EP3891507A1 - Marqueur pour la détermination de la fertilité de spermatozoïdes - Google Patents
Marqueur pour la détermination de la fertilité de spermatozoïdesInfo
- Publication number
- EP3891507A1 EP3891507A1 EP19813045.2A EP19813045A EP3891507A1 EP 3891507 A1 EP3891507 A1 EP 3891507A1 EP 19813045 A EP19813045 A EP 19813045A EP 3891507 A1 EP3891507 A1 EP 3891507A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sample
- mxi
- spermatozoa
- fertility
- per
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction
Definitions
- the present invention relates to a method for determining the fertility of spermatozoa comprising detecting mitogen-activated protein kinase 14 isoform 3 (Mxi-2).
- the method may be a method for determining the fertility of spermatozoa contained in a sample S, said method comprising detecting the total content of the Mxi-2 in the sample S.
- the present invention refers to a dipstick usable for this method.
- the present invention relates to further methods and uses in the context of the present invention.
- sperm abnormalities can be caused by different factors like e.g. inflammation of the testis, varicoceles (swollen veins in the scrotum), abnormally developed testis, genetic disorders, hormone problems.
- the insufficient male fertility may be considered as a pathologic condition such as a condition according to any of classes N46 or R86 of the 10 th revision of the International Statistical Classification of Diseases and Related Health Problems of the World Health Organization (WHO) in the version of 2016 (ICD-10).
- WHO World Health Organization
- ejaculates can be divided into several different groups:
- normozoospermia normal ejaculate (volume >2.0 ml, concentration >20x10 6 /ml, Motility >50% Morphology >30% with normal morphology)
- oligozoospermia oligospermia: sperm concentration fewer than 20x10 6 /ml;
- asthenozoospermia asthenozoospermia: fewer than 50% spermatozoa with forward progression or fewer than 25% spermatozoa with no movement;
- teratozoospermia (teratospermia): fewer than 30% spermatozoa with
- oligo-astheno-teratozoospermia signifies disturbance of all three variables.
- azoospermia (aspermia): (essentially) no spermatozoa in the ejaculate.
- Male infertility usually occurs because of sperms with abnormal shape, the sperm quality is not high enough or a problem with the ejaculation.
- exemplary such pathological condition may be oligozoospermia, asthenozoospermia, teratozoospermia or a combination of more than one of these conditions.
- Spermatozoa of particularly low fertility may be obtained from individuals suffering from oligo-astheno-teratozoospermia (OAT syndrome).
- a decreased level fertility of the spermatozoa plays a significant role when considering means for considering further therapeutic or non- medicinal steps.
- unintendedly childless couples are often interested to know whether the infertility is caused by the male and/or female body in order to consider suitable treatments or sperm or egg donation.
- one of the factors of particular interest for considering further steps is the determination of the fertility of spermatozoa.
- the determination of the fertility of spermatozoa is also of interest for sperm banks in order to sort sperm donations of low fertility out.
- an assessment of the fertility of spermatozoa also plays a significant role when breeding animals.
- Today the vast majority of larger sized farm animals, such as, e.g., bovines, is bred by means of artificial insemination of the female animals.
- numerous aliquots of sperm donations are provided to the farmers.
- farmers are on the one hand interested in good breeding success and on the other hand in well-fertile progeny.
- an assessment of the fertility of spermatozoa of the animals to be protected is of considerable interest, e.g., when zoos breed endangered species.
- the fertility of spermatozoa is mostly assessed by means of obtaining a fresh sperm donation and observing concentration and mobility of the spermatozoa comprised therein under a microscope. A lower sperm concentration or mobility is frequently correlated with a lower fertility.
- spermatozoa Like all body cells, also spermatozoa comprise a cell-type-typical proteome, i.e. , various molecular structures typical for this specific cell type, not excluding that other cell types may comprise partly the same molecular structures.
- Several molecular structures are known as biomarkers somewhat associated with fertility of spermatozoa. Almog et al. (Journal of Biol. Chem., 2008, 283:14479-14490) teaches that increased expression of ERK1 and phosphorylated p38 mitogen-activated protein kinase (MAPK) may indicate poor human sperm quality. Almog et al.
- WO 2018/185322 described a method for determining the fertility of spermatozoa contained in a sample S, said method comprising detecting the total content of the Vimentin variant 3 (Vim3) per spermatozoon or per sample volume and/or the spatial localization of the Vim3 within the stained spermatozoa.
- Vim3 Vimentin variant 3
- a decreased level of Vim3 typically indicates decreased fertility.
- Vim3 herein serves as a positive fertility marker.
- infertility marker a marker that indicates decreased fertility when it its level is increased
- Mxi-2 may serve as an infertility marker. Detection of Mxi-2 as biomarker provides statistically well-defined results and even enables distinguishing different types of spermatozoa aberrations from each other.
- An aspect of the present invention relates to a method for determining the fertility of spermatozoa comprising detecting mitogen-activated protein kinase 14 isoform 3 (Mxi-2).
- the method of the present invention is a method for determining the fertility of one or more spermatozoa contained in a sample S, said method comprising detecting the Mxi-2 in the sample S.
- the method comprises detecting the total content of the Mxi-2 in the sample S.
- the present invention also relates to a method for determining the fertility of spermatozoa contained in a sample S, said method comprising detecting the total content of the mitogen-activated protein kinase 14 isoform 3 (Mxi-2) in the sample, preferably per spermatozoon or per sample.
- Mxi-2 mitogen-activated protein kinase 14 isoform 3
- the spatial localization of Mxi-2 within the stained spermatozoa may also be detected.
- the method of the present invention may also be combined with the method of detecting Vimentin variant 3 (Vim3) as described in WO 2018/185322. Both methods may optionally also be conducted as a differential diagnosis.
- the method for determining the fertility of spermatozoa contained in a sample S may comprise detecting the total content of Mxi-2 and Vim3 per spermatozoon or per sample volume.
- the spatial localization of Vim3 and/ Mxi-2 within the stained spermatozoa may optionally also be detected.
- An increase in the Mxi-2:Vim3 ratio may indicate decreased fertility.
- the method is an in vitro method. Detecting may be understood in the broadest sense and may be conducted by any means. As described in more detail below, there are various means for detecting available in the art.
- detecting may be conducted by microscopic means such as, e.g., by microscopic imaging of spermatozoa in a sample S in which Mxi-2 (and optionally additionally Vim3) may be stained.
- the method comprises the following steps:
- detecting the total content of the Mxi-2 per spermatozoon or per sample volume may also be replaced by or supplemented by detecting the spatial localization of the Mxi-2 within the stained spermatozoa.
- the step of determining the total content of Mxi-2 per spermatozoon or per sample volume may be replaced by or supplemented by determining the degree of accumulation of Mxi-2 in the head or neck region of the spermatozoa.
- herein accumulation of Mxi-2 in the head or neck region of the spermatozoa may indicate decreased fertility. This is also exemplified in Figure 8 herein.
- the method comprises detecting the spatial localization of the Mxi-2 within one or more, optionally stained, spermatozoa.
- the method comprises determining the degree of accumulation of Mxi-2 in the head or neck region of the spermatozoa. In a preferred embodiment, an accumulation of Mxi-2 in the head or neck region of the spermatozoa indicates decreased fertility. In a preferred embodiment, an accumulation of Mxi-2 in the head or neck region of the spermatozoa indicates decreased fertility, wherein:
- the method may additionally comprise the following steps:
- step (iii’) detecting the total content of the Vim3 per spermatozoon or per sample volume and/or the spatial localization of the Vim3 within the stained spermatozoa, wherein the Vim3 is optionally stained Vim3 of step (ii’); and (iv’) determining the total content of Vim3 per spermatozoon or per sample volume and/or the degree of accumulation of Vim3 in the mid piece, more in particular the neck region of the spermatozoa.
- an increased content of the total content of Mxi-2 per spermatozoon or per sample volume contained in the sample S may indicate decreased fertility of the spermatozoa contained in the sample S.
- These contents may optionally be compared with one or more healthy spermatozoa (normozoospermia) and/or one or more control samples C+ of spermatozoa of high fertility of the same species and/or one or more control sample C- of spermatozoa of low fertility of the same species.
- control sample should be understood in the broadest sense.
- One, two or more control samples can, optionally, be measured in the same test series as one or more samples S.
- the control samples do, however, not have to be measured in the same test series.
- the values of the one or both control samples C+ and/or C- may also be already known values that may be, e.g., stored in and obtained from a data base.
- control sample C+ is a sample containing healthy spermatozoa (normozoospermia) from one or more ejaculates (or punctates or biopsies) obtained from one or more donors.
- control sample C- is a sample containing non-healty spermatozoa (oligo-astheno- teratozoospermia (OAT syndrome), teratozoospermia, azoospermia, in particular OAT syndrome).
- a punctate or a biopsy may be a such containing seminal fluid or a precursor thereof).
- the control sample C+ may also be an internal control. For instance, it may reflect the mean value of a set of samples mainly containing healthy spermatozoa (normozoospermia). Using such internal control may be of particular interest when the method of the present invention is conducted as high-throughput method and/or when using one or more multiwell plates (e.g., 6-well plates, 12-well plates, 96-well plates or 384-well plates).
- determining total content of Mxi-2 per spermatozoon or per sample volume may be conducted by various means. For example, this step may be conducted by means of microscopy, optionally combined with staining Mxi-2 (and optionally Vim3), but also by means of mass spectrometry and/or immunochemical means, etc., optionally combined with staining Mxi-2 (and optionally Vim3).
- the present invention relates to a method for determining the fertility of spermatozoa contained in a sample S, said method comprising the following steps:
- step (iii) detecting the total content of the Mxi-2 per spermatozoon or per sample volume, wherein the Mxi-2 is optionally stained Mxi-2 of step (ii);
- • a content of accumulation of Mxi-2 in the tail region of the spermatozoa contained in the sample S that is not higher than in the control sample C-, may indicate decreased fertility of the spermatozoa contained in the sample S.
- the method of the present invention additionally comprises the following steps:
- step (iii’) detecting the total content of the Vim3 per spermatozoon or per sample volume and/or the spatial localization of the Vim3 within the stained spermatozoa, wherein the Vim3 is optionally stained Vim3 of step (ii’);
- step (v’) comparing the total content of Vim3 per spermatozoon or per sample volume and/or the degree of accumulation of Vim3 determined in step (iv’) between the sample S and
- the method of the present invention may also comprise determining the degree of colocalization of Vim3 and mitochondria.
- an increased content of the total content of Mxi-2 (and optionally additionally a decreased content of the total content of Vim3) per spermatozoon or per sample volume contained in the sample S in comparison to the (essentially constant) control sample C+ and/or C- preferably indicates decreased fertility of the spermatozoa contained in the sample S.
- an increase in the Mxi-2:Vim3 ratio may indicate decreased fertility.
- the present invention may also comprise the further step of calculating a Mxi-2:Vim3 ratio, wherein an increase in the Mxi-2:Vim3 ratio may indicate decreased fertility.
- the present invention may also comprise the further steps of:
- control samples C+ and C- may serve as reference point(s) when the fertility of such sample(s) is known.
- the person skilled in the art will notice that the method of the present invention preferably is an in vitro method, i.e. , preferably a method not directly associated with the diagnosis of the human or animal body. The results may be used for medicinal or non-medicinal purposes.
- the sample S typically is an in vitro specimen, i.e., a specimen remote from the human and animal body.
- comparison is comparison between the sample S and at least one control sample C+ of spermatozoa of high fertility of the same species (option (a)).
- the method of the present invention may be conducted in that it comprises steps (i) and (ii) as above followed by steps (iii)-(v):
- Mxi-2 protein could be easily and predominantly detected in sperm of decreased fertility e.g. by immune staining (e.g., immunofluorescence), flow cytometry (also designated as fluorescence activated cell sorting (FACS) analysis, herein understood interchangeably), making the detection of Mxi-2 a specific marker for ejaculates (or punctates or biopsies) from man with decreased fertility.
- Mxi-2 protein was identified in sperms with different morphological aspects (i.e. normozoospermia, teratozoospermia, OAT syndrome, azoospermia).
- FACS fluorescence activated cell sorting
- Determining the fertility of spermatozoa may be understood in the broadest sense as any assessment of the degree of suitability of the spermatozoa for sexual reproduction by natural or artificial insemination. Accordingly, the degree of fertility also includes the mobility and vitality of the spermatozoa.
- the determination of spermatozoa as being infertile or less fertile does not exclude sexual reproductive capability by fusing a spermatozoon with an ovule artificially in vitro, in particular by injecting the genetic material into the ovule ⁇ in vitro fertilization in a test tube) because in such process no mobility and viability of the latter is required.
- Mxi-2 was found (significantly) lower in ejaculates (or punctates or biopsies) from patients with normozoospermia (control sample C+), whereas in ejaculates (or punctates or biopsies) from patients with oligo-astheno- teratozoospermia (OAT syndrome) and azoospermia the expression of Mxi-2 was significantly increased.
- spermatozoon “sperm”,“spermatozoon cell”,“sperm cell” and the like may be understood interchangeably in the common sense in the art, i.e. , as a, preferably matured, male gamete.
- spermatozoa may also be obtained from a swim-up (sperm) procedure (also: swim-up test).
- sperm swim-up procedure
- the fertile sperms survive and swim up.
- the upper fraction containing the fertile spermatozoa is investigated further.
- the control sample C- of spermatozoa of low fertility may preferably obtained from a patient of the same species suffering from oligozoospermia, asthenozoospermia, teratozoospermia, and/or oligo-astheno-teratozoospermia (OAT syndrome) and/or azoospermia and/or any other pathologic state accompanied by low fertility of the spermatozoa, in particular oligozoospermia, asthenozoospermia, teratozoospermia, and/or OAT syndrome. As indicated above, it may, however, also reflect a value determined before.
- a total content per spermatozoon may be understood in the broadest sense as the content of the respective polypeptide (e.g., Mxi-2 (and optionally additionally Vim3)) in the cells. It will be understood that typically not the content of polypeptide comprised in one spermatozoon is determined, but typically the average of a larger number of spermatozoa. As it is well-known that all spermatozoa of a sample S will generally each have (essentially) the same weight, the determination of the total content per spermatozoon is equivalent to the total content per spermatozoon weight.
- the reference “per spermatozoon” or“per spermatozoa weight” or the like normalizes different samples (e.g., samples S and C+ and/or C-) and makes them comparable with another, independent on the total number or concentration of spermatozoa comprised in the respective sample.
- samples S and C+ and/or C- e.g., samples S and C+ and/or C-
- the content of Mxi-2 (and optionally additionally Vim3) per milligram of the sample S can be determined, when the spermatozoa content is comparable in the different samples (e.g., samples S and C+ and/or C-) to be compared with another.
- the content of Mxi-2 (and optionally additionally Vim3) can be determined per volume and/or weight of ejaculate (or punctates or biopsies).
- a total content per sample volume may be understood in the broadest sense as content of the respective protein (e.g., Mxi-2 and optionally additionally Vim3) or intensity arising therefrom per volume.
- the volumes of samples S e.g., ejaculates, punctates or biopsies)
- the content of Mxi-2 (and optionally additionally Vim3) is preferably referred to the sample volume.
- azoospermia samples may provide comparably high contents of Mxi-2 which may be to cell fragments that may be contained in the sample S.
- the content e.g., Mxi-2 content
- ratios e.g., in comparison to a control sample C+ or C-
- the content may also be a relative dimension referring to signal intensity. This can be recalculated when compared to known intensities such as, e.g, by means of a dilution series of the respective protein of interest (e.g. Mxi-2) or a housekeeping protein that is stained in a comparable way.
- known intensities such as, e.g, by means of a dilution series of the respective protein of interest (e.g. Mxi-2) or a housekeeping protein that is stained in a comparable way.
- the content is defined as signal intensity per sample volume.
- the content may also be defined as mass content (e.g., in micrograms (pg), nanograms (ng) or picograms (pg)) or a mol content (e.g., micromol (pmol), nanomol (nmol), picomol (pmol) or femtomol (fmol)).
- mass content e.g., in micrograms (pg), nanograms (ng) or picograms (pg)
- a mol content e.g., micromol (pmol), nanomol (nmol), picomol (pmol) or femtomol (fmol)
- a mol content e.g., micromol (pmol), nanomol (nmol), picomol (pmol) or femtomol (fmol)
- a certain amount per spermatozoon per 10 5 , 10 6 , 10 7 , 10 8 , 10 10 spermatozoa or per sample volume e.g., in milliliters (ml)
- the content is defined as ng per (10 6 ) spermatozoa.
- the content when the content is referred to a volume, it may also be referred to a concentration content (e.g., in pg/ml, ng/ml, pg/ml, or a molar content (micromolar (mM), nanomolar (nM), picomolar (pM) or femtomolar (fM)).
- concentration content e.g., in pg/ml, ng/ml, pg/ml, or a molar content (micromolar (mM), nanomolar (nM), picomolar (pM) or femtomolar (fM)
- mM micromolar
- nM nanomolar
- pM picomolar
- fM femtomolar
- an increased content may be understood in the broadest sense as higher degree. Typically, an increased content is a degree that is at least 10%, at least 20%, at least 50%, at least 75%, at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 -fold, 8-fold, 9-fold or 10-fold higher than the value it is compared with (e.g., that of the control sample C+).
- control sample C- may be understood in the broadest sense as not being significantly lower, i.e. , not 10% lower, in particular equal or higher than the value it is compared with (e.g., that of the control sample C-).
- the increased content may refer to the increase of the intensity of the fluorescence signal (detectable under the same measurement conditions).
- fluorescence microscopy the increased content may refer to the increase of the intensity of the local fluorescence signal (detectable under the same measurement conditions for the head or neck or tail regains of the spermatozoa).
- flow cytometry also designated as fluorescence activated cell sorting (FACS), herein understood interchangeably
- FACS fluorescence activated cell sorting
- Analysis via flow cytometry may include washing the sperm cells (e.g. with a buffer, optionally combined with centrifugation) and staining cells with a Mxi-2- and/or a Vim3-specific antibody.
- a Mxi-2- and/or a Vim3-specific antibody may be used for this purpose.
- an unlabeled primary Mxi-2- and/or a Vim3-specific antibody may be bound to the sperm cells. These may be optionally washed (e.g. with a buffer, optionally combined with centrifugation).
- a secondary, preferably fluorescently labelled, antibody may be applied which specifically binds to the unlabeled Mxi-2- and/or a Vim3-specific antibody.
- the sperm cells may be optionally washed again (e.g. with a buffer, optionally combined with centrifugation).
- the stained sperm cells may be analyzed by means of flow cytometry. A higher signal intensity of a sperm cell indicates a higher content of the respective biomarker (e.g., Mxi-2 or Vim3) in said sperm cells.
- a shift to higher mean signal intensity indicates a higher mean content of the respective biomarker (e.g., Mxi-2 or Vim3) in the respective sample of sperm cells.
- the aliquot of the sample S containing the spermatozoa may be any specimen comprising one or more spermatozoa.
- the aliquot may comprise the whole sample S or may be, preferably, a part thereof.
- the spermatozoa may be viable or may be fixed, i.e. , dead.
- the sample S may be liquid, pasty or solid. It may be tissue sample, a solid sample, a liquid sample, a cell sample, tissue section etc. Suitable methods for obtaining a sample are known in the art and include the masturbation, a testis biopsy, or other common methods used in the art.
- a sample S comprising viable spermatozoa will typically be liquid or pasty and will (essentially) consist of spermatozoa and ingredients not harmful to the spermatozoa in the present concentrations.
- a fixed sample S may optionally also be solid.
- the sample S is placed on a specimen carrier, preferably a transparent specimen carrier, in particular a microscopic slide or a well of a multiwell plate.
- the sample S may be an ejaculate, a punctate or a biopsy or a processed ejaculate, punctate or biopsy or an aliquot thereof.
- a processed ejaculate may optionally be diluted in an aqueous buffer and/or in an organic liquid.
- the sample S may be a testis biopsy (e.g., obtained in the context of testicular sperm extraction (TESE)).
- TSE testicular sperm extraction
- This may exemplarily be stained (e.g., by means of a Mxi-2-specific antibody or antibody fragment that is labelled (e.g., by a fluorescence and/or color dye or a metal particle (e.g., a gold bead)) or an unlabeled Mxi-specific antibody or antibody fragment that is bound by a labeled secondary antibody).
- the method of the present invention may be an immunohistological method.
- the experimenter may compare the signal intensity found (in the relevant parts containing spermatozoa and precursors thereof) of the testis biopsy of a patient of interest with a comparable testis sample of a well- fertile individual of the same species. Such staining also allows the localization of Mxi in situ. In principle, the use of a radioactively labeled Mxi -specific antibody or antibody fragment may be even used in vivo to localize the Mxi-2 within the body.
- Mxi-2 is the naturally occurring Mxi-2 of the species of the individual of interest, i.e. , the Mxi-2 occurring in the respective sample S.
- Mxi-2 may be human Mxi-2 or Mxi-2 of a non-human animal, in particular a domestic mammal such as, e.g., a bovine, a pig, a horse, a donkey, a sheep, a goat, a dog, a cat, etc.).
- Mxi-2 is human Mxi-2.
- Mxi-2 has a homology of at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99% of SEQ ID NO: 3, in particular is identical with SEQ ID NO: 3:
- Mxi-2 is mammalian non-human Mxi-2 such as, e.g., bovine Mxi-2, pig Mxi-2, horse Mxi-2, donkey Mxi-2, sheep Mxi-2, goat Mxi-2, dog Mxi-2, or cat Mxi-2.
- Mxi-2 may be the gene expression product of Homo sapiens mitogen-activated protein kinase 14 (MAPK14, also MAPK p38 or p38 MAPK), transcript variant 3 (SEQ ID NO: 10):
- MAPK14 (MAPK p38, Mitogen- activated protein kinase 14) has a homology of at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99% or sequence identity of a polypeptide sequence of the UniProtKB database No. Q16539 (MK14_HUMAN).
- MAPK p38 has a homology of at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99% or sequence identity of SEQ ID NO: 11 :
- MAPK14 is a truncated version of MAPK14, e.g., truncated by one, two or more, up to ten, up to 20, up to 50 or up to 100 amino acid moieties or more than 100 amino acid moieties.
- MAPK14 is mammalian non-human MAPK14 such as, e.g., bovine MAPK14, pig MAPK14, horse MAPK14, donkey MAPK14, sheep MAPK14, goat MAPK14, dog MAPK14, or cat MAPK14.
- Vimentin variant 3 (also referred to as Vimentin3, Vimentin variant 3, Vimentin splice form 3) is a splice isoform of Vimentin.
- Vim3 in the context of the present invention may be any Vim3 compound.
- Vimentin itself is an intermediate sized filament that functions in signal transduction cellular function, structural integrity of cells and tissues and adhesion and migration.
- RSG Craig Venter Institute
- N01 -NV-48196 J. Craig Venter Institute, Rockville, MD 20850
- Vim3 is a spliced variant of Vimentin with a unique C-terminal ending.
- Vim3 is the naturally occurring Vim3 of the species of interest, i.e. , the Vim3 occurring in the spermatozoa comprised in the sample S.
- Vim3 may be human Vim3 or Vim3 of a non-human animal (e.g., a non-human mammal (e.g., an domestic mammal (e.g., a bovine, a pig, a horse, a donkey, a sheep, a goat, a dog, a cat, etc.) or another animal intended for propagation (e.g., an endangered species (e.g., a tiger, an elephant, etc.)).Vim3 has been found in numerous species so far.
- a non-human mammal e.g., an domestic mammal (e.g., a bovine, a pig, a horse, a donkey, a sheep, a goat, a dog, a cat, etc.)
- another animal intended for propagation e.g., an endangered species (e.g., a tiger, an elephant, etc.)
- Vim3 has been found in numerous species so far.
- Vim3 is human Vim3.
- the human splice variant Vim3 is has 431 amino acids and is 35 amino acids smaller than the full length protein. Its unique structure leads to a 10 kDa smaller protein.
- the amino acid sequence of human Vim3 has been published and is available at UniProt KB (http://www.uniprot.org/uniprot/B0YJC4) or at the National Center for Biotechnology Information under GenBank Accession number ACA06103.1 (http://www.ncbi.nlm.nih.gov/protein/167887751 ).
- Vim3 has a homology of at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99% of SEQ ID NO: 1 , in particular is identical with SEQ ID NO: 1 :
- Vim3 is mammalian non-human Vim3 such as, e.g., bovine Vim3, pig Vim3, horse Vim3, donkey Vim3, sheep Vim3, goat Vim3, dog Vim3, or cat Vim3.
- the Vimentin sequence of other species is also known, including e.g. Mus musculus (NCBI Accession: CAA39807.1 , NP_035831.2), Rattus norvegicus (NCBI Accession: NP_112402.1 ), Bos taurus (NCBI Accession: NP_776394.2), Gallus gallus (NCBI Accession: NP_001041541.1 ), Mesocricetus auratus (Accession: AAA37104.1 ),
- Oncorhynchus mykiss (Accession: CAA90601.1 ), Equus caballus
- NP_001230074.1 Salmo sa/ar (Accession: NP_001133947.1 ), Pan troglodytes (Accession: NP_001009148.1 ) and Cavia porcellus (Accession:
- NP_001166511.1 The splice variant corresponding to human Vim3 could be easily identified by sequence analysis and identification of homologues.
- the method of the present invention may be conducted immediately after obtaining the sample S (i.e. , within the first hour after the donor has ejaculated, and thus provided the sperm donation) or may be conducted later such as, e.g., between 1 and 5 hours, between 2 and 10 hours, between 5 and 24 hours, between 12 hours and 2 days, between 2 and 7 days, between 1 and 4 weeks, between 1 and 12 months or after more than 1 year.
- the method may be conducted in a sample S of fixed or viable spermatozoa.
- step (ii) of staining intracellular Mxi-2 comprises the fixation of the spermatozoa contained in the sample S prior to staining the intracellular Mxi-2 in the spermatozoa.
- Fixation may bear the technical advantage that the sample S may optionally be stored for longer still enabling sufficient readout. Optionally, then the sample S may also be stored in a dried state. Further, handling is considerably easier.
- compounds used for staining of (optional) step (ii) do not necessarily have to be cell-penetrating and non-toxic.
- the cell membranes may optionally also be rendered permeably for larger sized compounds such as, e.g., antibodies or antibody fragments.
- toxic agents may be used for staining without disturbing the results.
- Fixation may be performed by any means known in the art. Exemplarily, fixation may be performed by adding formaldehyde, methanol and/or ethanol to a solution comprising the spermatozoa of interest.
- Step (ii) of staining intracellular Mxi-2 may be understood in the broadest sense as labelling the intracellular Mxi-2 by any detectable means.
- detecting of step (iii) is determination of the localization of Mxi-2 in the spermatozoa. Therefore, detection is preferably detection by any imaging method (e.g., by microscopy). Alternatively, detection may also be detection of radioactivity.
- the Mxi-2 is preferably labeled. Therefore, Mxi- 2 is either detectably as such or, more preferably, is bound by a labeled marker.
- a labeled marker may be understood in the broadest sense as any compound specifically binding to Mxi-2 that is detectable by any means.
- a labeled marker comprises two moieties conjugates with another, i.e. , at least one binding moiety is conjugated with at least one label moiety. More preferably, a binding moiety and a label moiety are covalently conjugated with another, either directly of via a spacer.
- conjugated with may be understood in the broadest sense as any kind of covalent or non-covalent attachment or linkage of one component with another component, preferably via a covalent linkage. It is not limited to a specific kind of formation of a conjugate. Depending on the components conjugated with another, such conjugate can be obtained by chemical means and/or by genetic engineering and biotechnological means.
- a labeled marker may optionally comprise one or more binding moieties specifically binding to Mxi-2 and one or more label moieties.
- a labeled marker comprises one binding moiety specifically binding to Mxi-2 and one label moiety.
- the binding moiety is detectably by itself.
- the label moiety may be any moiety that is detectable, preferably detectable by an imaging method.
- step (ii) of staining intracellular Mxi-2 is staining with a fluorescently labeled marker.
- fluorescence labeled marker may be understood in the broadest sense as any compound specifically binding to Mxi-2 that is either fluorescent (by itself) or is conjugated with a fluorescence unit.
- a fluorescence marker comprises one or more binding moieties specifically binding to Mxi-2 and one or more fluorescent label moieties. More preferably, a fluorescently labeled marker comprises one binding moiety specifically binding to Mxi-2 and one fluorescence labeled moiety. More preferably, the binding moiety and the fluorescent label moiety are covalently conjugated with another, either directly of via a spacer. Alternatively, the binding moiety is fluorescent by itself.
- a fluorescent label moiety may be any fluorescent moiety known in the art.
- a fluorescent label moiety may be a fluorescent polypeptide moiety (e.g., cyan fluorescent protein (CFP), green fluorescent protein (GFP) or yellow fluorescent protein (YFP), red fluorescent protein (RFP), mCherry, etc.), a small-molecule dye moiety (e.g., an Atto dye moiety (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 520, ATTO 532, ATTO 550, ATTO 565, ATTO 590, ATTO 594, ATTO 610, ATTO 611X, ATTO 620, ATTO 633, ATTO 635, ATTO 637, ATTO 647, ATTO 647 N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), a Cy dye moiety (e.g.,
- VivoTag680, VivoTag750 an S dye (e.g., S0387), a DyLight fluorophore moiety (e.g., DyLight 750, DyLight 800), an IRDye moiety (e.g., IRDye 680, IRDye 800), a fluorescein dye moiety (e.g., fluorescein, carboxyfluorescein, fluorescein isothiocyanate (FITC)), a rhodamine dye moiety (e.g., rhodamine, tetramethylrhodamine (TAMRA)), a HOECHST dye moiety, a quantum dot moiety or a combination of two or more thereof.
- fluorescent label moiety may be used in fluorescence microscopy.
- the label moiety may be metal atom, metal ion or metal bead (e.g. , a (colloidal) gold such as a gold bead).
- metal bead may be used in electron microscopy.
- step (ii) of staining intracellular Mxi-2 comprises binding of a Mxi-2-specific antibody or antibody fragment, preferably a labeled Mxi- 2-specific antibody or antibody fragment, in particular a Mxi-2-specific antibody or antibody fragment labeled by a fluorescent label or a (colloidal) gold label.
- a Mxi-2-specific antibody or antibody fragment preferably a labeled Mxi- 2-specific antibody or antibody fragment, in particular a Mxi-2-specific antibody or antibody fragment labeled by a fluorescent label or a (colloidal) gold label.
- the label moiety may be radioactive label such as, e.g., 3 H, 14 C, 123 l, 124 l, 131 l, 32 P, 99m Tc or lanthanides (e.g., 64 Gd).
- a radioactive label may or may not be suitable for scintillation assays, computer tomography (CT), single-photon emission computed tomography (SPECT) or as a label suitable for Positron Emission Tomography (PET) (e.g., 11 C, 13 N, 15 0, 18 F, 82 Rb).
- the spermatozoa are optionally harvested, fragmented in their subunits and the subunits fragmented (e.g., by (ultra)centrifugation). Then, the content of radioactivity in the various fractions may be detected.
- detecting is performed by an imaging method.
- step (ii) of staining intracellular Mxi-2 may be staining with a fluorescently labeled marker and step (iii) is detecting the localization of the Mxi-2 conducted by fluorescence microscopy.
- the binding moiety (also: molecular or chemical entity or substance) may be any moiety specifically binding to Mxi-2.
- the binding moiety may be a high molecular weight compound of a molecular weight of 5 kDa or more or may be small molecule of a molecular weight of less than 5 kDa.
- the binding moiety is a high molecular weight compound of a molecular weight of 5 kDa or more, more preferably of more than 10 kDa, even more preferably of more than 20 kDa, even more preferably of more than 50 kDa and even more preferably of more than 100 kDa.
- the binding moiety may be a polypeptide, a polysaccharide or a synthetic agent (e.g., a small molecule drug) each optionally conjugated with a synthetic polymer (e.g., methacrylate (MA), hydroxypropyl methacrylate (HPMA), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol (PEG), polylysine (PL)) or a combination of two or more thereof.
- a synthetic polymer e.g., methacrylate (MA), hydroxypropyl methacrylate (HPMA), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol (PEG), polylysine (PL)
- the binding moiety is a polypeptide. More preferably, the binding moiety is an antibody or a mutant or fraction thereof.
- step (ii) of staining intracellular Mxi-2 comprises binding of a Mxi-2-specific antibody or antibody fragment, preferably a labeled Mxi- 2-specific antibody or antibody fragment, in particular a Mxi-2-specific antibody or antibody fragment labeled by a fluorescent label or a (colloidal) gold label.
- a Mxi-2-specific antibody or antibody fragment preferably a labeled Mxi- 2-specific antibody or antibody fragment, in particular a Mxi-2-specific antibody or antibody fragment labeled by a fluorescent label or a (colloidal) gold label.
- antibody may be understood in the broadest sense as any type of immunoglobulin or antigen binding fraction or mutant thereof known in the art.
- the antibody of the present invention may be an immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin Y (IgY) or immunoglobulin W (IgW).
- the antibody is an IgA, IgG or IgD. More preferably, the antibody is an IgG.
- the type of antibody may be altered by biotechnological means by cloning the gene encoding for the antigen-binding domains of the antibody of the present invention into a common gene construct encoding for any other antibody type.
- the binding between the antibody and its molecular target structure typically is a non-covalent binding.
- the binding affinity of the antibody to its antigen has a dissociation constant (Kd) of less than 1 mM, less than 500 nM, less than 200 nM, less than 100 nM, less than 50 nM, less than 40 nM, less than 30 nM or even less than 20 nM.
- antibody as used herein may be understood in the broadest sense and also includes what may be designated as an antibody mutant.
- an antibody mutant may be understood in the broadest sense as any antibody mimetic or antibody with altered sequence known in the art.
- the antibody mutant may have at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95% of the binding affinity of a corresponding antibody, i.e.
- Kd dissociation constant
- the term“antibody fragment” may be understood in the broadest sense as any fragment of an antibody that still bears binding affinity to its molecular target (i.e., its antigen, e.g., Mxi-2).
- the antibody fragment may be a fragment antigen binding (Fab fragment), Fc, F(ab') 2 , Fab', scFv, a truncated antibody comprising one or both complementarity determining region(s) (CDR(s)) or the variable fragment (Fv) of an antibody.
- Variable domains (Fvs) are the smallest fragments with an intact antigen-binding domain consisting of one V L and one V .
- Such fragments can be generated by enzymatic approaches or expression of the relevant gene fragments, e.g. in bacterial and eukaryotic cells.
- Different approaches can be used, e.g. either the Fv fragment alone or 'Fab'-fragments comprising one of the upper arms of the ⁇ ” that includes the Fv plus the first constant domains.
- These fragments are usually stabilized by introducing a polypeptide link between the two chains which results in the production of a single chain Fv (scFv).
- dsFv disulfide-linked Fv
- the binding domains of fragments can be combined with any constant domain in order to produce full length antibodies or can be fused with other proteins and polypeptides.
- a recombinant antibody fragment is the single chain Fv (scFv) fragment. Dissociation of scFvs results in monomeric scFvs, which can be complexed into dimers (diabodies), trimers (triabodies) or larger aggregates such as TandAbs and Flexibodies.
- the antibody may be a Fab, a Fab', a F(ab')2, a Fv, a disulfide-linked Fv, a scFv, a (scFv) 2 , a bivalent antibody, a bispecific antibody, a multispecific antibody, a diabody, a triabody, a tetrabody or a minibody.
- antibody may also include an antibody mimetic which may be understood in the broadest sense as organic compounds that, like antibodies, can specifically bind antigens and that typically have a molecular mass in a range of from approximately 3 kDa to approximately 25 kDa.
- Antibody mimetics may be, e.g., affibody molecules (affibodies), affilins, affitins, anticalins, avimers, DARPins, Fynomers, Kunitz domain peptides, single-domain antibodies (e.g., VHH antibodies or VNAR antibodies, nanobodies), monobodies, diabodies, triabodies, flexibodies and tandabs.
- the antibody mimetics may be of natural origin, of gene technologic origin and/or of synthetical origin.
- the antibody mimetics may also include polynucleotide-based binding units.
- the antibody may also be a CovX-body.
- the antibody may also be a cameloid species antibody.
- the antibody or antibody fragment in the context of Mxi-2, is selective for Mxi-2.
- the antibody or antibody fragment may bind to Mxi-2 with an at least 10-fold higher binding affinity than to full-length MAPK14.
- an Mxi-2 antibody usable in the context of the method of the present invention specifically binds to the epitope present at a region of Mxi-2 having the sequence GKLTIYPHLMDIELVMI (SEQ ID NO: 4).
- the antibody or antibody fragment is selective for Vim3.
- the antibody or antibody fragment may bind to Vim3 with an at least 10-fold, even more preferably at least 100-fold, even more preferably at least 1000-fold higher binding affinity than to full-length Vimentin (V9).
- the antibody or antibody fragment binds to an epitope comprising or consisting of the unique C-terminal 8 amino acids of Vim3 (RGKHFISL: SEQ ID No: 2) and/or unique C-terminal 10 amino acids of Vim3 (NLRGKHFISL: SEQ ID NO: 5). This is further exemplified in WO 2014/154686.
- the antibody according to the present invention is preferably a monoclonal antibody, a chimeric antibody or a humanized antibody.
- Monoclonal antibodies are monospecific antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell.
- a chimeric antibody is an antibody in which at least one region of an immunoglobulin of one species is fused to another region of an immunoglobulin of another species by genetic engineering in order to reduce its immunogenicity. For example murine V L and V regions may be fused to the remaining part of a human immunoglobulin.
- a particularly preferred type of chimeric antibodies are humanized antibodies. Flumanized antibodies are produced by merging the DNA that encodes the CDRs of a non-human antibody with human antibody-producing DNA.
- the resulting DNA construct can then be used to express and produce antibodies that are usually not as immunogenic as the non-human parenteral antibody or as a chimeric antibody, since merely the CDRs are non-human.
- the antibody or antibody fragment independent on its chemical nature, may optionally be dissolved in any medium suitable for storing said antibody such as, e.g., water, an aqueous buffer (e.g., a Hepes, Tris, or phosphate buffer(e.g. phosphate buffered saline (PBS)), an organic solvent (e.g., dimethyl sulfoxide (DMSO), dimethylformide (DMF)) or a mixture of two or more thereof.
- PBS phosphate buffer(e.g. phosphate buffered saline
- organic solvent e.g., dimethyl sulfoxide (DMSO), dimethylformide (DMF)
- the antibody or mutant thereof according to the present invention may be of any species or origin.
- the antibody or mutant thereof may recognize the naturally folded molecular target structure or a domain or fragment thereof (e.g., Mxi-2 in its natural environment inside the spermatozoa).
- the antibody or mutant thereof may be of any origin an antibody may be obtained from such as, e.g., natural origin, a gene technologic origin and/or a synthetic origin.
- the antibody may also be commercially available.
- the antibody may further comprise one or more posttranscriptional modification(s) and/or may be conjugated to one or more further structures such as label moieties or cell-penetrating peptides (CPPs).
- CPPs cell-penetrating peptides
- the antibody or antibody fragment may be added to a support, particularly a solid support such as an array, bead (e.g. glass or magnetic), a fiber, a film etc.
- a support particularly a solid support such as an array, bead (e.g. glass or magnetic), a fiber, a film etc.
- step (ii) of staining intracellular Mxi-2 comprises:
- indirect immunodetection comprising providing at least one Mxi-2-specific unlabeled antibody or antibody fragment (A2) and at least one labeled antibody or antibody fragment (A3) specifically binding to (A2),
- immunodetection is immunofluorescence and the labeled antibody or antibody fragment is fluorescently labeled.
- step (ii) is a sample S in which the Mxi-2 in the spermatozoa is stained, preferably by a dye (in particular a fluorescence dye) or a metal label (in particular a (colloidal) gold label), in particular a fluorescently labeled antibody.
- a dye in particular a fluorescence dye
- a metal label in particular a (colloidal) gold label
- detection of a bound Mxi-2-specific antibody or antibody fragment may be also conducted by any other method known in the art such as e.g., surface plasmon resonance (SPR) or related methods.
- SPR surface plasmon resonance
- the presence or absence of Mxi-2 is detected by detecting Mxi-2 protein expression in the sample.
- General protein detection methods in the art which are suitable for methods of rapid diagnosis of ejaculates (or punctates or biopsies) or from patients which are in the position to conceive children are immuno-electrophoresis, immuno-blotting, Western blot, SDS-PAGE, capillary electrophoresis (CE), spectrophotometry or enzyme assay for example, and dipsticks (lateral flow) but not limited to these.
- Detecting may also be an imaging step, more preferably a microscopic step, in particular a fluorescence microscopic step.
- fluorescence microscopy may comprise one or more of the following methods: laser scanning microscopy (LSM), two-photon fluorescence microscopy, fluorescence molecular imaging (FMI), fluorescence energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and/or fluorescence cross-correlation spectroscopy (FCCS). All these techniques as such are well-known to those skilled in the art.
- the Mxi-2 in the spermatozoa may fluorescently stained by a fluorescently labeled antibody (e.g., as exemplified herein), the excess fluorescently labeled antibody is washed away and the Mxi-2 localization and intensity is determined in a number of spermatozoa.
- a diminished fluorescence signal per spermatozoon or per sample volume, more preferably in the tail region, of the spermatozoa contained in the sample S in comparison to the control sample C+ may indicate decreased fertility of the spermatozoa contained in the sample S. This also applies to the detection of a signal per spermatozoon or per sample volume comparable to control sample C-.
- step (iii) may be performed by flow cytometry.
- step (ii) of staining intracellular Mxi-2 is staining with a fluorescently labeled marker and wherein the step (iii) is detecting the total content of the Mxi-2 per spermatozoon conducted by means of flow cytometry.
- the spermatozoa are stained by a Mxi-2-specific antibody or antibody fragment and populations of higher and lower fluorescence intensity are distinguished from another (e.g., as exemplified herein), the excess fluorescently labeled antibody may be washed away and the Mxi-2 per cell may be determined by flow cytometry.
- an increased fluorescence signal per spermatozoon or per sample volume in comparison to the control sample C+ indicates decreased fertility of the spermatozoa contained in the sample S. This also applies to the detection of a signal per spermatozoon or per sample volume comparable to control sample C-.
- FACS flow cytometer
- detecting may be performed by Western blot or ELISA (enzyme linked immunosorbent assay).
- Western blot or ELISA enzyme linked immunosorbent assay
- Mxi-2 may be concentrated in the sample S prior to being analyzed further.
- beads coated with a Mxi-2-specific antibody or antibody fragment may be used to obtain higher contents of Mxi-2 from the sample S.
- exemplarily agarose beads may be used, in particular agarose beads bearing an antibody-binding entity such as, e.g., protein A.
- an antibody-binding entity such as, e.g., protein A.
- any other kinds of beads usable from this purpose may be used in this context such as, exemplarily, silica or magnetic beads.
- the conjugation method may be freely chosen.
- a biotin-conjugated Mxi-2-specific antibody or antibody fragment may be immobilized on a streptavidin-bearing bead or a bead bearing maleinimidyl groups may be contacted with a cysteinyl-bearing Mxi-2-specific antibody or antibody fragment or the bead may bear amino group- binding residues such as succinimidyl esters.
- such bead may then be conjugated with a Mxi-2-specific antibody or antibody fragment.
- the beads coated with a Mxi-2-specific antibody or antibody fragment may be contacted with the sample S and incubated in order to allow the binding of the antibody to its molecular target Mxi-2.
- the beads may be removed from the liquid sample S (e.g., by means of centrifugation, filtration, crossflow filtration, or the like, or, in case of using magnetic beads, by means of magnetic forces) and may be optionally washed with a buffer.
- the Mxi-2 may then be cleaved off and investigated further such as e.g., by means of Western blot, flow cytometry, or any other method described herein, or a combination of two or more of these methods. If a defined content of beads coated with a Mxi-2-specific antibody or antibody fragment and a defined content of sample S is used, this procedure may provide (semi-)quantitative results by comparing the content of Mxi-2 in the sample S with the sample S+.
- such beads coated with a Mxi-2-specific antibody or antibody fragment may also be labelled by itself (e.g., by means of a dye (e.g., a fluorescence dye) or a metal label (e.g., gold beads)).
- a dye e.g., a fluorescence dye
- a metal label e.g., gold beads
- such beads coated with a Mxi-2-specific antibody or antibody fragment which have been bound to Mxi-2 may also be subjected to a labeled Mxi-2-specific antibody or antibody fragment. Then, the intensity of label per bead may be detected such as, e.g., by means of flow cytometry. Also this may provide (semi- )quantitative results by comparing the content of Mxi-2 in the sample S with the sample S+.
- detecting may be performed by means of lateral flow (e.g., dipstick) analysis.
- dipstick usable in the context of the present invention preferably comprises a Mxi-2-specific antibody or antibody fragment. Examples for such dipsticks are provided in detail herein.
- the sample S is typically liquid, semi-liquid or liquefied so that it can be soaked by a carrier of the dipstick.
- the sample S comprises an aqueous liquid.
- the sample S usable by the dipstick analysis may be a semen sample.
- a dipstick can be a fast and reliable result of man fertility.
- detecting and determining steps may also be conducted by polymerase chain reaction (PCR) as described in WO 2014/154686 for Vim3, which can be likewise also be conducted for Mxi-2. Then, preferably, specific primers are used.
- PCR polymerase chain reaction
- Such PCR may provide information on the integrity of the respective gene in the spermatozoa and indirectly provide information on the total content of Mxi-2 (optionally additionally Vim3) per spermatozoon or per sample volume.
- RT-PCR real time PCR
- control value or background value may be obtained by carrying out the method of the present invention additionally and simultaneously with one or more controls (e.g., a sample of low fertility C-), a background or blank sample (CO). Alternatively, it may be a value determined previously, e.g. a value provided by a third person, e.g. the manufacturer of laboratory equipment or a published value known from the art. As described above, a control sample C+ representing normozoospermia may be used. This also applies to the detection of a signal per spermatozoon or per sample volume comparable to control sample C-
- the readout may be performed manually or via a computer-assisted automated manner.
- the analysis of the determined accumulation may be performed manually or via a computer-assisted automated manner.
- the method of the present invention may be a high-throughput method. For instance it may be conducted on one or more multiwell plates, such as, e.g., 6-well plates, 12-well plates, 96-well plates or 384-well plates.
- steps (iv) and (v) are performed by a computer- assisted automated manner.
- the method may optionally also be combined with one or more classical means for determining the fertility of spermatozoa, such as light microscopic analysis (spermiogram) of spermatozoa movability and/or morphology.
- classical means for determining the fertility of spermatozoa such as light microscopic analysis (spermiogram) of spermatozoa movability and/or morphology.
- the method of the present invention is combined with the further step
- a decreased content of movability and/or a deviation in morphology of the spermatozoa in the sample S indicates decreased fertility of the spermatozoa contained in the sample S.
- step (vi) may be performed in a separate aliquot of in the same aliquot as steps (i)-(v) of the method of the present invention.
- step (vi) is performed in a separate aliquot.
- step (vi) may optionally be performed before, simultaneous or subsequent to the steps (i)-(v) of the method of the present invention.
- step (vi) in a separate aliquot may bear the advantage that the staining (step (ii)) does not interfere and using toxic agents for staining, including fixation of the spermatozoa, are not excluded from the method.
- step (vi) may optionally be performed before steps (i)-(v) of the method of the present invention or it may be performed simultaneously with steps (iv) and (v) or it may be performed subsequent to steps (i)-(v) of the method of the present invention.
- step (vi) is performed before steps (i)-(v) of the method of the present invention, then it may be performed with unstained spermatozoa.
- the sample S may be an fixed specimen. However, preferably, the sample S comprises viable spermatozoa still suitable of sexual reproduction.
- the sample S is of interest for sexual reproduction of a human or non-human animal, preferably wherein the sample S is or is derived from an ejaculate (or punctates or biopsies) of a male human or male non-human animal.
- the sample S is a male human or male non-human animal sperm donation of interest for artificial insemination.
- a non-human animal preferably is a non-human mammal (e.g., a domestic mammal (e.g., a bovine, a pig, a horse, a donkey, a sheep, a camel, a goat, a dog, a cat, etc.) or another mammal intended for propagation (e.g., an endangered species (e.g., a tiger, an elephant, etc.)).
- a domestic mammal e.g., a bovine, a pig, a horse, a donkey, a sheep, a camel, a goat, a dog, a cat, etc.
- another mammal intended for propagation e.g., an endangered species (e.g., a tiger, an elephant, etc.)).
- a sample S derived from an ejaculate may be an ejaculate (or punctate or biopsy) or aliquot thereof that may optionally be diluted in a liquid maintain the viability of the viability of the spermatozoa (e.g., by means of an aqueous buffer and/or an organic solvent (e.g., dimethyl sulfoxide)). It will be understood that also such sample S derived from an ejaculate (or punctate or biopsy) may optionally be stored under suitable conditions maintaining viability (e.g., by means of shock-freezing (e.g., in liquid nitrogen)).
- shock-freezing e.g., in liquid nitrogen
- the method of the present invention may have particular benefit on improving the sexual reproduction of humans and non-human animals.
- the sample S is a male human or male non-human animal sperm donation of interest for artificial insemination.
- the fertility of man is an interesting factor for achieving pregnancy.
- couples facing undesired childlessness are interested in determining any hindsight of why pregnancy is not achieved.
- the male sterility that is often associated with a comparably low level of spermatozoa fertility is of interest.
- the method of the present invention may provide information. From the finding of being aware of a comparably low level of spermatozoa fertility, suitable treatments may be considered.
- the patient from whom the sample S is obtained is treated for having an increase in fertility, when a decreased fertility is determined in his/its spermatozoa.
- the method comprises the further step of treating the patient from whom the sample S has been obtained from in order to increase fertility of the spermatozoa in his/its semen. This can be performed by any means such as, e.g., by means of administering a sufficient content of a therapeutic agent that increases fertility to the patient.
- ejaculates or punctates or biopsies
- ejaculates or punctates or biopsies
- two, three, four, five or more than five ejaculates (or punctates or biopsies) may be pooled.
- the sequential order to pooling and storage is variable. Storage may be performed by any means, for instance in a fridge (e.g., at approximately 4°C) or in a freezer (e.g., around -20°C or -80°C or in liquid nitrogen).
- spermatozoa from the patient from whom the sample S has been obtained are used for in vitro fertilization. This may be or particular interested when the spermatozoa have a decreased mobility.
- the method comprises the further step selected from the group consisting of
- spermatozoa fertility is diminished to such degree that it is considered as a pathologic condition.
- a pathologic condition may be understood in the broadest sense as any health state deviating from healthy state. Depending on the severity of the pathologic condition, it may also be designated as disease, illness, malady or the like.
- decreased fertility of the spermatozoa contained in the sample S is associated with at least one pathologic condition of the donor of the spermatozoa contained in the sample S according to at least one of classes N46 and R86 of the 10 th revision of the International Statistical Classification of Diseases and Related Health Problems of the World Health Organization in the version of 2016 (ICD-10), in particular indicates at least one pathologic condition selected from the group consisting of oligozoospermia, asthenozoospermia, teratozoospermia and oligo-astheno-teratozoospermia (OAT syndrome).
- a sperm donation of a donor of unknown fertility in particular a sperm donation comprised in a sperm bank, may be tested by the method of the present invention in order to access fertility thereof. This may avoid using per se infertile sperm donations for artificial insemination.
- a portion A comprising spermatozoa of high fertility may be selected from such sperm bank.
- the method comprises the following steps:
- step (iii) detecting the spatial localization of the Mxi-2 within the stained spermatozoa of step (ii) by fluorescence microscopy;
- an increased content of accumulation of Mxi-2 in the head or neck region of the spermatozoa in the sample S and/or a decreased content of accumulation of Mxi-2 in the tail region of the spermatozoa in the sample S as determined in step (v) may indicate decreased fertility of the spermatozoa contained in the sample S.
- the method may also comprise a comparable step for detecting Vim3 as described in WO 2018/185322, comprising the following steps:
- (ii’) optionally staining intracellular Vim3 the spermatozoa with a fluorescently labeled marker, in particular by binding of (a labeled antibody or antibody fragment (A1 ) or a combination of an unlabeled antibody or antibody fragment (A2) and a labeled secondary antibody or antibody fragment (A3) ) specifically binding to (A2);
- step (iii’) detecting the localization of the Vim3 within the stained spermatozoa, wherein the Vim3 is optionally stained Vim3 of step (ii’), by fluorescence microscopy;
- a decreased content of accumulation of Vim3 in the mid piece, more in particular the neck region, of the spermatozoa in the sample S as determined in step (v’) indicates decreased fertility of the spermatozoa contained in the sample S.
- the method of the present invention comprises the following steps:
- step (iii) detecting the total content of the Mxi-2 per spermatozoon or per sample volume, wherein the Mxi-2 is optionally stained Mxi-2 of step (ii), in particular by flow cytometry and/or ELISA;
- This method may optionally also be combined with a comparable method based on the detection of Vim3 as described in WO 2018/185322.
- the method of the present invention may also be supported by additional steps.
- the method further comprises the additional step:
- the method of the present invention may optionally further comprise the step of treating a patient whose spermatozoa have been found to bear decreased fertility. Additionally or alternatively, the method may optionally further comprise the step of artificial insemination or in vitro fertilization with the spermatozoa of a patient whose spermatozoa have been found to bear sufficient fertility.
- Another aspect of the present invention relates to the use of Mxi-2 as a marker for fertility of spermatozoa.
- the terms“marker and“biomarker” may be understood interchangeably in the broadest sense as generally understood in the art as a measurable indicator of some biological state or condition.
- the present invention also relates to the use of Mxi-2 as a biomarker for determining the fertility of spermatozoa contained in a sample S.
- the present invention relates to the use of Mxi-2 as a marker for fertility of spermatozoa, wherein an increased content of the total content of Mxi-2 (preferably per spermatozoon or per sample volume) indicates decreased fertility of the spermatozoa.
- the present invention relates to the use of Mxi-2 as a marker for fertility of spermatozoa, wherein an increased content of accumulation of Mxi-2 in the head or neck region of the spermatozoa and/or a decreased content of accumulation of Mxi-2 in the tail region of the spermatozoa indicates decreased fertility of the spermatozoa.
- the present invention relates to the use of Mxi-2 and Vim3 as markers for fertility of spermatozoa, wherein an increased content of the total content of Mxi-2 and a decreased content of the total content of Vim3 (preferably per spermatozoon or per sample volume) indicates decreased fertility of the spermatozoa.
- this use comprises one or more of the method steps as laid out above, in particular the use is conducted according to a method of the present invention as laid out herein.
- the method of the present invention is a method that may also be used in a medicinal and diagnostic context.
- a further aspect of the present invention relates to a Mxi-2-specific antibody or antibody fragment for use in a method of diagnosing a pathologic condition associated with decreased fertility in a patient, wherein said method is conducted as described herein, wherein the sample S is a semen sample obtained from the patient, and wherein the pathologic condition associated with decreased fertility preferably is a pathologic condition according to at least one of classes N46 and R86 of the ICD-10, in particular wherein the pathologic condition selected from the group consisting of oligozoospermia, asthenozoospermia, teratozoospermia and OAT syndrome.
- the pathologic condition is selected from the group consisting of oligozoospermia, asthenozoospermia, teratozoospermia and oligo- astheno-teratozoospermia (OAT syndrome).
- a dipstick analysis lateral flow analysis
- a further aspect of the present invention relates to a dipstick (preferably usable for the method of or the use of the present invention) comprising, placed in the direction of flow of the sample S, on a carrier that is suitable for soaking the sample S, the following:
- a stripe (2) comprising immobilized unlabeled MAPK14-specific, in particular Mxi-2-specific, antibodies or antibody fragments;
- stripe (3) optionally a stripe (3) of immobilized unlabeled antibodies or antibody fragments specifically binding the labeled Mxi-2-specific antibodies or antibody fragments of stripe (1 );
- a stripe (1’) comprising labeled Vim3-specific antibodies or antibody fragments which are not immobilized and freely movable when the sample S passes through this stripe (1’);
- a stripe (2’) comprising immobilized unlabeled vimentin-specific, in particular Vim3-specific, antibodies or antibody fragments;
- stripe (3’) optionally a stripe (3’) of immobilized unlabeled antibodies or antibody fragments specifically binding the labeled Vim3-specific antibodies or antibody fragments of stripe (1’).
- a dipstick according to the present invention (preferably usable for the method of or the use of the present invention) comprise at least, placed in the direction of flow of the sample S, on a carrier that is suitable for soaking the sample S, the following:
- a stripe (2) comprising immobilized unlabeled MAPK14-specific, in particular Mxi-2-specific, antibodies or antibody fragments.
- the terms “dipstick”, “dip-stick”, “test strip”, “control strip”, “diagnostic/medical dipstick” may be understood interchangeably in the broadest sense as any device that is usable to test a sample S in the context of the present invention (according to the lateral flow technique).
- the sample S is typically liquid, semi-liquid or liquefied so that it can be soaked by a carrier of the dipstick.
- the sample S comprises an aqueous liquid.
- the sample S usable by the dipstick may be a semen sample (e.g., ejaculate, punctate or biopsy).
- the sample S is preferably premixed with a labeled Mxi-2-specific antibody or antibody fragment.
- the volume and molar ratios will be adapted accordingly in order to optimize binding efficiency.
- the volume of the sample S (optionally diluted and/or premixed with a labeled Mxi- 2-specific antibody or antibody fragment) added to the dipstick will be adapted to the size and material of the dipstick.
- Typical volumes for adding to a segment suitable for soaking the sample S are in the range of from 10 to 1000 pi, preferably 50 to 500 mI, in particular 75 to 300 mI, exemplarily (approximately 200 mI).
- the carrier may be a (hydro) gel or a piece of paper board, and may be optionally film laminated.
- the dipstick will be stored in dry state and is moistened by the sample S.
- the edge or segment suitable for soaking the sample S (0) may be contacted with the sample S. This is preferably conducted long enough to enable the sample liquid to be soaked in the carrier of the dipstick.
- the other parts of the dipstick are preferably not directly contacted with the sample S.
- the sample S flows through the carrier of the dipstick at least until the stripes (1 ) (if present) and (2) and optionally (3) have been passed by the sample S or parts thereof.
- the sample S is of a first species and the antibodies or antibody fragments of each of stripe (1 ) (if present) or the antibodies or fragments used for premixing with the sample S (in particular if stripe (1 ) is not present) on the one hand and (2) and optionally (3) of the other hand are each of different species.
- the immobilized unlabeled antibodies or antibody fragments of stripe (3) specifically bind to the Fc fragment of the labeled Mxi-2- specific antibodies or antibody fragments of stripe (1 ) (if present) or premixed with the spermatozoa in solution (in particular if stripe (1 ) is not present).
- the Mxi-2-specific antibodies or antibody fragments which are not immobilized are (preferably monoclonal) rabbit antibodies.
- the immobilized antibodies of stripe (3) may be (preferably monoclonal) antibodies directed against the Fc part of the antibodies provided in stripe (1 ) or premixed with the spermatozoa in solution (in particular if stripe (1 ) is not present).
- the label may be a fluorescence label, a visible dye label or, particularly preferably, a (colloidal) gold label.
- a (colloidal) gold label may be added to an antibody or antibody fragment bay any means, exemplarily by means of a GOLD Conjugation Kit.
- Mxi-2-containg sample S+
- the labeled Mxi-2-specific antibodies may bind to Mxi-2 in the spermatozoa and form a spematoza:Mxi-2-specific antibody conjugate. This conjugate will then binding to the unlabeled Mxi-2-specific antibodies of stripe.
- the ratio between signal intensity of the label in stripe (2) and (3) indicates infertility or fertility.
- a lower (2): (3) ratio may indicate higher fertility, whereas a higher (2): (3) ratio may indicate lower fertility in the sense of the method of the present invention laid out above.
- the dipstick (preferably usable for the method of or the use of the present invention) comprises, placed in the direction of flow of the sample S, on a carrier that is suitable for soaking the sample S, the following:
- the dipstick (preferably usable for the method of or the use of the present invention) comprises, placed in the direction of flow of the sample S premixed with labeled Mxi-2-specific antibodies or antibody fragments (which are not immobilized and freely movable) on a carrier that is suitable for soaking the sample S premixed with labeled Mxi-2-specific antibodies or antibody fragments, the following:
- a stripe (2) comprising immobilized unlabeled MAPK14-specific (either directed against MAPK14 in general or Mxi-2-specific) antibodies or antibody fragments;
- stripe (3) optionally a stripe (3) of immobilized unlabeled antibodies or antibody fragments specifically binding the labeled Mxi-2-specific antibodies or antibody fragments of stripe (1 ).
- the MAPK14-specific antibody used in stripe (2) is directed to both forms.
- the premixing of the labeled Mxi-2-specific antibodies or antibody fragments may be followed by an incubation to allow and optimize binding of the Mxi-2-specific antibodies or antibody fragments to its molecular target Mxi-2. This may exemplarily be performed by incubating for 10 to 60 min at a temperature of from 2 to 25 °C.
- a dipstick according to the present invention may be may be prepared according to Preechakasedkit et al. , 2012, Biosens Bioelectron 31 (1 ):562-566; Tao et al., 2014, Lett Appl Microbiol 59(2):247-251 or Wang et al., 2010, J Virol Methods 2010, 170(1 -2):80-85.
- the method of the present invention also enable the selective choice of a portion A comprising spermatozoa of high fertility, e.g., from a sperm bank comprising a variety of human and non-human ejaculate (or punctate or biopsy) aliquots.
- a further aspect of the present invention relates to a method for obtaining a portion A sufficient for sexual reproduction of a human or non-human animal containing spermatozoa of high fertility, said method comprising the following steps: (1 ) providing one or more samples S containing spermatozoa potentially suitable for sexual reproduction;
- step (2) classifying the fertility of the sample S determined by step (2) as:
- step (3) selecting and obtaining a sample S+ containing spermatozoa of higher fertility above the threshold fertility according to step (3) as portion A.
- the portion A is or is derived from a sperm donation of interest for artificial insemination obtained from a male human or a male non human animal.
- this may be particularly beneficial for improving the selection of sperm donations, e.g., from a sperm bank. This is often particularly desired for breeding non-human animals, e.g., for agricultural use.
- the portion A is or is derived from a sperm donation of interest for artificial insemination obtained from a male non-human animal which is a mammal intended for breeding.
- such method may also be employed for selecting and breeding animals of high fertility.
- a still further aspect of the present invention relates to a method for obtaining a non-human male animal bearing spermatozoa of high fertility, said method comprising the steps:
- samples S containing spermatozoa potentially suitable for sexual reproduction in particular wherein said samples S are derived from ejaculates (or punctates or biopsies) obtained from non-human male animals each of the same species SP;
- step (III) identifying a sample S+ of high fertility suitable for sexual reproduction based on the findings of step (II);
- step (IV) inseminating a non-human female animal of species SP susceptible for pregnancy with the selected sample S+ as identified in step (III) by means of artificial insemination or copulation with the male non-human animal from which sample S+ has been derived from;
- step (V) enabling the gestation of the progeny obtained from step (IV) in the female animal, subsequent birth and obtaining the non-human male animal of high fertility.
- this method is in principle employable independent on the species of the animal as long as the animal spermatozoa express Mxi-2.
- the method is independent on the race of the animal.
- the present invention also relates to a test kit comprising one or more reagents useful for practicing the method according to the present invention.
- a kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as a mixture if reagents are compatible.
- the kit may also include other material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in sample processing, washing, or conducting any other step of the assay.
- a kit according to the present invention may include a solid phase and a capture agent affixed to the solid phase, wherein the capture agent is an antibody specific for the analysis (e.g., a Mxi-2-specific antibody) being assessed in the test sample.
- the solid phase may comprise a material such as a magnetic or paramagnetic particle including a microparticle, a bead, a test tube, a microtiter plate, a cuvette, a membrane, a scaffolding molecule, a quartz crystal, a film, a filter paper, a dipstick a disc or a chip.
- a Test kit according to the present invention may preferably further comprise user instructions for carrying out one or more of the methods of the invention.
- kits of the invention can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, for example, computer media including, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
- electronic storage media e.g., magnetic discs, tapes, cartridges, chips
- optical media e.g., CD ROM
- Figure 1 shows an exemplary setup of a dipstick usable for the method of the present invention.
- Fig. 5A shows the dipstick before use.
- (1 ) indicates a stripe comprising labeled Mxi-2-specific antibodies (depicted as stars), which are not immobilized and freely movable when the sample S passes through this stripe.
- (2) indicates a stripe comprising immobilized unlabeled Mxi-2-specific antibodies.
- (3) indicates a stripe of immobilized unlabeled antibodies specifically binding the labeled Mxi-2-specific antibodies (depicted as stars).
- S indicates the sample S to be added to the dipstick.
- (4) indicates the flow direction of the moisten sample S.
- FIG. 5b shows the results when a Mxi-2-containg sample (S+) is added to the dipstick. Then, upon flowing through the dipstick (4), the labeled Mxi-2-specific antibodies are binding to Mxi-2 in the spermatozoa and form a spematoza: Mxi-2 - specific antibody conjugate. This conjugate is then binding to the unlabeled Mxi-2- specific antibodies of stripe (2).
- Fig. 5c shows the results when a sample lacking Mxi-2 (S-) is added to the dipstick. Then, upon flowing through the dipstick (4), the labeled Mxi-2-specific antibodies are not bound until the stripe (3).
- the ratio between signal intensity of the label in stripe (2) and (3) indicates fertility. A higher (2): (3) ratio indicates lower fertility, whereas a higher (2): (3) ratio indicates lower fertility.
- Figure 2 shows another setup of a dipstick usable for the method of the present invention as an alternative to the one shown in Figure 5.
- Fig. 6A shows the dipstick before use.
- the sample S e.g. ejaculate, punctate or biopsy
- a labeled Mxi-2-specific antibodies (depicted as stars).
- (2) indicates a stripe comprising immobilized unlabeled MAPK14-specific antibodies.
- (3) indicates a stripe of immobilized unlabeled antibodies specifically binding the labeled Mxi-2-specific antibodies (depicted as stars).
- (4) indicates the flow direction of the moisten sample S.
- FIG. 6b shows the results when a Mxi-2- containg sample (S+) premixed with a labeled Mxi-2-specific antibodies (depicted as stars) is added to the dipstick. Then, the a labeled Mxi-2-specific antibodies (depicted as stars) and the Mxi-2 in the spermatozoa form a spematoza: Mxi-2 - specific antibody conjugate. After adding this sample to the dipstick, it flows through the dipstick (4). The spematoza:Mxi-2-specific antibody conjugate is then binding to the unlabeled Mxi-2-specific antibodies of stripe (2).
- 6c shows the results when a sample lacking Mxi-2 (S-) premixed with a labeled Mxi-2-specific antibodies (depicted as stars) is added to the dipstick. Then, the labeled Mxi-2- specific antibodies (depicted as stars) are not binding to the spermatozoa. Thus, upon flowing through the dipstick (4), the labeled Mxi-2-specific antibodies are not bound until the stripe (3). Thus, the ratio between signal intensity of the label in stripe (2) and (3) indicates fertility. A lower (2): (3) ratio indicates higher fertility, whereas a higher (2): (3) ratio indicates lower fertility.
- Figure 3 shows results from an ELISA assay showing the content of Mxi-2 in spermatozoa detected with a labeled antibody at 450 nm.
- a control sample (normozoospermia, Normo) is compared to sperm cells from and a patient suffering from oligo-astheno-teratozoospermia (OAT) syndrome, sperm cells from and a patient suffering from teratozoospermia (Terato) and sperm cells from and a patient suffering from azoospermia (Azoo).
- Figure 4 shows the localization of the nuclei (DAPI stain, in Fig. 4A), Mxi-2 (Fig. 4B), MAPK14 full length (Fig. 4C) and a merged image (Fig. 4D) in spermatozoa from a patient suffering from teratozoospermia.
- Figure 5 shows the localization of the nuclei (DAPI stain, in Fig. 5A), Mxi-2 (Fig. 5B), MAPK14 full length (Fig. 5C) and a merged image (Fig. 5D) in spermatozoa from a further patient suffering from teratozoospermia.
- Figure 6 shows an overlay of flow cytometry data.
- the corresponding single samples analyzed by flow cytometry are depicted in Figure 7.
- unstained controls were compared with Mxi-2 stained samples containing healthy sperm cells (normozoospermia, Normo) and Mxi-2 stained samples containing sperm cells from patients suffering from oligo-astheno-teratozoospermia (OAT) syndrome, sperm cells from patients suffering from asthenozoospermia (Astheno), sperm cells from patients suffering from teratozoospermia (Terato), and sperm cells from patients suffering from oligozoospermia (Oligo).
- the samples were stained with an Mxi-2-specific antibody as described in the example section.
- Figure 8 shows immunostaining of a control sample of healthy sperm cells (normozoospermia, Normo) and of containing sperm cells from patients suffering from oligo-astheno-teratozoospermia (OAT).
- the sperm cells are obtained from the swim-up fraction of a swim-up procedure, where the fertile sperms survive and swim up.
- the DNA-containing head of the sperm cells were stained with DAPI (A).
- the sperm cells were stained with a MAPK p38-specific antibody (B) and with a Mxi-2-specific antibody (C).
- D merge of the staining with MAPK p38-specific antibody and with a Mxi-2-specific antibody is shown (D).
- Figure 9 shows the content of Vim3 in sperm cells determined via an ELISA assay.
- samples containing healthy sperm cells were compared with samples containing sperm cells from patients suffering from oligo-astheno-teratozoospermia (OAT) syndrome, from patients suffering from azoospermia (Azoo), from patients suffering from teratozoospermia (Terato), from patients suffering from asthenozoospermia (Astheno), and from patients suffering from oligozoospermia (Oligo).
- OAT oligo-astheno-teratozoospermia
- Figure 10 shows the content of Mxi-2 in sperm cells determined via an ELISA assay.
- samples containing healthy sperm cells were compared with samples containing sperm cells from patients suffering from oligo-astheno-teratozoospermia (OAT) syndrome, from patients suffering from teratozoospermia (Terato), from patients suffering from azoospermia (Azoo), from patients suffering from oligozoospermia (Oligo) and from patients suffering from asthenozoospermia (Astheno).
- OAT oligo-astheno-teratozoospermia
- the ejaculates were collected from patients and analyzed according to the WHO regulations. Sample quality was controlled and assessment of sperm quality for reference purposes was performed by QuaDeGa (Mtinster, Germany).
- MAPK14 MAPK p38
- r38a/b antibody h-147 of Santa Cruz Biotechnology (Dallas, USA) was used.
- Vim3 monoclonal using the last 8 amino acids (RGKHFISL: SEQ ID No: 2) of the unique C-terminal ending of Vim3, Clone 51 , Davids Biotechnologie (Regensburg, Germany) may be used.
- sc-6260 of Santa Cruz may be used as primary Vimentin V9 antibody.
- the primary antibodies for use in the context of the method of the present invention are exemplarily also specified by binding to the following epitopes:
- An Alexa488-labeled anti-mouse antibody (Ref. A11001 ) of Life Technologies (Carlsbad, California, USA) was used to bind the primary Mxi-2 antibody.
- secondary antibodies usable are such as, e.g., a secondary anti-rabbit antibody (Santa Cruz, Heidelberg, Germany), a secondary anti-mouse antibody (Santa Cruz, Heidelberg, Germany) or a secondary goat-anti mouse antibody (1 :50000) (Columbia Biosciences, Maryland, USA HRP112).
- This antibodies can be labelled by a flurophore (e.g., Alexa488) or an enzyme (e.g., horseradish peroxidase)
- Example I Analysis via enzyme-linked immunosorbent assay (ELISA) a) ELISA Assay showing correlation between the content of Mxi-2 and aberrations in sperm cells
- the ejaculate was washed 3x with phosphate buffered saline (PBS) buffer and was then suspended in PBS buffer.
- ELISA plates (Corning Costar® 96-Well EIA/RIA StripwellTM Plates) were washed before start with PBS buffer. 50 pi of the ejaculate suspension were added to the ELISA plate and were incubated for 1 hour at room temperature (RT). Samples were washed 2x with PBS and subsequently incubated with the Mxi-2 primary antibody (diluted 1 :500) for 1 hour at RT. The samples were washed again 2x with PBS.
- the ELISA assay was conducted as described above. Samples containing healthy sperm cells (normozoospermia, Normo) were compared with samples containing sperm cells from patients suffering from oligo-astheno-teratozoospermia (OAT) syndrome, from patients suffering from teratozoospermia (Terato), from patients suffering from azoospermia (Azoo), from patients suffering from oligozoospermia (Oligo) and from patients suffering from asthenozoospermia (Astheno).
- the ELISA assay was conducted as described above, wherein the Mxi-2-specific primary antibody was replaced by the Vim3-specific primary antibody.
- Samples containing healthy sperm cells were compared with samples containing sperm cells from patients suffering from oligo-astheno- teratozoospermia (OAT) syndrome, from patients suffering from teratozoospermia (Terato), from patients suffering from azoospermia (Azoo), from patients suffering from oligozoospermia (Oligo) and from patients suffering from asthenozoospermia (Astheno).
- a comparable staining protocol was performed for staining the MAPK14 with a primary antibody in a 1 :1000 dilution and a respective secondary antibody (Alexa700-labeled anti-rabbit in a 1 :5000 dilution). Further, the nuclei in the samples were stained with 4',6-diamidino-2-phenylindole (DAPI).
- DAPI 4',6-diamidino-2-phenylindole
- a swim-up sperm procedure (also: swim-up test) was conducted. Flerein, only the fertile sperms survive and swim up. This fraction is further investigated. In a further microscopic imaging analysis, the sperm cells were stained with DAPI (staining the DNA-comprising head region), a MAPK p38-specific antibody and a Mxi-2-specific antibody. The sperm cells were treated and immunostained as described above. This may also be designated as a“swim-up test”.
- PCR is usable in the context of the present invention.
- primers are exemplarily usable:
- the levels found in a sample S of interest are comparable with predetermined threshold level(s) and/or with those levels determined in one or more control samples.
- Sperm cells may also be analysed by means of flow cytometry. 100 pi of each ejaculate are washed as described above with 1x PBS. Cell Fixation and Cell permeabilization kit (Thermo Scientific) are used according to the manufactures protocol. The incubation with the Vim3 antibody is performed for 1 hour at room temperature; sperms are washed with PBS twice and incubated with a secondary Alexa 488 antibody for 20 mins. As control 293t cells are used to proof the signal intensity. The appropriated controls are performed. Flow cytometry is performed by FACSCanto I (Becton Dickinson) and obtained data is analyzed using FlowJo (Tree Star). b) Protocol for Flow cytometry B
- Ejaculates were each washed three times (3x) by alternating centrifugation and resuspension in PBS buffer to obtain the purified sperm cells. These were incubated with an Mxi-2-specific antibody for 1 h at room temperature (RT). Subsequently, the sperm cells were washed 3x by alternating centrifugation and resuspension in PBS buffer. Then, the sperm cells were incubated with the secondary staining antibody as described above for 1 h at RT (Mxi-2 stained). The stained sperm cells were washed 3x by alternating centrifugation and resuspension in PBS buffer. Then, each sperm cell sample was analyzed by a flow cytometry in a FACS device (Attune NxT, Invitrogen).
- Western blot analysis may be used for determining the content of Mxi-2 in the spermatozoa. All samples are neutralized to beta-actin (b-actin) used as housekeeping gene. For densitometry, each 10 patient samples are measured. The background of the blot is measured separately and calculated against the densitometry of the protein lane. Afterwards, the results from the housekeeping gene are determined as 100% and the Mxi-2 signal is calculated in regard to the 100% of the housekeeping. Calculation is performed by 1 st way ANOVA. Western blot is performed as followed: Proteins are separated according to their molecular weight.
- b-actin beta-actin
- SDS polyacrylamide gel electrophoresis SDS-PAGE
- the discontinuous gel system is composed of a stacking and a separating gel which differs in salt concentration, pH and acrylamide concentration.
- a 10% separating gel is used and performed as followed: the separating gel contains 0.4 M Tris-HCI pH 8.8, 0.1 % SDS, 10-12% acrylamide/bis-acrylamide (29:1 ), 0.5% ammonium persulfate and 0.06% TEMED.
- the stacking gel contains 0.125 M Tris pH 6.8, 0.1 % SDS, 3% acrylamide/bisacrylamide (29:1 ), 0.5% ammonium persulfate and 0.12 % TEMED.
- 50 to 1000 pi sample are centrifuged at full speed and resuspended with PBS and“5 x Western loading dye” and heated for 5 min at 99°C.
- the gel run is performed in“Laemmlis running” buffer. The running time is between 1.5 h to 2 h (15 mA until dye front reached separating gel, then 30 mA) depending on the protein size.
- After separation in the SDS-PAGE the proteins are transferred onto PVDF membrane using a semidry blotting system in Towbin buffer.
- the membrane is incubated with the secondary antibody in blocking solution (5% milk in TBST) for 1 hour, shaking at room temperature and then washed again for 30 min in TBST stock solution. Finally the membrane is incubated 1.5 min with ECL reagent and developed in a chemiluminescence reader (ChemiDoc, Biorad).
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EP18210761 | 2018-12-06 | ||
PCT/EP2019/084015 WO2020115291A1 (fr) | 2018-12-06 | 2019-12-06 | Marqueur pour la détermination de la fertilité de spermatozoïdes |
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US5512473A (en) | 1993-01-29 | 1996-04-30 | Brent; Roger | Max-interacting proteins and related molecules and methods |
US5723305A (en) | 1996-04-18 | 1998-03-03 | Sullivan; Robert | Marker for male infertility and/or fertility |
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