Method for in vitro or ex vivo diagnosis of male infertility

Abstract

The invention relates to a method for in vitro or ex vivo diagnosis of male infertility, resulting from a dysfunction of the lipid metabolism of spermatozoa, in an individual, as well as a kit and the use of one or more specific marker(s) for this diagnosis.

Description

[0001] DESCRIPTION

[0002] TITLE: METHOD FOR THE IN VITRO OR EX VIVO DIAGNOSIS OF MALE INFERTILITY

[0003] The present invention relates to the context of male infertility and relates to a new method for the in vitro or ex vivo diagnosis of certain causes of male infertility in an individual. The invention also relates to a kit and the use of specific marker(s) for such a diagnosis.

[0004] In France, infertility currently affects one in seven couples and, as such, constitutes a major public health issue with significant medical and financial implications. In more than 50% of cases, infertility in couples is linked to male factors (so-called male infertility), and mainly to factors that impact the fertilization potential of spermatozoa. The fertilization potential of spermatozoa depends on their production in sufficient quantity and their efficient progression through the female genital tract to reach and fertilize the oocyte. This latter fundamental property is mediated by the flagellum, which provides the mechanical force for the propulsion and mobility of spermatozoa.It is also conditioned by sufficient energy production and adaptation of the energy metabolism of spermatozoa according to the nutrients available throughout their journey from the male genital tract to the fallopian tubes.

[0005] The factors impacting male fertility are multiple and varied and lead to equally varied sperm pathologies, such as azoospermia, oligozoospermia, asthenozoospermia, teratozoospermia or necrozoospermia. The most frequently found pathology in infertile men is asthenozoospermia, defined by the reduction or absence of sperm motility. Two categories of asthenozoospermia can be distinguished: so-called structural asthenozoospermia resulting from morphological and structural defects of the sperm flagellum and so-called functional asthenozoospermia resulting from dysfunctions of sperm activity.

[0006] Over the last decade, major progress has been made in the discovery of genes whose mutations are responsible in men for sperm flagellum abnormalities leading to infertility due to structural asthenozoospermia. However, the conditions of so-called functional asthenozoospermia have been very little studied and remain poorly defined, with, to date, only a few genes identified that mainly encode ion channels and transporters. Surprisingly, while ATP production appears crucial for sperm motility, functional deficiency of metabolic pathways regulating energy production has never been formally established as a cause of male infertility. Furthermore, sperm bioenergetics has mainly been studied through glycolysis and mitochondrial oxidative phosphorylation (OxPhos) (Ford, WCL. 2006; Ferramosca & Zara.2014; du Plessis et al 2015), while lipid metabolism, particularly fatty acid oxidation, has been very little studied by the scientific community. In this context, the inventors have highlighted a new category of infertile patients with lipid metabolism dysfunction in their spermatozoa. These patients present asthenozoospermia associated with necrozoospermia.

[0007] Although the clinical and genetic diagnosis of male infertility has improved considerably, there is currently no treatment capable of restoring fertility in men that would allow natural procreation; patients must resort to assisted reproductive technologies (ART) and most often to in vitro fertilization (IVF) by intracytoplasmic injection of their spermatozoa (ICSI) into the oocytes of their partners. In France, ART has an estimated cost of 300 million euros per year, and involves invasive medical procedures for the partners of infertile men: hormonal injections to induce super ovulation, anesthesia and ovarian puncture to collect the oocytes, and embryo transfer into the uterus. It is important to note that currently, in more than 50% of cases, the ART process ends in failure even after several attempts.There is therefore a real need to better understand the causes of male infertility in order to be able to offer appropriate curative solutions, offering men the possibility of being active in the treatment of their pathology and thus freeing their female partners from restrictive and invasive medical procedures, not without side effects and risks.

[0008] In this sense, there is a need to better understand the dysfunctions that cause male infertility, in order to allow a more precise classification of patients according to their type of infertility, and to allow, where appropriate, the development of therapeutic solutions adapted to each type of infertility.

[0009] In this context, the inventors have developed a novel method for proposing markers capable of specifically diagnosing in an asthenozoospermic individual, a dysfunction of the lipid metabolism of spermatozoa leading to severe asthenozoospermia or astheno-necro-zoospermia. In addition, these anomalies can also be combined with oligozoospermia defined by a reduced number of spermatozoa in the ejaculate and / or teratozoospermia defined by an increased number of spermatozoa presenting morphological anomalies in the ejaculate. This innovative diagnostic approach and the associated innovative tool allow a simple, rapid and effective identification of patients presenting such infertility caused by a dysfunction of the lipid metabolism of spermatozoa, with the aim of being able to offer them specific and adapted therapeutic solutions.The subject of the present invention is therefore a method for in vitro or ex vivo diagnosis of asthenozoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in an asthenozoospermic individual, comprising a step of detecting, in at least one biological sample of said individual, at least one marker chosen from: an accumulation of lipid droplets greater than a reference value, a content of carnitine and / or carnitine derivatives lower than a reference value, a content of ACSBG2 protein lower than a reference value, a content of ACSBG2 transcripts lower than a reference value, an alteration of the sequence of the ACSBG2 transcripts compared to a reference sequence, and an alteration of the sequence of the ACSBG2 gene.

[0010] The detection in a biological sample of an individual of at least one of these markers makes it possible to quickly and simply identify a patient with asthenozoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa from a patient suffering from asthenozoospermia resulting from other cause(s) (such as structural asthenozoospermia). From then on, it becomes possible to determine in a targeted manner, specific therapeutic solutions adapted to each patient, in order to allow them either a restoration of sperm motility with the aim of procreating naturally, or an improvement in the quality of their spermatozoa, in particular their vitality, which would facilitate the realization of ART by IVF / ICSI.

[0011] The present invention therefore makes a real contribution to the diagnosis and management of certain functional male infertilities characterized in particular by asthenozoospermia or astheno-necrozoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa. This diagnostic advance opens the way to the development of treatments aimed at restoring or improving the fertilizing power of spermatozoa, with a view to obtaining a pregnancy naturally or by medically assisted procreation.

[0012] In the context of the present invention:

[0013] - "dysfunction of sperm lipid metabolism" means an anomaly affecting the signaling pathways and / or enzymatic pathways involved in the biosynthesis and / or degradation of lipids, and / or the pathways of lipid energy metabolism such as mitochondrial oxidation of fatty acids,

[0014] - "in vitro diagnosis" and "ex vivo diagnosis" mean tests carried out outside an individual, after taking the biological sample from said individual. The tests carried out on the biological samples taken from the individual(s) and kept in the laboratory, generally under sterile conditions, without modifications over a short period, are part of an ex vivo diagnosis according to the invention. The tests carried out on such samples and over a longer period of time are generally part of an in vitro diagnosis according to the invention,

[0015] - "detection of at least one marker chosen from" means the detection of one or more markers chosen indifferently from the list of markers. Thus, in all the objects of the invention described in the present text, at least one of said markers, at least two of said markers, at least three of said markers, and so on until the detection of all the markers envisaged,

[0016] - "asthenozoospermia" means the absence or reduction of sperm motility with a rate of less than 42% of motile sperm and / or a rate of less than 30% of progressive sperm in the ejaculate according to WHO standards (5 èmepercentile; WHO laboratory manual for the examination and processing of human semen - Sixth edition, 2021 ). It is the predominant anomaly found in infertile men. Asthenozoospermia can result from structural defects in the sperm flagellum (hereinafter "structural asthenozoospermia"). Asthenozoospermia can also result from dysfunctions in sperm activity due to deregulation, particularly of membrane and intracellular signaling pathways (hereinafter "functional asthenozoospermia"). Patients with functional asthenozoospermia have sperm without severe abnormalities in the structure of their flagella but which, however, fail to move correctly or are even completely immobile.Severe asthenozoospermia is defined as absence or reduction of sperm motility with a rate of less than 35% motile sperm and / or a rate of less than 24% progressive sperm in the ejaculate according to WHO standards (2.5. ème percentile; WHO laboratory manual for the examination and processing of human semen - Sixth edition, 2021),

[0017] - “asthenozoospermic individual” means a male individual with impaired sperm motility as defined in the preceding paragraph and / or diagnosed as suffering from asthenozoospermia,

[0018] - "necrozoospermia" means a rate of vitality of spermatozoa present in the ejaculate, less than 54%, according to WHO standards,

[0019] - "oligozoospermia" means a number of spermatozoa present in the total volume of ejaculate, less than 39 million, according to WHO standards,

[0020] - "astheno-necro-zoospermia" means the absence or reduction of sperm motility with a motile sperm rate of less than 42% combined with a sperm vitality rate of less than 54%, according to WHO standards, - "biological sample" means a tissue, a fluid, as well as components of said tissue and fluid (e.g. cells). Depending on the scope of the method, and by way of non-exhaustive examples, the sample may be semen and its components such as seminal fluid, sperm, immature germ cells, or blood, urine, saliva, testicular tissue taken by biopsy or any other biological tissue likely to contain genomic DNA,

[0021] - "reference value" means a value for a given parameter, defined for a "control" individual (also called a "control" individual) not having a fertility problem. The reference value for a given parameter therefore corresponds to that defined for the same parameter for a fertile individual. Such a value may come directly from the analysis of a biological sample from a fertile control individual or be the result of several values ​​from several biological samples from the same fertile individual and / or from several fertile individuals,

[0022] - "accumulation of lipid droplets" means the presence of droplets containing lipid esters in the cytoplasm of spermatozoa, mainly at the level of the sperm head and the connective tissue; these droplets can be visualized, for example, by specific cytological staining such as OiIRedO (non-fluorescent) or Nile Red (fluorescent and also called "Nile Red"),

[0023] - "carnitine or carnitine derivative content" means the quantity of 3-hydroxy-4-trimethylammonio-butanoate (or the quantity of its derivatives) present in the biological sample analyzed, this quantity being able to be measured by any technique known to those skilled in the art, for example by liquid chromatography coupled with mass spectrometry. Carnitine is particularly known for its properties of transporting the acylated chains of fatty acids to the mitochondrial matrix. Among the carnitine derivatives, we find in particular the carnitine derivatives linked to an acyl group, the acylcarnitines, these derivatives being able to contain several carbon chains (C2, C3, C4, etc.),

[0024] - "ACSBG2 protein content" means the amount of Acyl-coenzyme A synthetase enzyme (UniProtKB identifier: Q5FVE4) in the biological sample analyzed. This enzyme is derived from the translation by ribosomes of one of the transcripts of the ACSBG2 gene (Acyl-CoA Synthetase Bubblegum 2 - Gene ID: 81616 and Ensembl: ENSG00000130377). The ACSBG2 protein is known for its role in the modification of long-chain fatty acids, which conditions their transport to the mitochondrial matrix,

[0025] - "ACSBG2 transcript content" means the quantity of transcripts resulting from the transcription of the ACSBG2 gene (Acyl-CoA Synthetase Bubblegum 2 - Gene ID: 81616 and Ensembl: ENSG00000130377) in the biological sample analyzed, - "alteration of the sequence of ACSBG2 transcripts compared to a reference sequence" means at least one difference in said sequence of transcripts such that their splicing is abnormal or their translation by ribosomes is made impossible or leads to an absence of ACSBG2 protein or to ACSBG2 proteins carrying an alteration of their amino acid composition.The reference sequence to which the altered sequence of the transcripts can be compared is that of one of the transcripts of the ACSBG2 gene (Acyl-CoA Synthetase Bubblegum 2 - Gene ID: 81616 and Ensembl: ENSG00000130377), taking into account the degeneracy of the genetic code, said ACSBG2 gene having the nucleotide sequence SEQ ID NO: 1 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 1.

[0026] - “Alteration of the ACSBG2 gene sequence” means at least one difference in said sequence compared to the known sequence of the ACSBG2 gene (Acyl-CoA Synthetase Bubblegum 2) located on chromosome 19 (Gene ID: 81616 and Ensembl: ENSG00000130377) and that this difference: either negatively impacts the transcription of the gene so that it can no longer be transcribed, or results in the alteration of ACSBG2 transcripts, as defined in the previous point. This alteration of the gene can be located in the coding region (such as missense or nonsense mutations), in exon / intron junctions (such as splice site mutations) or in non-coding regions (such as deletions or duplications). Said known sequence of ACSBG2 has as its nucleotide sequence the sequence SEQ ID NO: 1 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95% or even 99% identity with the sequence SEQ ID NO: 1,

[0027] - “fructose content” means the quantity of fructose, in all its forms known to those skilled in the art, in particular its pD-Fructopyranose and pD-fructofuranose forms, present in the biological sample analyzed,

[0028] - “zinc content” means the quantity of zinc, in all its forms known to those skilled in the art, present in the biological sample analyzed,

[0029] - “citric acid content” means the quantity of 2-hydroxypropane-1,2,3-tricarboxylic acid or all its derived forms known to those skilled in the art, present in the biological sample analyzed,

[0030] - "prostatic acid phosphatase content" means the quantity of isoenzyme-2 of acid phosphate (PAC), mainly synthesized in the prostate, present in the biological sample analyzed, - "neutral alpha-glucosidase content" means the quantity of neutral alpha-glucosidase enzyme (NaG or "Neutral a-glucosidase" according to its English name (EC 3.2.1.20)), present in the biological sample analyzed.

[0031] Preferably, the subject of the present invention is an in vitro or ex vivo diagnostic method as defined above in which the following characteristics are chosen alone or in combination:

[0032] - the biological sample of said individual is a sperm sample and said at least one detected marker is chosen from: o the accumulation of lipid droplets, in the spermatozoa and / or the seminal fluid, greater than a reference value, o the content of carnitine and / or carnitine derivatives, in the seminal fluid and / or in the spermatozoa, less than a reference value, o the content of ACSBG2 protein, in the spermatozoa, less than a reference value, o the content of ACSBG2 transcripts, in the spermatozoa, less than a reference value, o the alteration of the sequence of the ACSBG2 transcripts, in the spermatozoa, compared to a reference sequence, o the alteration of the sequence of the ACSBG2 gene, in the spermatozoa,

[0033] - the biological sample of said individual is a semen sample, in which the carnitine content in the seminal fluid is lower than a reference value and in which at least one of the following markers is detected in the seminal fluid: a fructose content, a zinc content, a citric acid content, a prostatic acid phosphatase content or a neutral alpha-glucosidase content greater than or equal to a reference value,

[0034] - the carnitine content detected in seminal fluid is less than 390 nmol / ejaculate,

[0035] - the carnitine content detected in the seminal fluid and / or in the spermatozoa is zero,

[0036] - the ACSBG2 protein content detected in the spermatozoa is zero,

[0037] - the accumulation of lipid droplets in spermatozoa and / or seminal fluid is detected via a technique for staining said lipid droplets, preferably via the Oil-RedO technique,

[0038] - the biological sample of said individual is a sample of blood or saliva of said individual, the at least one marker detected being the alteration of the sequence of the ACSBG2 gene,

[0039] - the detection step comprises the detection of at least two of said markers, preferably at least three of said markers, preferentially at least four of said markers, even more preferentially at least five of said markers and even more preferentially all six markers,

[0040] - the method comprises an additional step of detecting a marker, said marker being a rate of dead spermatozoa greater than 42%, preferably the rate of dead spermatozoa detected being greater than 70%,

[0041] - the method is a method for the in vitro or ex vivo diagnosis of astheno-necro-zoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in said individual.

[0042] The invention also relates to a kit for the in vitro or ex vivo diagnosis of asthenozoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in an asthenozoospermic individual, comprising detection means configured for the detection, in a biological sample of said individual, of at least one marker chosen from: an accumulation of lipid droplets greater than a reference value, a content of carnitine or carnitine derivatives lower than a reference value, a content of ACSBG2 protein lower than a reference value, a content of ACSBG2 transcripts lower than a reference value, an alteration of the sequence of the ACSBG2 transcripts compared to a reference sequence, and an alteration of the sequence of the ACSBG2 gene.

[0043] Preferably, the detection means comprise means for staining said lipid droplets in the spermatozoa and / or the seminal fluid, preferably the staining means being means according to the Oil-RedO technique.

[0044] Advantageously, the kit according to the invention further comprises detection means configured for the detection of a level of dead spermatozoa in said biological sample greater than a reference value.

[0045] The invention also relates to a use of at least one marker chosen from: an accumulation of lipid droplets greater than a reference value, a content of carnitine or carnitine derivatives less than a reference value, a content of ACSBG2 protein less than a reference value, a content of ACSBG2 transcripts less than a reference value, an alteration of the sequence of the ACSBG2 transcripts compared to a reference sequence, and an alteration of the sequence of the ACSBG2 gene, for the in vitro or ex vivo diagnosis of asthenozoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in an individual, said marker being detected in a biological sample of said asthenozoospermic individual.

[0046] Preferably, said at least one detected marker is chosen from: the accumulation of lipid droplets, in the spermatozoa and / or the seminal fluid, greater than a reference value, the content of carnitine and / or carnitine derivatives, in the seminal fluid of the patient and / or in the spermatozoa, less than a reference value, the content of ACSBG2 protein, in the spermatozoa, less than a reference value, the content of ACSBG2 transcripts, in the spermatozoa, less than a reference value, the alteration of the sequence of the ACSBG2 transcripts, in the spermatozoa, compared to a reference sequence, the alteration of the sequence of the ACSBG2 gene, in the spermatozoa, the biological sample of said individual is a sperm sample.

[0047] Advantageously, the use according to one of the preceding variants is for the in vitro or ex vivo diagnosis of astheno-necro-zoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in said individual.

[0048] The present invention is illustrated in the following example and with the support of the figures according to which:

[0049] [Fig. 1]: represents the alteration of the ACSBG2 gene sequence observed in patient P1;

[0050] [Fig. 2]: represents (A) the quantitative and qualitative analysis of ACSBG2 transcripts detected in patients P1 and P2 and (B) the sequencing of said transcripts from patient P1;

[0051] [Fig. 3]: represents (A) the detection by immunofluorescence or (B) by western blot of the ACSBG2 protein in patients P1 and P2;

[0052] [Fig. 4]: represents (A) the detection of the accumulation of lipid droplets in patient P1 and (B) the quantitative analysis of said lipid droplets in patients P1 and P2;

[0053] [Fig. 5]: represents (A) the detection of carnitine and acylcarnitine in the spermatozoa of patients P1 and P2 and (B) the detection of carnitine in the seminal fluid of patients P1 and P2, in relation to other markers normally associated with carnitine and present in seminal fluid.

[0054] 1 / Example 1 - Material and method 7.7 - Ethical authorizations

[0055] The study was conducted in accordance with ethical guidelines (Declaration of Helsinki). The collection and use of semen samples from control individuals and patients, as well as genetic analyses of patients, were authorized by the Comité de Protection des Personnes CPP Ile de France III (CPP n° SC2748. A. Touré, E. Dulioust, AP-HP Cochin). Informed consent was obtained from all patients and control individuals before their inclusion in the study.

[0056] 7.2 - Patient identification

[0057] Patients P1 and P2 agreed to participate in the inventors' research program during their infertility management. Patient P1 presented with severe astheno-necrozoospermia (mean motility 0% and viability 6%; 3 spermograms) and was identified by exome sequencing as carrying a homozygous truncating mutation in the ACSBG2 gene. The inventors characterized the consequences of this mutation and in particular highlighted an accumulation of lipid droplets in the spermatozoa of patient P1 compared with the spermatozoa of control subjects.

[0058] Following this unprecedented observation, the inventors used the lipid droplet staining technique on sperm smears from 15 patients with functional asthenozoospermia for whom no genetic information was available. Patient P2 was thus identified and also presented severe asthenonecrozoospermia (mean mobility 0% and viability 2%; 3 spermograms) with a similar accumulation of lipid droplets in his spermatozoa. The characterization of the two patients P1 and P2, presented in this invention application, was then carried out in parallel.

[0059] [Table 1]

[0060] Patient P1 Patient P2 Normal values

[0061] Volume (ml) 2.3 2.1 > 1.4 pH 7.7 7.7 > 7.2

[0062] High Viscosity High

[0063] Numbering (10 6 ) 63 301.6 > 39 [35-40]

[0064] Total mobility 0 0.3 > 42 [40-43]

[0065] Progressive mobility 0 0 > 30 [29-31]

[0066] Vitality 6 2 > 54 [50-56]

[0067] Typical forms 17.7 19.3 > 23 [20-26]

[0068] Sperm parameters of patients P1 and P2 (average of 3 spermograms) 1.3 - Sperm samples

[0069] Semen samples were obtained by masturbation after 2 to 7 days of sexual abstinence. The evaluation of semen parameters was carried out according to the procedures established by the World Health Organization (WHO). Sperm viability was assessed by eosin-nigrosin staining and sperm morphology was analyzed on sperm smears stained by the Schorr method, according to the David classification (Auger et al. 2016). Semen samples from so-called “control” individuals (hereinafter “control individual(s)”) were selected on the basis of normal values ​​for volume, pH, count, motility, viability and morphology, in accordance with WHO criteria.

[0070] 1.4 - Exomic sequencing

[0071] Whole-exome sequencing of patients was performed on genomic DNA extracted from blood using the Oragen DNA Extraction Kit (DNAgenotech®, Ottawa, Canada). Coding regions and intron / exon junctions were sequenced on the China-based Novogen platform (agilent v6, HiSeqX) after enrichment with Agilent kits (Agilent Technologies, Wokingham, UK).

[0072] Sequencing data were analyzed by alignment with the GRCh38 reference genome, using a previously published bioinformatics pipeline (Lorès et al. Am J hum genet 2019). Variants were validated by Sanger sequencing on ABI 3130XL equipment (Applied Biosystems).

[0073] 1.5 - Analysis of transcripts by RT-PCR

[0074] Total RNA (800–1,000 ng) was extracted from 5–10 million human spermatozoa using a NucleoSpin RNA kit (Macherey-Nagel; Düren, Germany) and processed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific; Waltham, MA, USA) following the manufacturer's protocol. PCR reactions were performed from 50 ng of reverse-transcribed mRNA with GoTaq DNA polymerase (Promega) using ACSBG2 gene-specific primers with 40 amplification cycles (95°C, 30 s; 55°C, 30 s; 72°C, 1 min).

[0075] Amplicons were analyzed by agarose gel electrophoresis or purified using the GeneJET Gel Extraction Kit (Fermentas, Thermo Fisher Scientifics; Waltham, MA, USA) for sequencing (Eurofins Scientific, France). Sequencing results were analyzed using BioEdit software (Ibis Therapeutics; Carlsbad, CA, USA).

[0076] 1.6 - Protein Analysis by Western Blot 2 to 10 million spermatozoa were washed with M2 medium (Sigma-Aldrich) and then with PBS buffer by centrifugation at 10,000 xg at room temperature for 10 min. The resulting sperm pellets were resuspended and denatured in Laemmli buffer and incubated at 95°C for 5 min. After centrifugation at 13,000 xg at 4°C for 10 min, the supernatant was collected and 5% p-mercaptoethanol was added before further incubation at 95°C for 5 min. The denatured protein samples were loaded onto SDS-PAGE gel (8% acrylamide / bisacrylamide [40% 37.5:1]) and transferred to nitrocellulose membranes. Membranes were blocked with PBS-0.1% Tween-5% milk solution and immunoblot analysis was performed using the primary antibodies ACSBG2 (Sigma-Aldrich) and Tubulin (Sigma-Aldrich).The revelation was made by chemiluminescence (Millipore kit, Immobilion Forte Western HRP Substrate).

[0077] 1.7 - Protein analysis by immunofluorescence

[0078] 10 μL of sperm samples were spread on a Superfrost Plus slide (Menzel Glasbearbeitungswerk, GmbH & Co. KG) and fixed by incubation in PBS / 4% PFA for 10 min. The slides were incubated for 20 min at 95°C in citrate buffer (H-3300, VectorLabs) and then treated with 0.2% Triton in PBS buffer for permeabilization. A blocking step was performed by incubation in PBS / 1% BSA for 1 h. The slides were then incubated overnight with the primary antibodies rabbit ACSBG2 (Sigma-Aldrich) and mouse Tubulin (Sigma-Aldrich) at 4°C. After washing in PBS buffer, the slides were incubated with anti-mouse Alexa Fluor 488 (Invitrogen) and anti-rabbit Alexa Fluor 568 (Invitrogen) secondary antibodies for 1 h at room temperature. The slides were washed in PBS buffer and mounted in Vectashield medium (Vector Laboratories) supplemented with 0.5 pg / mL DAPI.Slides were analyzed with a Nikon Eclipse E600 epifluorescence microscope (Nikon, Japan). Digital images were acquired with a cooled charge-coupled device (CCD) camera (Hamamatsu Co.) with identical instrument settings, and using MetaMorph software (Molecular Devices).

[0079] 1.8 - Detection of lipid droplets

[0080] 10 μL of sperm samples were spread onto a Superfrost Plus slide (Menzel Glasbearbeitungswerk, GmbH & Co. KG) and fixed by incubation in PBS / 4% paraformaldehyde buffer for 10 min. The slides were then treated with 50 mM NH4Cl / PBS solution for 10 min. After permeabilization in 0.5% tritonX-100 solution for 4 min, the slides were incubated in 60% Oil-red-O / Triethylphosphate (Sigma Aldrich) solution for 45 min. Nuclei were counterstained with Mayer hematoxylin and the slides were mounted with Vectashield medium (Vector Laboratories). Digital images were acquired with a cooled charge-coupled device (CCD) camera (Hamamatsu Co.) with identical instrument parameters, and using MetaMorph software (Molecular Devices).

[0081] 1.9 - Carnitine and acylcarbamitines dosage in spermatozoa

[0082] Sperm lysates were prepared by sonication in distilled water. 20 μL aliquots were spotted onto Whatman membrane filters until completely absorbed. Dried spots were processed for tandem mass spectrometry analysis as previously described (REF: PMID9365395). Data were acquired using a Micromass Quattro Micro API spectrometer equipped with a 2795 high-performance liquid chromatography module and a data system controlled by the MassLynx 4.1 operating system (Waters, Milford, MA).

[0083] 1.10 - Carnitine dosage in seminal fluid

[0084] 500pL of sperm were centrifuged for 15 minutes at 3000g and the supernatant corresponding to the seminal fluid was used for biochemical assays of fructose, zinc, citric acid, prostatic acid phosphatase, carnitine and neutral alpha-glucosidase, following the procedures established by the WHO.

[0085] 21 Example 2 - Results

[0086] 2. 1 - Alteration of the ACSBG2 gene sequence as a marker for in vitro or ex vivo diagnosis of asthenozoospermia linked to dysfunction of sperm lipid metabolism (figure 1)

[0087] Figure 1 highlights the homozygous variant c.1089-2A>G, affecting an intronic residue of the splice acceptor site by sequencing the genomic DNA of patient P1 and its comparison with the reference sequence ACSBG2 (Acyl-CoA Synthetase Bubblegum 2; Gene ID: 81616). Comparison of the electropherogram of patient P1 with that of a control individual shows the substitution at the splice acceptor site of the nucleotide Adenosine (A) by the nucleotide Guanosine (G) in patient P1.

[0088] 2.2 - Reduction in the content and / or alteration of the sequence of ACSBG2 transcripts as marker(s) for the in vitro or ex vivo diagnosis of asthenozoospermia related to dysfunction of sperm lipid metabolism (Figure 2) Figure 2(A) highlights a reduced content of ACSBG2 transcripts in the spermatozoa of patients P1 and P2 compared to spermatozoa of control individuals. The amplification of ACSBG2 transcripts was carried out using oligonucleotides specifically targeting different regions of the ACSBG2 transcripts (exons 9-11 for patient P1 and exons 7-10 for patient P2). The amplification of HPRT transcripts, carried out as a quality control of the transcripts, shows no difference between patients and control individuals. In addition to reduced transcript content, patient P1 shows abnormal amplicon size, indicative of altered transcript sequence.

[0089] Figure 2(B) highlights the alteration of the sequence of ACSBG2 transcripts from patient P1. Comparison of the electropherogram of transcripts amplified in a control individual with that of transcripts amplified in patient P1 indicates the absence of exon 10, confirming that the c.1089-2A>G gene variant leads to a transcript splicing defect in patient P1.

[0090] 2.3 - Reduction in ACSBG2 protein content as a marker for in vitro or ex vivo diagnosis of asthenozoospermia linked to dysfunction of sperm lipid metabolism (Figure 3)

[0091] Figure 3(A) highlights the reduction in ACSBG2 protein content by immunofluorescence on sperm smears from patients P1 and P2 compared to sperm from control individuals. ACSBG2 protein is detected using specific antibodies. This detection is significant and visible for sperm from control individuals only. Conversely, it is very weakly detected in sperm from patients P1 and P2, thus reflecting a reduction in ACSBG2 protein content in sperm from patients P1 and P2. Tubulin is detected as a qualitative control of the analysis.

[0092] Figure 3(B) highlights the reduction of ACSBG2 protein content by western blot on protein extracts of spermatozoa from patients P1 and P2 compared with spermatozoa from control individuals. In addition, spermatozoa from an asthenozoospermic patient (AST1), not presenting alteration of the ACSBG2 gene sequence, are used as a control of the analysis. In patient AST 1, the ACSBG2 protein is well detected. Tubulin is also analyzed in all individuals (patients and control individuals) as a qualitative and quantitative control of the experiment. Like the immunofluorescence analysis, detection by western blot reveals a reduction of ACSBG2 protein content in spermatozoa from patients P1 and P2. 2.4 - Accumulation of lipid droplets in spermatozoa as a marker for in vitro or ex vivo diagnosis of asthenozoospermia linked to dysfunction of sperm lipid metabolism (figure 4).

[0093] Figure 4(A) highlights the accumulation of lipid droplets in a spermatozoon from patient P1 compared to the spermatozoon from a control individual, by OiIRedO histological staining. There is a significant accumulation of lipid droplets in the spermatozoon from patient P1 compared to the spermatozoon from the “control” individual.

[0094] Figure 4(B) demonstrates the accumulation of lipid droplets in the spermatozoa of patients P1 and P2 by counting the presence or absence of droplets in at least 100 spermatozoa from said patients and control individuals. The ratio [number of spermatozoa containing droplets in patients P1 and P2] / [number of spermatozoa containing droplets in control individuals] indicates the level of accumulation and thus reveals a number of lipid droplets more than twice the normal in the spermatozoa of patient P2 and more than three times the normal in the spermatozoa of patient P1.

[0095] 2.5 - Reduction in the content of carnitine and / or acylcarnitines in spermatozoa and / or in seminal fluid as marker(s) for the in vitro or ex vivo diagnosis of asthenozoospermia linked to a dysfunction of the lipid metabolism of spermatozoa (figure 5)

[0096] Figure 5(A) highlights the reduction in the content, quantified by the concentration (pM / L), of carnitine (CO) and acylcarnitine (carnitine derivative, illustrated by C2 acylcarnitines), in the spermatozoa of patients P1 and P2, compared to the spermatozoa of control individuals and to the spermatozoa of an asthenozoospermic patient (AST3). The comparison with patient AST3 highlights that lipid defects are not systematically found in situations of asthenozoospermia, thus allowing the identification of a new category of functional asthenozoospermia.

[0097] Figure 5(B) highlights the reduction in carnitine content in the seminal fluid of patients P1 and P2. The concentration of the various biochemical markers present in the seminal fluid and secreted by the seminal vesicles (fructose), the prostate (zinc, citric acid, prostatic acid phosphatase) and the epididymis (carnitine, neutral alpha-glucosidase) is plotted against reference values ​​corresponding to normal concentrations in "control" individuals. For example, these reference values ​​can be: 2.4 pmol / ejaculate for zinc, 47 pmol / ejaculate for citric acid, 1665 IU / ejaculate for prostatic acid phosphatase, 13 pmol / ejaculate for fructose, 390 nmol / ejaculate for carnitine and 20 ml / ejaculate for neutral alpha-glucosidase (IU (or U): unit corresponding to the quantity of enzyme which catalyzes the transformation of 1 pmol of substrate per minute).A ratio lower than 1 is indicative of a decrease in the content of the analyzed marker. It is noted that carnitine shows a significant reduction in its content in the seminal fluid in both patients P1 and P2. This tendency towards reduction is not observed for all the other markers in patients P1 and P2. The levels of these other markers remain present either at slightly reduced levels or equivalent to the values ​​of the “control” individuals (for patient P1), or at levels higher than the said values ​​of the “control” individuals (for patient P2).

[0098] 2.6 - Conclusions

[0099] It is clear from these experimental data that a method, a kit and a use according to the invention allow the diagnosis of a new category of infertile patients suffering from asthenozoospermia, the cause of which results from a dysfunction of the lipid metabolism of spermatozoa. Thanks to the invention, it is therefore possible to identify such patients in a simple, rapid and effective manner and to offer them specific therapeutic solutions adapted to this dysfunction. This invention makes it possible to open the way to a direct therapeutic treatment of this type of male infertility. In other words, the invention opens the way to a therapeutic treatment intended only for male patients suffering from this particular type of asthenozoospermia, and therefore not relying on their female partners, as is currently the case for ART by IVF / ICSI.This is therefore a real step forward in this area, since to date there is no therapeutic treatment for male infertility, whatever the type of infertility.

[0100] These experimental data demonstrate the effectiveness of the invention for each of the markers identified by the inventors. Of course, these markers can be combined with each other, in any conceivable combination, in order to strengthen and / or consolidate the associated diagnosis.

Claims

CLAIMS 1. Method for in vitro or ex vivo diagnosis of asthenozoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in an asthenozoospermic individual, comprising a step of detecting, in at least one biological sample of said individual, at least one marker chosen from: an accumulation of lipid droplets greater than a reference value, a content of carnitine and / or carnitine derivatives lower than a reference value, a content of ACSBG2 protein lower than a reference value, a content of ACSBG2 transcripts lower than a reference value, an alteration of the sequence of the ACSBG2 transcripts compared to a reference sequence, and an alteration of the sequence of the ACSBG2 gene.

2. Method according to the preceding claim, wherein the biological sample of said individual is a sperm sample and said at least one detected marker is chosen from: the accumulation of lipid droplets, in the spermatozoa and / or the seminal fluid, greater than a reference value, the content of carnitine and / or carnitine derivatives, in the seminal fluid and / or in the spermatozoa, less than a reference value, the content of ACSBG2 protein, in the spermatozoa, less than a reference value, the content of ACSBG2 transcripts, in the spermatozoa, less than a reference value, the alteration of the sequence of the ACSBG2 transcripts, in the spermatozoa, compared to a reference sequence, the alteration of the sequence of the ACSBG2 gene, in the spermatozoa.

3. Method according to the preceding claim, in which the biological sample of said individual is a sperm sample, in which the carnitine content in the seminal fluid is lower than a reference value and in which at least one of the following markers is detected in the seminal fluid: a fructose content, a zinc content, a citric acid content, a prostatic acid phosphatase content or a neutral alpha-glucosidase content greater than or equal to a reference value.

4. A method according to any one of claims 2 or 3, wherein the carnitine content detected in seminal fluid is less than 390 nmol / ejaculate.

5. Method according to any one of claims 2 to 4, in which the carnitine content detected in the seminal fluid and / or in the spermatozoa is zero.

6. Method according to any one of claims 2 to 5, in which the content of ACSBG2 protein detected in the spermatozoa is zero.

7. Method according to any one of the preceding claims, wherein the accumulation of lipid droplets in the spermatozoa and / or seminal fluid is detected via a technique for staining said lipid droplets, preferably via the Oil-RedO technique.

8. Method according to claim 1, wherein the biological sample of said individual is a blood or saliva sample of said individual, the at least one detected marker being the alteration of the sequence of the ACSBG2 gene.

9. Method according to any one of the preceding claims, wherein the detection step comprises the detection of at least two of said markers, preferably at least three of said markers, preferentially at least four of said markers, even more preferentially at least five of said markers and even more preferentially all six markers.

10. Method according to any one of the preceding claims, comprising an additional step of detecting a marker, said marker being a rate of dead spermatozoa greater than 42%, preferably the rate of dead spermatozoa detected being greater than 70%.

11. Method according to any one of the preceding claims, for the in vitro or ex vivo diagnosis of astheno-necro-zoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in said individual.

12. Kit for the in vitro or ex vivo diagnosis of asthenozoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in an asthenozoospermic individual, comprising detection means configured for the detection, in a biological sample of said individual, of at least one marker chosen from: an accumulation of lipid droplets greater than a reference value, a content of carnitine or carnitine derivatives lower than a reference value, a content of ACSBG2 protein lower than a reference value, a content of ACSBG2 transcripts lower than a reference value, an alteration of the sequence of ACSBG2 transcripts compared to a reference sequence, and an alteration of the sequence of the ACSBG2 gene.

13. Kit according to the preceding claim, in which the detection means comprise means for staining said lipid droplets in the spermatozoa and / or the seminal fluid, preferably the staining means being means according to the Oil-RedO technique.

14. Kit according to any one of claims 12 or 13, further comprising detection means configured for the detection of a level of dead spermatozoa in said biological sample greater than a reference value.

15. Use of at least one marker chosen from: an accumulation of lipid droplets greater than a reference value, a content of carnitine or carnitine derivatives less than a reference value, an ACSBG2 protein content less than a reference value, an ACSBG2 transcript content less than a reference value, an alteration of the sequence of the ACSBG2 transcripts compared to a reference sequence, and an alteration of the sequence of the ACSBG2 gene, for the in vitro or ex vivo diagnosis of asthenozoospermia resulting from a dysfunction of the lipid metabolism of spermatozoa in an asthenozoospermic individual, said marker being detected in a biological sample of said individual.

16. Use according to the preceding claim, wherein said at least one detected marker is chosen from: the accumulation of lipid droplets, in the spermatozoa and / or the seminal fluid, greater than a reference value, the content of carnitine and / or carnitine derivatives, in the seminal fluid of the patient and / or in the spermatozoa, less than a reference value, the content of ACSBG2 protein, in the spermatozoa, less than a reference value, the content of ACSBG2 transcripts, in the spermatozoa, less than a reference value, the alteration of the sequence of the ACSBG2 transcripts, in the spermatozoa, compared to a reference sequence, the alteration of the sequence of the ACSBG2 gene, in the spermatozoa, and wherein the biological sample of said individual is a sperm sample.

17. Use according to claim 15 or 16, for the in vitro or ex vivo diagnosis of astheno-necro-zoospermia resulting from a dysfunction of the lipid metabolism of the spermatozoa in said individual.

Images