kit for rapid lip cancer diagnosis |
| CN103308693A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Application of secretory protein 1 rich in cysteine in medical apparatus |
| CN103308691A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Application of secretory protein 1 rich in cysteine in preparation of product for diagnosing cheilocarcinoma |
| CN103308695A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Cysteine-rich secretory protein 1 (CRISP1) kit for cheek carcinoma diagnosis |
| CN103308692A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | CRISP1 (Cysteine-Rich Secretory Protein 1) gold-labeled kit for rapid oral floor carcinoma diagnosis |
| CN103308694A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Gold-labeled kit for salivary gland carcinoma diagnosis |
-
2010
- 2010-02-18 CA CA 2693546 patent/CA2693546A1/en not_active Abandoned
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| CN103257242A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | Application of CRISP (cysteine-rich secretory protein)1 kit for diagnosing carcinoma of buccal mucosa |
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| CN103257228A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | Novel application of CRISP1 (cysteine-rich secretory protein 1) kit for diagnosis on carcinoma of palate |
| CN103257241A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | New application of CRISP (cysteine-rich secretory protein)1 to medical instrument |
| CN103257240A (en) * | 2013-05-22 | 2013-08-21 | 苏州市马尔泰新材料有限公司 | Application of CRISP (cysteine-rich secretory protein)1 kit for diagnosing carcinoma of tongue |
| CN103267859A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of CRISP1 (cystein-rich secretory protein 1) test paper for diagnosing gingival carcinoma |
| CN103267848A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | CRISP1 (cystein-rich secretory protein 1) kit for quickly diagnosing gingival carcinoma |
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| CN103267856A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of kit for diagnosing jaw cancer |
| CN103267862A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing gold label kit for diagnosing maxillary sinus carcinoma |
| CN103267857A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | CRISP1 (cystein-rich secretory protein 1) test paper for diagnosing tongue cancer |
| CN103267861A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of cysteine-rich secretory protein 1 in manufacturing product for diagnosing palate carcinoma |
| CN103267858A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | Application of CRISP1 (Cysteine-Rich Secretory Protein 1) in preparing gold label test paper for diagnosing salivary gland carcinoma |
| CN103267863A (en) * | 2013-05-22 | 2013-08-28 | 苏州市马尔泰新材料有限公司 | CRISP1 (cystein-rich secretory protein 1) test paper for quickly diagnosing carcinoma of maxillary sinus |
| CN103278629A (en) * | 2013-05-22 | 2013-09-04 | 苏州市马尔泰新材料有限公司 | Gold-labelled kit for quickly diagnosing oropharynx carcinoma |
| CN103278645A (en) * | 2013-05-22 | 2013-09-04 | 苏州市马尔泰新材料有限公司 | Application of gold-labelled test strip for quickly diagnosing carcinoma of lip |
| CN103278644A (en) * | 2013-05-22 | 2013-09-04 | 苏州市马尔泰新材料有限公司 | Gold-labelled CRISP (cysteine-rich secretory protein)1 kit for quickly diagnosing carcinoma of palate |
| CN103278646A (en) * | 2013-05-22 | 2013-09-04 | 苏州市马尔泰新材料有限公司 | Application of gold-labelled cysteine-rich secretory protein (CRISP)1 kit for diagnosing mouth floor carcinoma |
| CN103293319A (en) * | 2013-05-22 | 2013-09-11 | 苏州市马尔泰新材料有限公司 | Gold-labeled test paper for rapidly diagnosing carcinoma jaw |
| CN103293320A (en) * | 2013-05-22 | 2013-09-11 | 苏州市马尔泰新材料有限公司 | Application of cysteine-rich secretory protein 1 (CRISP 1) in preparation of gingival cancer diagnosis products |
| CN103293296A (en) * | 2013-05-22 | 2013-09-11 | 苏州市马尔泰新材料有限公司 | Application of cysteine-rich secretory protein 1 (CRISP 1) in preparation of kit for diagnosing carcinoma of floor of mouth |
| CN103308682A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Kit for rapid lip cancer diagnosis |
| CN103308693A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Application of secretory protein 1 rich in cysteine in medical apparatus |
| CN103308691A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Application of secretory protein 1 rich in cysteine in preparation of product for diagnosing cheilocarcinoma |
| CN103308695A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Cysteine-rich secretory protein 1 (CRISP1) kit for cheek carcinoma diagnosis |
| CN103308692A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | CRISP1 (Cysteine-Rich Secretory Protein 1) gold-labeled kit for rapid oral floor carcinoma diagnosis |
| CN103308694A (en) * | 2013-05-22 | 2013-09-18 | 苏州市马尔泰新材料有限公司 | Gold-labeled kit for salivary gland carcinoma diagnosis |
| CN103257238B (en) * | 2013-05-22 | 2015-01-28 | 上海市第十人民医院 | Application of CRISP (cysteine-rich secretory protein)1 test paper for diagnosing carcinoma of maxillary sinus |
| CN103257240B (en) * | 2013-05-22 | 2015-03-25 | 上海市第十人民医院 | Application of CRISP (cysteine-rich secretory protein)1 kit for diagnosing carcinoma of tongue |
| CN103308691B (en) * | 2013-05-22 | 2015-04-29 | 上海市第十人民医院 | Application of secretory protein 1 rich in cysteine in preparation of product for diagnosing cheilocarcinoma |
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