WO2018092170A1

WO2018092170A1 - Method for in vitro diagnosis of male infertility

Info

Publication number: WO2018092170A1
Application number: PCT/IT2017/000253
Authority: WO
Inventors: Salvatore Longo
Original assignee: Lta-Biotech S.R.L.
Priority date: 2016-11-15
Filing date: 2017-11-14
Publication date: 2018-05-24
Also published as: IT201600115036A1

Abstract

The present invention relates to a method for in vitro diagnosis of male infertility or for in vitro evaluation of the quality of cryopreserved spermatozoa by the determination, in the seminal fluid, of the content of antioxidant, vitamin and representative of oxidative/nitrosative stress compounds and purine, pyrimidine and energy metabolism compounds.

Description

Method for in vitro diagnosis of male infertility

The present invention regards a method for in vitro diagnosis of male infertility. ^!

In more detail, the invention regards a method for in vitro diagnosis of male infertility or for the evaluation of spermatozoa quality in cryopreserved seminal fluid by determining in the seminal fluid the content of antioxidant, vitamin compounds, representative of oxidative/nitrosative stresses and purine, pyrimidine and energy metabolism.

As it is known, according to recent statistics, about 240,000 new marriages/year are celebrated in Italy: at 2 years, around 48,000 couples find difficulties to conceive, so over 20,000 new couples per year seek medical advice for infertility and about half of them are subjected to assisted fertilization treatments (1 , 2). Also in the Western world it is estimated that infertility affects about 15-20% of couples (3). Epidemiological data (4) also indicate that infertility of the couple is attributed to:

• a male cause, for 30-40%;

• a female pathology, for 30-40%;

• a health problem of both, for 20%;

• an idiopathic cause for the remaining 10-20%.

Referring to male infertility, it is possible to obtain epidemiological data indicating the percentage ranges of the causes of this pathology (4):

Infections 6.6 -26.0

Varicocele 12.3 -39.0

Chromosomal abnormalities 3.0-12.0

Cryptorchidism 3.0- 8.5

Endocrinopathies 0.6 - 8.9

Obstructions 1.5-7.0

Idiopathic 30 - 48.5

Sexual 1.7 - 5.7

K. testicle 0.0-2.3 Systemic Diseases 0.0-5.0

It is immediately evident that a very high percentage (about 35%) is of unknown (idiopathic) etiology, that is, the causes are unknown and the diagnosis is based exclusively on the impossibility of the affected individual to procreate. The obvious consequence is that, for all these cases, there is no valid therapeutic approach that is capable of modifying the pathological picture.

If the cases of male infertility diagnosable through simple objective examination are excluded, the diagnostic pathway provides spermiogram as central laboratory exam (4). This exam is conducted following precise indications given by the World Health Organization (WHO) guidelines (5), which can be summarized as follows.

First of all, the pre-examination instructions must be scrupulously observed; it is essential that prior to the investigation a 3- to 5-day sexual abstinence be maintained to reduce casual variations, as the production of the seminal fluid and spermatozoa concentration are subject to considerable variability and for this reason, furthermore, it is useful to repeat it 2 or 3 times. Preferably, the collection should be done at the lab, for reading within one hour the issue; exceptionally, on a reasoned request, it can be made in a domestic environment, protecting the sample from thermal excursions (20 °C -> 37 °C) and in a proper container, such as that sterile for urine. The parameters to be considered in the evaluation of seminal fluid, whose alteration is often indicative of specific pathologies, are:

1. volume; must be> 2 ml; volume reductions (hyposia) can be found in retrograde ejaculation, in the absence of seminal vesicles, in hypogonadism or in alterations of the sympathetic system;

2. pH; it is between 7.2 and 8.0;

3. fluidization; physiologically it occurs within a time ranging from 10 to 60 minutes;

4. viscosity; when increased it can be defined by graduation (+,

++, +++); 5. spermatozoa count; they are indicated for number/ml (normal> 20 millions/ml) and for concentration/ejaculation (normal> 40 millions);

6. motility; it is evaluated freshly in Makler's room, after one hour from the emission and distinguishing the spermatozoa movement in: a. linear fast; b. linear slow; c. diskinetic or non-linear; d. agitation in locu or in situ; e. motionless. Reference values for normality are "a + b"≥ 50% or "a"≥ 25%. Motility abnormalities are often determined by phlogosis, anti-spermatozoa antibodies, structural alterations in the spermatozoa, varicocele, partial obstruction of the seminal ways, etc.;

7. morphology; it will indicate, in addition to the spermatozoa with a typical form (> 30%), also the distinct atypia among those of the head, neck and tail of the spermatozoa;

8. evaluation of the non-nemaspermic cellular component; erythrocytes, germ line elements, cleavage epithelial cells, sperm agglutination areas, prostatic corpuscles; leukocytes, when increased (> 1 million/ml), are expression of phlogosis of the seminal ways;

9. Optional tests; among the most important are the vitality test (with eosin coloration to detect the percentage of live spermatozoa, normal for values>70%) and the MAR (Mixed Anti-globulin Reaction) test for the research for anti-permeant antibodies.

In the final evaluation, it should be taken into account that spermatogenesis lasts about 85 days and therefore febrile episodes (> 38.5 °C), surgical treatments, and a certain amount of drugs may, even transiently, alter the outcomes of a spermiogram.

The terminology used in describing the spermiogram outcomes is shown below.

Normozoospermia: result of values within the norm.

Oligozoospermia: nemaspermic concentration <20 millions/ml.

Asthenozoospermia: <50% spermatozoa with fast and slow (a + b) motility or <25% with progressive rapid motility (a).

Teratozoospermia: <30% of spermatozoa with normal form. Oligo-astheno-teratozoospermia: Anomaly of all of three previous variables.

Azoospermia: absence of spermatozoa in ejaculation.

Hyposia: decreased ejaculate volume (<1-1.5 ml).

Aspermia: absence of ejaculation.

From the above, it is evident that laboratory analysis dedicated to the search for male infertility causes does not provide any type of biochemical and biochemical-clinical investigation for the research of specific biological indicators related to infertility. It should also be underlined that the spermiogram is based mainly on the evaluation of parameters determined by the operator. That is, the subjective component of the analyst is predominant in the final result of the examination, making the variability of the analysis between laboratory to laboratory very wide, and also operator to operator. It should be further noted that abnormal results in the spermiogram analysis do not always identify the cause of male infertility and therefore do not address any therapeutic treatments (4). Lastly, a large percentage of idiopathic male infertility has a normal spermiogram indicating the need to introduce analysis of biochemical type on semen fluid to detect alterations of specific compounds linked to known biochemical functions.

In the last few years, a series of experimental trials have been undertaken aimed to the biochemical characterization of the seminal fluid of healthy donors and subjects with infertility problems in order tohighlight one or more substances present in altered concentrations in subjects with infertility and those, therefore, be used as biomarkers in the diagnosis of male infertility (6). In all of these studies, the various substances in the seminal fluid were analyzed with high reproducibility and sensitivity methods in order to have objective diagnostic tests (not dependent on the operator's evaluation) that could fill the existing analytic gaps in the traditional spermiogram. Some of these studies have adopted the so- called proteomic approach for which protein profiles of normal and pathological seminal fluid were measured by searching for one or more protein molecules with abnormal values in seminal fluid of subjects with infertility (6-14). Other studies, on the contrary, are based on the use of the so-called "untargeted" metabolomic approach, for which concentrations of a large number of low molecular weight compounds are measured regardless of their biochemical role and their biological function. Even in this case, the purpose was to highlight one or more molecules present in anomalous concentrations in pathological seminal fluid compared to normal ones. The results of these studies have highlighted some potentially useful substances for the diagnosis of male infertility (15- 20). In some studies, the strategy of measuring specific oxidative stress compounds (21-24) was utilized evaluating, in some cases, concentrations of antioxidant compounds such as coenzyme Qio and alpha-tocopherol (25).

The limitation of the proteomic approach is linked to the high analytical cost (very expensive instrument and reagents required for analyzes), to the difficult standardization of results and to the necessity for highly qualified personnel both for performing analyses and for the interpretation of analytical results.

Similar limitations are found in the "untargeted" metabolomic approach for the seminal fluid analysis (26), for which very high cost instrumentation (NMR, LC LC/MS-MS, GC/MS-MS) and hardly to find in biochemical and biochemical-clinical laboratories are required, as well as for the use of microRNAs as infertility biomarkers (27, 28) for which limitations of both analytical quality (poor reproducibility and high variability between subjects) and analytical costs exist.

It is also known the patent application WO 2008/142396 which bases the diagnosis of male infertility exclusively on uridine concentration determination in seminal fluid. However, this method of diagnosis could not be considered reliable because uridine is not always altered in the infertile subjects.

In light of the outlined above, it is therefore evident that there is a need to have new biomarkers for the measurement of infertility measurable in seminal fluid with reliable analytical methods, characterized by low cost and ease of execution. In this context, the solution according to the present invention is introduced, which is intended to provide a method for diagnosis of male infertility by biochemical analysis of seminal fluid, or for evaluating the fertility of cryopreserved spermatozoa by means of a "targeted" metabolomic approach and devoted to the evaluation of specific antioxidant, vitamin compounds, representative of oxidative/nitrosative stresses, representative of purine, pyrimidine and energy metabolism.

According to the present invention, the concentrations of a series of compounds belonging to the above-mentioned chemical classes have been determined in seminal fluid samples of healthy donors and with infertility problems and pathological spermiogram analysis. More specifically, the population of subjects with infertility problems was represented by a first group of individuals with normozoospermia (idiopathic infertility), a second group with oligo-astheno-teratozoospermia, and a third group comprising the sum of those with oligozoospermia, asthenozoospermia and teratozoospermia, on the basis of the spermiogram analysis conducted and evaluated according to WHO criteria.

The results showed that many (twenty-one) of the measured compounds are related to anomalies in the spermiogram and, more importantly, they are altered in cases of idiopathic infertility with almost normal spermiogram.

The analysis was carried out using an analytical method such as High Performance Liquid Chromatography (HPLC) characterized by sensitivity, reproducibility, specificity, low analytical costs, easiness of execution, wide diffusion in biochemical and biochemical-clinical laboratories.

Based on the results obtained, it is immediately relevant that, with respect to the values found in fertile controls, ascorbic acid, all-trans retinoic acid, all-trans retinol, alpha-tocopherol, gamma tocopherol, total carotenoids (calculated as sum of astaxanthin, lutein, zeaxanthin, beta- apo-trans-carotenal, beta-cryptoxanthin, lycopene, alfa-carotene, beta- carotene), malondialdehyde, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), nitrites (-N0₂ ^"), nitrates (N0₃ ^"), creatinine, cytosine, cytidine, uracil, beta- pseudouridine, hypoxanthine, xanthine, uridine, guanine, guanosine are compounds resulting in altered concentration in the seminal fluid of all patients with infertility. It can be stated that this panel of twenty-one substances, belonging to water soluble (ascorbic acid) and liposoluble antioxidants (all-trans retinol, all-trans retinoic acid, alpha-tocopherol, gamma-tocopherol, total carotenoids), to compounds representative of oxidative/nitrosative stress (malondialdehyde, 8-OHdG, nitrites and nitrates), to pyrimidine metabolism (beta-pseudouridine, cytidine, cytosine, uracil and uridine), purine metabolism (hypoxanthine, xanthine, inosine, guanine, guanosine) and energy metabolism) allows to discriminate healthy subjects from those with infertility, hence assuming the biochemical and biochemical-clinical significance of objective biomarkers for the diagnosis of male infertility. This differentiated diagnosis, based on the profiles of the different metabolites, was made more easily executable by unifying the data of the differences between the groups for the various compounds in a highly predictive Biomarker Score to distinguish the different groups of male patients with infertility.

In addition, experimental data show that not all of the above twenty one mentioned parameters are altered in the various patient groups with infertility. For example, patients with normozoospermia (idiopathic infertility) differ from those with spermiogram abnormalities (oligo-astheno- teratozoospermia, oligozoospermia, asthenozoospermia, teratozoospermia) for ascorbic acid, beta-pseudouridine, cytidine, inosine (p <0.05) content. Moreover, there is a differentiation for many parameters (see Tables) as shown by comparing the controls and normozoospermic, and the controls with patients with spermiogram abnormalities (this group being the sum of oligo-astheno-teratozoospermic, oligozoospermic, asthenozoospermic and teratozoospermic). In practice, this indicates the possibility of performing a differentiated infertility diagnosis based on different profiles of interest metabolites, quantified in the seminal fluid of different infertile donors. This differentiated diagnosis, based on the profiles of the different metabolites (which assume the meaning of male infertility biomarkers), has been made more easily executable by unifying the differences between the groups for the different compounds into a highly predictive Biomarker Score to distinguish the different groups of male patients with infertility.

Based on the experimental results reported below (Figure 1 ), it is understood that to have a statistical certainty greater than 99% to discriminate against fertile and infertile subjects, avoiding false positives, the minimum number of biomarkers needed to undergo infertility diagnosis according to the present invention, selected from the twenty-one mentioned above, corresponds to eleven.

The results, obtained through the measurement of this panel of low molecular weight compounds are not only relevant for a diagnostic but also for a therapeutic point of view. In fact, because of the biochemical significance of the various compounds that play a role as biomarkers, it can be stated that all subjects with infertility (including idiopathic individuals with normozoospermia) exhibit a deficiency of specific antioxidant substances (ascorbic acid, all-trans retinol, all-trans retinoic acid, alpha-tocopherol, gamma-tocopherol, total carotenoids) which can be corrected by targeted administration of appropriate cocktails containing the defective antioxidants. Likewise, the altered concentrations of purine compounds (hypoxanthine, xanthine, inosine, guanine, guanosine), pyrimidine compounds (beta-pseudouridine, cytidine, cytosine, uracil and uridine) and those related to energy metabolism (creatinine) could be appropriately corrected through ad hoc food regimens or through the administration of drugs, nutraceuticals, supplements, etc. Correction of the profile of the different metabolites, measured in the seminal fluid of subjects with infertility, may thus result in a resolution of the problem of procreation, greatly increasing the value of biochemical analysis according to the present invention.

It has to be underlined that compared to the proteomic (6-14), metabolomic (15-24, 26) molecular (27-28) approaches, the analytical panel according to the present invention can be determined with highly sensitive methods that need instrumentation currently in use in biochemical and biochemical-clinical laboratories and at low costs. In addition, the usable methods are easy to execute and the equipment requires very low maintenance costs. Finally, the cost of a single analysis, compared to proteomic, metabolomic, and molecular analyzes, is 10 to 20 times lower, making the method of the present invention applicable on a large scale.

In the study according to the present invention, the substances have been analyzed by HPLC methods (29-38). However, it should be mentioned that various alternative methods of colorimetric, enzymatic, immunochemical, mass spectrometry, gas chromatography can be used, which allow quantification of the compounds of interest in the seminal fluid. Regardless of the analytical method used for their determination, the values of the compounds dosed in the seminal plasma allow the biochemical diagnosis of infertility also by computing the Biomarker Score (derived from values of metabolic interest measured in the seminal fluid).

Referring to the above, with respect to the patent application WO 2008/142396, on the basis of the experimental results reported below, it is possible to observe that the uridine value in the seminal fluid of fertile subjects was 2,152.04 ± 333.03 pmol/l, while in the total of infertile subjects, the value was 1 ,818.99 ± 851.20. Although the uridine of patients is significantly lower than normal, data indicates that not all of the infertile subjects have uridine value lower than that of fertile. This occurs only in 30% of cases, therefore, in 70% of cases of infertility, it is not possible to carry out a male infertility diagnosis by only determining uridine.

Therefore, the specific object of the present invention is a method for in vitro diagnosis of male infertility or in vitro evaluation of the quality of cryopreserved spermatozoa (or cryopreserved seminal fluid), said method comprising or consisting of determining, by biochemical analysis of a seminal fluid or seminal plasma sample of a subject, an unique Biomarker Score of said subject based on the concentration of at least eleven or at least fifteen of twenty-one compounds or at least all twenty-one compounds selected from ascorbic acid, beta-pseudouridine, cytidine, cytosine, creatinine, guanine, guanosine, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), inosine, hypoxanthine, malondialdehyde, nitrite, uracil, uridine, xanthine, all-trans retinol, all-trans retinoic acid, alpha-tocopherol, gamma- tocopherol, coenzyme Qi₀, total carotenoids calculated as sum of astaxanthin, lutein, zeaxanthin, beta-apo-trans-carotenal, beta- cryptoxanthin, lycopene, alpha-carotene, beta -carotene, wherein the Biomarker Score is the sum of the numerical values assigned to the concentration of each of said compounds of the subject relative to the maximum and minimum concentration cut-off values of each compound, determined in fertile subjects, which statistically discriminates against fertile subjects infertile, for example maximum and minimum confidence intervals of 95% of each compound, determined in normal fertile subjects. In particular, the numerical values resulting out of the concentrations of 95% confidence interval of the same compounds determined in fertile subjects, are added.

The concentration of ascorbic acid, guanosine, guanine, uridine, all-trans retinol, uridine, all-trans retinoic acid, alpha-tocopherol, total carotenoids in infertile subjects is tendentially lower than the concentration of the same compounds in fertile controls, while the beta- pseudouridine, cytidine, cytosine, creatinine, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), inosine, hypoxanthine, malondialdehyde, nitrites, uracil, xanthine gamma tocopherol, coenzyme Q10, in infertile subjects is tendentially greater than concentration of the same compound in fertile controls.

According to a form of realization of the present invention, said at least eleven compounds may be ascorbic acid, alpha-tocopherol, total carotenoids, malondialdehyde (MDA), 8-hydroxy-2'deoxy-guanosine (8- OHdG), nitrites, creatinine, cytosine, cytidine, guanine, guanosine.

The method according to the present invention can also be utilized to monitor the therapeutic efficacy of drugs for the treatment of male infertility or the improvement of said compounds in the seminal fluid.

According to a form of realization of the present invention, the method can include or consist of: (a) determining the concentration of each compound of said at least eleven, at least fifteen or at least all of the twenty-one compounds in a seminal fluid or seminal plasma biological sample of a subject;

b) associating each concentration of each compound with a first numeric value, such as 0, when the concentration of the compound is equal to or include selected values, respectively maximum and minimum, of the concentration of the cut-offs of the corresponding compound in fertile subjects, and with a second numeric value, different from the first one, as for example 1 , when the concentration of the compound is greater or lower than the maximum and minimum cut-off concentration values respectively of the corresponding compound in fertile subjects;said cut-off values of the maximum and minimum concentrations are the values of maximum and minimum concentrations of each compound determined in fertile subjects, which statistically discriminate fertile and infertile subjects, such as the maximum and minimum values of the 95% confidence interval for each compound, determined in normal fertile subjects;

c) calculating the personal Biomarker Score of the subject by adding the numerical values associated to the concentrations of said at least eleven, at least fifteen or at least all of the twenty-one compounds of said subject;

d) obtaining the probability of the said subject of being a fertile subject, or being infertile and/or being infertile with normozoospermia, or infertile with any of the oligo-astheno-terato-zoosospermia, oligozoospermia, asthenozoospermia and teratozoospermia, by comparing the personal Biomarker Score with control references that associate each Biomarker Score of selected subjects in at least one group selected among fertile subjects, infertile subjects and/or infertile subjects with normozoospermia, and/or infertile subjects with any of the oligo-astheno- terato-zoosospermia, oligozoospermia, asthenozoospermia and teratozoospermia with the posterior probability that each Biomarker Score will belong to at least one said group.

In particular, control references may be obtained by a method which includes or consists of: (a) selecting one or more groups of subjects chosen in a group of fertile subjects, and/or a group of infertile subjects and/or a group of infertile subjects with normozoospermia and/or infertile subjects with any of the oligo-astheno-terato-zoospermia, oligozoospermia, asthenosospermia and teratozoospermia;

(b) determining the concentration, in a biological sample of the liquid or seminal plasma of each of the subjects of said one or more groups of at least eleven or at least fifteen of the twenty-one compounds or at least all of the twenty compounds selected from ascorbic acid, beta- pseudouridine, cytosine, cytidine, creatinine, guanine, guanosine, 8- hydroxy-2'-deoxy-guanosine (8-OHdG), inosine, hypoxanthine, malondialdehyde, nitrites, uracil, uridine, xanthine, all-trans retinol, all- trans retinoic acid, alpha-tocopherol, gamma-tocopherol, coenzyme Qio, total carotenoids calculated as sum of astaxanthin, lutein, zeaxanthin, beta-apo-trans-carotenale, beta-cryptoxanthin, lycopene, alpha-carotene, beta-carotene;

c) selecting, in the group of fertile subjects, maximum and minimum concentrations cut-off values of each of said at least eleven, at least fifteen or at least twenty-one compounds, said maximum and minimum concentration cut-off values are the concentration values maximum and minimum of each compound, determined in fertile subjects, which statistically discriminate against fertile and infertile subjects, for example, are the maximum and minimum values of the 95% confidence intervals of each compound determined in normal fertile subjects;

d) associating the concentration of each of said at least eleven, at least fifteen or at least twenty-one compounds of each subject of said one or more groups to a first numerical value such as 0, when the concentration of the compound is equal to or between the maximal and minimal values of concentration cut-off and to a second numeric value, different from the first, such as 1 , when the concentration of the compound is greater or lower to the maximum and minimum concentrations cut-off values respectively; e) calculating a Biomarker Score of each subject of said one or more groups of subjects summing the numeric values associated to the concentrations of said at least eleven compounds, at least fifteen, or at least all the twenty one compounds of each subject;

f) calculating the posterior probability associated to each Biomarker Score of each subject of each group, said posterior probability being the probability of each Biomarker Score of belonging to said one or more groups of subjects, with the aim to obtain a referring control of fertile subjects and/or infertile subjects and/or infertile subjects with normozoospermia and/or infertile subjects with any of the oligo-astheno- terato-zoospermia, oligozoospermia, asthenosospermia and teratozoospermia.

According to an embodiment of the present invention, said at least eleven compounds may be ascorbic acid, alpha-tocopherol, malondialdehyde (MDA), 8-hydroxy-2'-deoxy-guanosine (8-OHdG), nitrites, creatinine, cytosine, cytidine, guanine, guanosine, total carotenoids calculated as sum of astaxanthin. lutein, zeaxanthin, beta- apo-trans-carotenale, beta-cryptoxanthin, lycopene, alpha-carotene, beta- carotene.

According to an embodiment of the present invention, when the first numeric value is 0 and the second numeric value is 1 , Biomarker Score from 0 to 4 and from 5 to 21 respectively discriminate fertile and infertile subjects respectively; Biomarker Scores from 5 to 12 are associated to a probability of infertility with normozoospermia greater than the probability of infertility with any of the oligo-astheno-teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia, while Biomarker Score from 13 to 21 are associated with a probability of infertility with any of the oligo-astheno-terato-zoospermia, oligozoospermia, asthenozoospermia and teratozoospermia greater than the probability of infertility with normozoospermia.

The present invention also concerns a method for providing one or more control references for in vitro diagnosis of male infertility or in vitro evaluation of the quality of cryopreserved spermatozoa, said method comprising or consisting of

(a) selecting one or more groups of subjects chosen in a group of fertile subjects and/or a group of infertile subjects and/or a group of infertile subjects with normospermia and/or infertile subjects with any of the oligo-astheno-teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia;

(b) determining the concentration, in a biological sample of the liquid or seminal plasma of each of the subjects of said one or more groups of subjects, of at least eleven or at least 15 of the twenty-one compounds or at least all twenty-one compounds chosen between ascorbic acid, beta-pseudouridine, cytidine, cytosine, creatinine, guanine, guanosine, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), inosine, hypoxanthine, malondialdehyde, nitrites, uracil, uridine, xanthine, all-trans retinol, all-trans retinoic acid, alpha-tocopherol, gamma-tocopherol, coenzyme Qi₀, total carotenoids calculated as sum of astaxanthin, lutein, zeaxanthin, beta-apo-trans-carotenal, beta-cryptoxanthin, lycopene, alpha- carotene, beta-carotene;

c) selecting in the group of fertile subjects the maximum and minimum values of cut-off concentration of each of said at least eleven, at least fifteen or twenty-one compounds, said maximum and minimum values of concentration cut-off are the maximum and minimum concentration values of each compound, determined in fertile subjects, which statistically discriminate against fertile and infertile subjects, for example the maximum and minimum confidence intervals of 95% of each compound determined in normal fertile subjects;

d) associating the concentration of each of said at least eleven, at least fifteen or at least twenty-one compounds of each subject of said one or more groups of subjects at a first numeric value, such as 0, when the concentration of the compound is equal to or between the maximum and minimum concentration values of cut-off and to a second numerical value, different from the first, such as 1 , when the concentration of the compound is greater or lower than to the maximum and minimum concentration values of cut-off, respectively;

e) calculating a Biomarker Score of each subject of said one or more groups of subjects by summing said first and second numerical values associated with the concentrations of said at least eleven of, at least fifteen of, or at least all the twenty-one compounds of each subject; f) calculating the posterior probability associated with each Biomarker Score of each subject in each group, said posterior probability being the probability that each Biomarker Score belongs to said one or more groups of subjects, with the aim to obtain a control reference of fertile subjects and/or of infertile subjects and/or of infertile subjects with normospermia and/or infertile subjects with any of the oligo-astheno- teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia.

In particular, said at least 11 compounds may be ascorbic acid, alpha-tocopherol, malondialdehyde (MDA), 8-hydroxy-2'deoxy-guanosine (8-OHdG), nitrites, creatinine, cytosine, cytidine, guanine, guanosine, total carotenoids calculated as the sum of astaxanthin, lutein, zeaxanthin, beta- apo-trans-carotenal, beta-cryptoxanthin, lycopene, alpha-carotene, beta- carotene.

Therefore, the present invention also provides a reference tool of healthy (fertile) subjects for the in vitro male infertility diagnosis by evaluating the biochemical quality of cryopreserved seminal plasma or for in vitro diagnosis and/or prognosis and/or monitoring of subjects affected by male infertility categorized into the spermiogram as normozoospermic, or any of the oligo-astheno-teratozoospermic, astheno-spermic, teratozoospermic, oligozoospermic, said reference instrument being obtained by the above-defined method.

In addition, the present invention provides a reference tool for subjects suffering from male infertility categorized by the spermiogram as normozoospermic, or any of the oligo-astheno-teratogosospermic, asthenozoospermic, teratozoospermic, oligozoospermic said reference instrument obtained by the above defined method. In particular, the reference toolt for fertile controls and subjects suffering from male infertility categorized to the spermiogram as normozoospermic, or any of the oligo-astheno-teratogosospermic, asthenozoospermic, teratozoospermic, oligozoospermatic, may include the following values in the aftermath probability for each Biomarker Score belonging to the fertile control group (C) and to the group of subjects suffering from male infertility, categorized at the spermiogram as normozoospermic, or astheno-terato-oligozoospermic, or asthenozoospermic or teratozoospermic or oligozoospermic:

where each value of the posterior probability can have a variability of ± 10%.

Moreover, the reference tool for controls and subjects suffering from male infertility categorized at the spermiogram as normozoospermic, or any of the oligo-astheno-teratozoospermic, oligozoospermic, asthenozoospermic and teratozoospermic, may include the following values of in the aftermath probability for each Biomarker Score belonging to the fertile control group (C) and to one of the at least two of male infertile groups, in which the first group is categorized into the spermiogram as normozoospermic (N), and the second group constitutes the sum of the patients categorized as any Astheno-Terato- Oligozoospermic (ATO), Asthenozoospermic (A), Teratozoospermic (T), oligozoospermic (O):

where each value of the posterior probability can have a variability of ± 10%.

Based on the above, the present invention also provides a reference tool of subjects suffering from male infertility categorized by the spermiogram as normozoospermic, or any of the oligo-astheno- teratozoospermic, oligozoospermic, asthenozoospermic and teratozoospermic, the reference instrument being obtained by the method as above defined.

Since the present invention, by the determination of the 21 biomarkers, allows to effectan evaluation of the biochemical quality of the seminal plasma, it is also possible to utilize the values of the Biomarker Score to subdivide infertile patients into five categories attesting to the severity of alterations in the biochemical quality of plasma, as follows:

Biomarker Score Values between 0 and 4 = normality

Biomarker Score Value between 5 and 8 = low gravity;

Biomarker Score Value between 9 and 13 = moderate gravity;

Biomarker Score Value between 13 and 17 = high gravity;

Biomarker Score Value between 18 and 21 = very high gravity;

This subdivision results particularly useful for evaluating the efficacy of a particular therapeutic treatment for improving the biochemical quality of the seminal plasma and aimed at the care of male infertility.

Methods according to the present invention can be conducted by HPLC with a spectrophotometric or fluorimetric or mass spectrometry detector, by direct spectrophotometric measurements or coupled to colorimetric reactions and enzymatic assays by immunochemical tests, by nuclear magnetic resonance. May, also, be used any other method or analytical technique suitable for determination in the seminal fluid/plasma of one or all of the compounds of interest, such as the microfluidic applied to the lab-on-chip technology, to be used for the diagnosis of male infertility or for the monitoring of the therapeutic efficacy of drugs for the treatment of male infertility or for the improvement of said compounds in the seminal fluid.

It is a further object of the present invention a computer program comprising code means configured in such a way that, when executed on a computer, they perform the steps of each of the above defined methods.

It is a further object of the present invention a pharmaceutical composition or a food supplement comprising or consisting of one or more vitamins of group B, such as B1 , B2, B3, B4, B5, B6, B7, B8, B9, B10, B11 , B12 as active principles, in association with one or more excipients and/or adjuvants pharmaceutically acceptable, said pharmaceutical composition or food supplement being for the use in infertility therapy.

The pharmaceutical composition may further comprise at least one or all of the compounds selected from ascorbic acid, vitamin E (alpha- tocopherol), vitamin A (all-trans retinol), guanosine, guanine, uridine, inosine, or precursors of said compounds, or stimulants of biosynthesis of said compounds (such as a mixture of tocopherols and tocotrienols to supplement vitamin E), antioxidantsreduced glutathione, ascorbic acid, all- trans retinol, lutein, lycopene, coenzyme Q₁₀ and alpha tocopherol, or as their precursors such as for example beta carotene or analogs such as astaxanthin, zeaxanthin, cantaxanthin, capsanthin, citranaxanthin, gamma tocopherol, delta tocopherol, tocopherol acetate, tocotrienols.

The invention will be described in the following for illustrative purposes, but not limited to, with particular referring to some illustrative examples and the following figures, wherein:

Figure 1 shows the percentage distribution of Biomarker Score values in 50 fertile controls (in white) and 140 subjects with infertility (in black);

Figure 2 shows the percentage distribution of Biomarker Score values in 50 fertile controls (in white), in 30 normozoospermic (N) (striped) and 1 10 astheno-teratozoospermic (ATO) + asthenozoospermic (A) + teratozoospermic (T) + oligozoospermic (O) (in black).

Example 1 : Study on the correlation between the concentrations of seminal fluid compounds and male infertility

Materials and Methods

Population studied. Fifty healthy donors with no infertility problems (all donors had procreated) were recruited, aged between 28 and 55 years. The population of subjects with infertility problems was represented by 30 individuals with normozoospermia (idiopathic infertility), 40 with oligo-astheno-terato-zoospermia, 25 with oligozoospermia, 25 with asthenozoospermia and 20 with teratozoospermia, on the basis of the spermiogram analysis conducted and evaluated according to WHO criteria. The fresh ejaculate, after 30 minutes at 37 °C to facilitate liquefaction, was centrifuged for 15 minutes at 1 ,860 x g, in order to obtain seminal fluid without spermatozoa and any other cell species.

Deproteinization of seminal fluid samples has been carried out with a new method that allows to obtain a protein-free extract to be used to analyze low molecular weight compounds of both liposoluble and water- soluble nature (29). To this aim, 500 pi of seminal fluid was added to 1 ml of acetonitrile (HPLC-grade), vortexed vigorously for 60 seconds and incubated at 37 °C for 1 hour in a thermostatic bath. At the end of the incubation, the sample was centrifuged at 21 ,680 xg for 15 minutes at 10 °C to allow sedimentation of the protein precipitate. The supernatant was collected and divided into two aliquots. One was directly analyzed for the quantitative determination of liposoluble low molecular weight compounds, the other (300 μΙ) was added with 1.5 ml of chloroform (HPLC-grade), agitated vigorously for 90 seconds and centrifuged at 21,680 x g for 15 minutes at 10 °C. This step permits the transfer of the organic phase (acetonitrile) into chloroform, leaving, after centrifugation, a higher aqueous phase containing low molecular weight compounds of water- soluble nature (30).

By way of example, but not for limiting purposes, in the study carried out the liposoluble compounds of interest present in the organic extract of seminal fluid (gamma-tocopherol, alpha-tocopherol, coenzyme Qio, all-trans-retinal, all-trans retinoic acid, total carotenoids (as sum of astaxanthin, lutein, zeaxanthin, beta-apo-trans-carotenal, beta- cryptoxanthin, lycopene, alpha-carotene, beta-carotene) were separated and quantified by reverse phase HPLC using a chromatographic method set up by the inventors (29). The chromatographic column was a Gold ODS C-18 (ThermoFisher Scientific, Milan, Italy), of 4.6 mm inner diameter and 10 cm of lenght with particles of 5 m in diameter and 120 A pore, provided with a pre-column of 2 cm and containing the same phase of the separating column.

The separation of the compounds of interest was carried out using two eluents: eluent A, composed by 70% methanol + 30% water; eluent B composed by 100% acetonitrile. A gradient separation was also adopted, constituted as following: 0.5 minutes to 100% of A; 8 minutes at up to 100% of B; 17 minutes with 100% B. The flow of chromatographic run was 1 ml/minute and the temperature of the chromatographic column was steadily maintained at 37 °C (29).

Water-soluble compounds present in the aqueous extract of deproteinized seminal fluid (reduced glutathione, ascorbic acid, malondialdehyde, 8-OHdG, nitrite, nitrate, hypoxanthine, xanthine, uric acid, inosine, adenosine, guanine, guanosine, uracil, beta-pseudouridine, uridine, cytidine, cytosine, orotic acid, creatinine) were separated and quantified by ion-coupled HPLC using a method previously described (30) and applied in numerous experimental and clinical trials to separate the aforementioned substances (31-38) in tissue samples and biological fluids for diagnostic and clinical monitoring purposes. The column used was a Hypersil ODS C-18, 4.6 mm in diameter, 25 cm in length, 5 Mm particles and 120 A pores, equipped with a 2 cm pre-column filled with the same stationary phase. Separation of the compounds was carried out in isocratic form with an eluent consisting of a quaternary ammonium ion (tetrabuthylammonium) at a concentration of 12 mM, a polar organic solvent (methanol) at 0.125%, a salt of weak acid (disodium phosphate) at the concentration of 10 mM, pH 7.00. At the end of each chromatographic run, the column was washed with an eluent composed of acetonitrile (40%), methanol (30%) and water (30%). The flow of chromatographic runs was 1.2 ml/minute and the column was thermostated at 10 °C.

For both separations, the chromatographic column was connected to a HPLC system formed by a 4-ways pump with low pressure eluent mixing, an eluent membrane degasser, and a thermostatic autosampler. The detector used was a spectrophotometric type diode arrays with high sensitivity (equipped with 5 cm flow cell), selected to acquire the chromatographic data between the wavelengths of 200 and 550 nm. The HPLC system was controlled by a PC equipped with software for the management of instruments and the acquisition and processing of chromatographic data. In the case of the analysis of the liposoluble compounds (29), 200 μΙ of the deproteinized seminal fluid extract in acetonitrile were loaded in the column, without any dilution. In the case of the analysis of water- soluble compounds, the samples were diluted 20 times with water and subsequently 200 μΙ were loaded into the column.

The concentration of the various quantized compounds was calculated by correlating the areas of the chromatographic peaks of the seminal fluid samples with the corresponding areas of the chromatographic peaks of ultrapure standard runs at well-known high pureness.

Statistical Analysis

The statistical analysis was carried out using the Statistical Office of Social Sciences (SPSS) version 15.0. All data were analyzed first for normal distribution using the Kolmogorov-Smimov test. Continuous variables were expressed as mean ± SD, categorical variables displayed as frequencies and appropriate parametric tests (t-test Student or ANOVA) or non-parametric (Mann-Whitney U-test, Kruskal-Wallis ANOVA, test χ²) were used to evaluate the significance of differences between subgroups. A value of P less than 0.05 was considered statistically significant.

Within the panel of 26 different metabolites, 21 satisfied the characteristics to be significantly different in the 140 infertile subjects, when compared to values measured in the 50 fertile controls.

In control subjects, for each of these 21 compounds, the 95% percentile confidence intervals were determined. Subsequently, the whole population (controls and patients) was layered into two categories for each biomarker: 1) Negative (0) = subjects with concentration equal or included in the confidence interval 95% percentile of controls and 2) Positive ( 1) = subjects with concentration higher or lower than maximum and minimum values of the confidence interval at 95% percentile of the controls. Finally, each subject was associated to a specific value of biochemical positivity and a "Biomarker Score" (sum of the number of Positive categories) with a range of 0 (none of biomarkers higher or lower than the corresponding 95% percentile of controls) to 21 (each biomarker higher or lower than the corresponding 95% percentile of the controls). The schematization to obtain the Biomarker Score starting from the analysis of the selected circulating compounds, is reported below:

26 compounds dosed in seminal plasma samples of 50 fertile controls and 140 patients with infertility, including 30 individuals (according to the analysis of spermiogram) with normozoospermia (idiopathic infertility), 40 with oligo-astheno-teratozoospermia, 25 with oligozoospermia, 25 with asthenozoospermia and 20 with teratozoospermia;

21/26 compounds were significantly different in infertile subjects with respect to fertile controls;

For each of these 21 compounds (seminal plasma biomarkers), the confidence interval at 95% percentile in the controls was determined;

For each biomarker of seminal plasma, each subject of fertile and infertile is classified as Normal, score 0 (the value of that biomarker equal to or included in the corresponding confidence interval at 95% percentile of the controls) or Positive, score 1 (value of that biomarker higher or lower than the maximum and minimum values of the corresponding confidence interval at 95% percentile of controls);

For each subject, the sum of all the Positive values obtained for the various biomarkers, allowed to calculate the Biomarker Score.

Every fertile control and infertile patient was characterized by a Biomarker Score comprised between 0 (no biomarker higher or lower than the maximum and minimum values of the corresponding confidence interval at 95% percentile of controls) and 21 (all biomarkers higher or lower than the maximum and minimum values of the corresponding confidence interval at 95% percentile of controls).

The Biomarker Score was initially used to distinguish fertile controls from infertile subjects and, subsequently, to distinguish infertile individuals with normozoospermia (idiopathic infertility) from the sum of those with oligo-astheno-terato-zoospermia, oligozoospermia, asthenozoospermia and teratozoospermia. In addition, on the basis of distributions of the Biomarker Score values of fertile controls and infertile patients, the distribution curves of the two groups were calculated and, subsequently, of infertile patients categorized as normozoospermic and the sum of those with oligo-astheno-terato-zoospermia, oligozoospermia, asthenozoospermia and teratozoospermia. In this way it was possible to calculate the posterior probability that a value of Biomarker Score belonged to one of the three different categories: fertile controls, infertile subjects with normozoospermia, infertile subjects with any of the astheno- terato-oligozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia.

RESULTS

In Table 1 are reported mean values ± standard deviation (D.S) and reference intervals of compounds of interest determined in seminal fluid samples of 50 healthy donors

Table 1. Mean values ± (S.D.) and reference intervals to 95% of the compounds of interest determined in seminal fluid samples of 50 healthy donors, expressed in pmol/L

Uracil 4.11 ± 1.86 1.50 - 8.00

Beta-pseudouridine 4.47 ± 2.61 2.00 - 10.00

Hypoxanthine 5.04 ± 2.30 2.00 - 10.00

Xanthine 39.69 ± 10.38 25.00 - 65.00

Uridine 2152.04 ± 333.03 1500.00 - 3500.00

Uric acid 213.52 ± 53.19 120.00 - 350.00

Inosine 2.61 ± 0.75 1.00 - 4.50

Guanine 58.57 ± 15.59 30.00 - 90.00

Guanosine 69.31 ± 12.06 30.00 - 90.00

Orotic Acid 38.97 ± 8.33 20.00 - 60.00

Adenosine 42.37 ± 8.35 20.00 - 60.00

Data obtained on healthy volunteers allowed to determine the concentration of compounds of interest in semen samples, that could be defined "physiological conditions of normality" as they were obtained on donors without any problems with procreation. The concentrations of the individual substances in these samples were used to calculate the 95% percentile confidence intervals (shown in the table as REFERENCE INTERVALS), to calculate the Biomarker Score of the controls and infertile subjects as described above, and to compare them with values determined in seminal fluid samples of 140 donors with infertility problems. The first statistical comparison was conducted without dividing the infertile subjects according to the abnormalities found in the spermiogram, and it is reported in Table 2. Table 2. Mean values ± (S.D.) of the compounds of interest determined in seminal fluid samples of 50 healthy donors and 140 patients with infertility problems, expressed in pmol/L.

CONTROLS PATIENTS n = 50 n = 140

MEAN + S.D. MEAN ± S.D. μΐτιοΙ/L μΐτιοΙ/Ι_

Reduced glutathione (GSH) 15.56 ± 5.17 15.91 ± 9.18

Ascorbic acid 300.19 ± 76.03 186.10 ± 121.71 a//-fransretinoic acid 0.003 ± 0.001 0.001 ± 0.001 a//-fra/7sretinol 0.078 ± 0.027 0.04 ± 0.02

Alfa-tocopherol 3.38 ± 1.1 1 1.50 + 0.91

Gamma-tocopherol 0.064 ± 0.020 0.029 ± 0.021

Total carotenoids 0.28 ± 0.13 0.058 ± 0.046

Coenzyme Q10 0.038 ± 0.018 0.027 ± 0.022

Malondialdehyde (MDA) 0.01 ± 0.01 0.29 ± 0.18

8-hydroxy-2'-deoxy- absent 0.094 ± 0.066 guanosine (8-OHdG)

Nitrites (-N0₂ ^") 2.89 ± 0.84 10.78 ± 7.95

Nitrates (-N0₃ ^") 24.59 ± 6.11 33.15 ± 27.04

Creatinine 117.68 ± 32.04 244.22 ± 92.28

Cytosine 0.80 ± 0.37 2.84 ± 0.70

Cytidine 1.66 ± 0.61 5.79 ± 3.70

Uracil 4.1 1 ± 1.86 6.75 ± 3.96

Beta-pseudouridine 4.47 ± 2.61 7.29 ± 3.59

Hypoxanthine 5.04 ± 2.30 12.67 ± 6.27

Xanthine 39.69 ± 10.38 51.09 ± 28.82

Uridine 2152.04 ± 333.03 1818.99 ± 851.20

Uric acid 213.52 ± 53.19 227.79 ± 131.08

Inosine 2.61 ± 0.75 4.26 ± 3.27

Guanine 58.57 ± 15.59 35.25 ± 18.62

Guanosine 69.31 ± 12.06 35.15 ± 16.56

Orotic Acid 38.97 ± 8.33 39.90 ± 22.64

Adenosine 42.37 ± 8.35 43.46 ± 17.02

In the column of infertile patients, values in bold resulted significantly different with respect to the corresponding concentrations found in controls (p < 0.01). These 21 compounds assume the significance of biochemical markers of male infertility and are available to determine the biochemical quality of seminal fluid and to perform, in conjunction with the spermiogram, the biochemical diagnosis of male infertility.

At this point, at each of the following 21 metabolites ascorbic acid, beta-pseudouridine, cytidine, cytosine, creatinine, guanine, guanosine, 8- hydroxy-2'-deoxy-guanosine (8-OHdG), inosine, hypoxanthine, malondialdehyde, nitrites , uracil, uridine, xanthine, all-trans retinol, all- trans retinoic acid, alpha-tocopherol, gamma-tocopherol, coenzyme Q10, total carotenoids (calculated as astaxanthin, lutein, zeaxanthin, beta-apo- frans-carotenal, beta-cryptoxanthin, lycopene, alpha-carotene, beta- carotene) with the meaning of male infertility biomarkers, of each subject with infertility, it has been assigned a value of 0 when the concentration of the metabolite was lower or equal to, and higher or equal to the selected values, respectively, maximum and minimum cut-off of concentration of the corresponding metabolite in healthy subjects that statistically discriminates healthy concentration values from unhealthy concentration values, such as the 95% percentile of the corresponding metabolite in healthy subjects, or with a value of 1 when the concentration of the metabolite is higher or lower than above-mentioned concentration cut-off values. Calculating the Biomarker Score as described above, it was found that the 50 fertile control subjects had an average Biomarker Score of 1.6 ± 1.2 whilst the 140 infertile subjects had an average Biomarker Score of 12.7 ± 5.9. In Figure 1 are reported the Biomarker Score distributions in fertile controls and infertile subjects, from which it is possible evaluate that the Biomarker Score values of all fertile control subjects were included between 0 and 4, whilst those of the 140 infertile subjects were included between 5 and 21.

Subsequently, infertile subjects, basing on the results of spermiogram analysis, were subdivided into 2 subgroups: normozoospermic (N) and the sum of astheno-terato-oligozoospermic (ATO), asthenozoospermic (A), teratozoospermic (T), oligozoospermic (O).

The aim was to highlight both biochemical anomalies common to all of the infertilities, and to evidence biochemical profiles specific to one of the two subgroups. The results of these statistical comparisons are summarized in Table 3. Table 3. Mean values ± (S.D.) expressed in pmol/L of the compounds of interest determined in seminal fluid samples of fertile donors (CONTROLS), normozoospermic subjects (N) and all astheno- terato-oligozoospermic (ATO), asthenozoospermic (A), teratozoospermic (T) and oligozoospermic (O) subjects.

Also in this case, values reported in bold were significantly different with respect to controls (p < 0.01). It is possible to observe that the statistical comparison underlines the biochemical value of the 21 infertility biomarkers, measured in the seminal plasma, whose concentration, for some of them, is altered only in patients classified by the spermiogram as astheno-terato-oligozoospermic (ATO), asthenozoospermic (A), teratozoospermic (T), oligozoospermic (O).

Calculating the Biomarker Score for fertile controls and for the two groups of infertile subjects (normozoospermics (N) and the sum of astheno-terato-oligozoospermic (ATO), asthenozoospermic (A), teratozoospermic (T), oligozoospermic (O), as shown in Table 3), it is possible to underline that the respective frequency distribution curves partially distinguish the normozoospermics subjects from the sum of astheno-terato-oligozoospermic (ATO), asthenozoospermic (A), teratozoospermic (T), oligozoospermic (0). Figure 2 shows the percent frequency distribution curves as a function of the value of the Biomarker Score in the fertile controls and the two groups of infertile subjects, subdivided as indicated above. Mathematical calculations allow to calculate at the aftermath the probability based on the percentage frequency.

EXAMPLE 2: Clinical case

As an example of the usefulness of the analysis of the biochemical quality of seminal fluid in infertility cases, here is reported the case of XX patient, apparently healthy subject of 43 years of age, who reported procreation difficulties for more than ten years, with three failed attempts to homologous artificial fertilization. The seminal fluid examination, by means of the spermiogram, resulted in a condition of oligo-teratozoospermia, with a percentage of normoconform spermatozoa less than 1 %.

Biochemical parameters in the seminal plasma also clearly indicated the metabolic alterations characteristic of patients with the same classification of the spermiogram (both for energy and antioxidant compounds).

Based on the results of biochemical analysis on seminal fluid, a three-month period of treatment with specific supplements and a diet rich mainly in food of plant origin was recommended to patient XX. Supplements were based on vitamin B complex (B1-B12). Moreover, ascorbic acid, vitamin A, vitamin E were administered. In addition, the patient followed a Mediterranean diet, rich in antioxidant vegetable-based compounds.

Tables 4, 5, 6 and 7 show the results of the seminal fluid analysis of patient XX, performed according to the method described in the present invention. The column titled RESULT 1 shows values determined before treatment, while the column titled RESULT 2 reports values of the analysis carried out after three months of dietary supplementation. In bold, values that are not within the confidence interval of 95% (as indicated in the Tables as REFERENCE INTERVALS).

It is possible to observe that at the first analysis, the Biomarker Score of patient XX was equal to 16, which corresponds to a high severity of biochemical alterations in seminal plasma (Biomarker Score between 13 and 17).

It is also appears evident that, after selective treatment, aimed to improve the biochemical alterations present in the seminal plasma by analyzing its biochemical quality through the method described in the present invention, under the name Redox-Energy Test, none of the 21 biomarkers came out of the confidence interval at 95% percentile, including the 16 compounds altered at the first analysis, and the Biomarker Score was = 0. This value indicates a normal biochemical quality of the seminal fluid. At the time of this second biochemical analysis, a new spermiogram was also performed with these results: Spermatozoa: 10 millions/ml (before 1 million/ml) (normal value≥ 15 million/ml).

Spermatozoa for ejaculate: 25 millions (before 2 millions) (normal value≥ 39 millions)

Normal forms: 3% (before 0%) (normal value≥ 4%)

Also in this case it is evident a clear improvement in the spermiogram with values of the parameters examined almost equal to the normal values according to WHO.

It is very important to point out that patient XX, at the time of this second control check, announced the start of his wife's natural pregnancy.

It appears evident from this clinical case that the use of the Redox-

Energy Test, according to the method described in the present invention, has allowed to identify the biochemical alterations of the seminal fluid and, through a personalized treatment, based on specific supplements and selective diet, above all allowed modifications to these alterations, ultimately allowing the patient XX to return fertile and to naturally fertilize his companion. Table 4

RESULT 1 RESULT 2 REFERENCE μηιοΙ/L μηΊθΙ/L INTERVALS μιτιοΙ/L

Reduced glutathione (GSH) 3.94 28.76 10.00 - 30.00

Vitamin C (ascorbic acid) 28.95 288.33 150.00 - 450.00

Table 5

RESULT 1 RESULT 2 REFERENCE Mmol/L μιτιοΙ/L INTERVALS

ηηοΙ/L

Table 6

RESULT 1 RESULT 2 REFERENCE μΓηοΙ/L μΓηοΙ/L INTERVALS

ηιοΙ/L

Table 7

RESULT 1 RESULT 2 REFERENCE pmol/L pmol/L INTERVALS μηΊθΙ/L

Orotic acid 13.66 23.33 20.00 - 60.00

Uric acid 56.29 223.19 120.00 - 350.00 Adenosine 29.92 32.07 20.00 - 60.00

Beta-pseudouridine 6.33 5.14 2.00 - 10.00

Cytidine 0.87 0.68 0.50 - 3.00

Cytosine 033 0.76 0.10 - 2.00

Creatinine 241.72 244.63 60.00 - 250.00

Guanine 34.01 38.26 30.00 - 90.00

Guanosine 24.76 39.64 30.00 - 90.00

Inosine 0.00 3.30. 1.00 - 4.50

Hypoxanthine 0.91 8.06 2.00 - 10.00

Uracil 2.08 6.28 1.50 - 8.00

Uridine 1100.32 2633.41 1500.00 - 3500.00

Xanthine 51.12 32.95 25.00 - 65.00

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32. Di Pietro V, Perruzza I, Amorini AM, Balducci A, Ceccarelli L, Lazzarino G, Barsotti P, Giardina B, Tavazzi B. Clinical, biochemical and molecular diagnosis of a compound homozygote for the 254 bp deletion-8 bp insertion of the APRT gene suffering from severe renal failure. Clin Biochem. 2007;40:73-80. 33. Romitelli F, Santini SA, Chierici E, Pitocco D, Tavazzi B, Amorini AM, Lazzarino G, Di Stasio E. Comparison of nitrite/nitrate concentration in human plasma and serum samples measured by the enzymatic batch Griess assay, ion-pairing HPLC and ion-trap GC-MS: the importance of a correct removal of proteins in the Griess assay. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;851:257-67.

34. Lazzarino G, Amorini AM, Eikelenboom MJ, Killestein J, Belli A, Di Pietro V, Tavazzi B, Barkhof F, Polman CH, Uitdehaag BM, Petzold A. Cerebrospinal fluid ATP metabolites in multiple sclerosis. Mult Scler. 2010;16:549-54.

35. Lazzarino G, Amorini AM, Di Pietro V, Tavazzi B. HPLC analysis for the clinical-biochemical diagnosis of inborn errors of metabolism of purines and pyrimidines. Methods Mol Biol. 2011 ;708:99- 117.

36. Amorini AM, Giorlandino C, Longo S, D'Urso S, Mesoraca A, Santoro ML, Picardi M, Gullotta S, Cignini P, Lazzarino D, Lazzarino G, Tavazzi B. Metabolic profile of amniotic fluid as a biochemical tool to screen for inborn errors of metabolism and fetal anomalies. Mol Cell Biochem. 2012;359:205-16.

37. Tavazzi B, Batocchi AP, Amorini AM, Nociti V, D'Urso S, Longo S, Gullotta S, Picardi M, Lazzarino G. Serum metabolic profile in multiple sclerosis patients. Mult Scler Int. 2011 ;2011 :167156. doi: 10.1155/2011/167156.

38. Amorini AM, Lazzarino G, Di Pietro V, Signoretti S, Lazzarino G, Belli A, Tavazzi B. Metabolic, enzymatic and gene involvement in cerebral glucose dysmetabolism after traumatic brain injury. Biochim Biophys Acta. 2016;1862:679-87.

Claims

1) Method for in vitro diagnosis of male infertility or in vitro evaluation of the quality of cryopreserved spermatozoa, said method comprising or consisting of

determining, by biochemical analysis of a seminal fluid or seminal plasma sample of a subject, a single Biomarker Score of that subject based on the concentration of at least eleven or at least fifteen of twenty- one compounds or at least all the twenty-one compounds selected between ascorbic acid, beta-pseudouridine, cytidine, cytosine, creatinine, guanine, guanosine, 8-hydoxy-2'-deoxy-guanosine (8-OHdG), inosine, hypoxanthine, malondialdehyde, nitrites, uracil, uridine, xanthine, all-trans retinol, all-trans retinoic acid, alfa-tocopherol, gamma-tocopherol, coenzyme Qi₀, total carotenoids calculated as the sum of astaxanthine, lutein, zeaxanthine, beta-apo-frans-carotenal, beta-cryptoxanthine, lycopene, alfa-carotene, beta-carotene,

wherein the Biomarker Score is the sum of the numerical values assigned to the concentration of each of the said compounds of the subject compared to the cut-off values of maximum and minimum concentrations of each compound, determined in fertile subjects, which statistically discriminate fertile subjects from infertile subjects, e.g. maximum and minimum values of the confidence interval of 95% of each compound, determined in normal fertile subjects.

2) Method according to claim 1 , wherein said at least eleven compounds are ascorbic acid, alpha-tocopherol, total carotenoids, malondialdehyde (MDA), 8-hydroxy-2'-deoxy-guanosine (8-OHdG), nitrites, creatinine, cytosine, cytidine, guanine, guanosine.

3) Method according to any one of the previous claims, said method comprising or consisting of:

a) determining the concentration of each compound of said at least eleven, at least fifteen or at least all of the twenty-one compounds in a biological sample of seminal fluid or seminal plasma of a subject;

b) associating each concentration of each compound with a first numerical value, such as 0, when the concentration of the compound is equal to or comprised between the values chosen, respectively, the maximum and minimum of the concentration cut-off of the corresponding compound in fertile subjects, and with a second numerical value different from the first one, such as 1 , when the concentration of the compound is higher or lower respectively to the chosen values maximum and minimum of the cut-off concentration of the corresponding compound in fertile subjects;

said maximum and minimum concentration cut-off values are the maximum and minimum concentration values of each compound determined in fertile subjects, which statistically discriminate fertile subjects from infertile subjects, such as the maximum and minimum values of the confidence interval to 95% of each compound, determined in healthy fertile subjects;

c) calculating the personal Biomarker Score of the subject by summing the numerical values of said at least eleven, fifteen, or all of the twentyone compounds of said subject;

d) obtaining the probability of the said subject of being fertile, or infertile and/or infertile with normozoospermia, or infertile with any of the oligo- astheno-teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia by the comparison of the personal Biomarker Score with control references that associate each Biomarker Score of selected subjects in at least one group chosenamong fertile, infertile and/or infertile subjects with normozoospermia, and/or infertile subjects with any of the oligo-astheno-teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia with the posterior probability of each Biomarker Score being from at least one group.

4) Method according to any one of the preceding claims, wherein the control references are obtained by a method comprising or consisting of: a)selecting one or more groups of subjects chisen from a group of fertile subjects, and/or a group of infertile subjects and/or a group of infertile subjects with normozoospermia and/or infertile subjects with any of the oligo-astheno-teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia; b) determining the concentration, in a biological sample of seminal fluid or seminal plasma of each subject of said one or more groups, at least eleven or at least fifteen of the twenty-one compounds or all twenty- one compounds selected from ascorbic acid, beta-pseudouridine, cytidine, cytosine, creatinine, guanine, guanosine, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), inosine, hypoxanthine, malondialdehyde, nitrites, uracil, uridine, xanthine, all-trans retinol, all-trans retinoic acid, alfa-tocopherol, gamma- tocopherol, coenzyme Qi₀, total carotenoids calculated as the sum of astaxanthine, lutein, zeaxanthine, beta-apo-frans-carotenal, beta- cryptoxanthine, lycopene, alfa-carotene, beta-carotene;

c) choosing select, in the group of fertile subjects, the maximum and minimum values of the concentrations cut-off of each of the said at least eleven, at least fifteen or twenty-one compounds, said maximum and minimum values of the concentrations cut-off being values that statistically discriminate concentration values of each of said compounds in fertile subjects from concentration values in infertile subjects, such as 95% percentile of the corresponding compound in fertile subjects;

d) associating the concentration of each of said at least eleven, at least fifteen or twenty-one compounds of each subject of said one or more groups at a first numerical value, such as 0, when the concentration of the compound is equal to or comprised between the maximum and minimum of the concentration cut-off values and a second numerical value, different from the first one, such as 1 , when the concentration of the compound is higher or lower respectively to maximum and minimum of the cut-off concentration values;

e) calculating a Biomarker Score of each subject of said one or more groups of subjects by summing the numerical values associated with the concentrations of said at least eleven, at least fifteen, or all twenty-one compounds of each subject;

f) calculating the posterior probability associated with each Biomarker Score of each subject in each group, said posterior probability being the probability of each Biomarker Score to belong to said one or more groups of subjects, in order to obtain a control reference of fertile subjects and/or infertile and/or infertile subjects with normozoospermia and/or infertile subjects with any of the following: oligo-astheno-terato- zoospermia, oligozoospermia, asthenozoospermia and teratozoospermia.

5) Method according to any one of the preceding claims, wherein said at least eleven compounds are ascorbic acid, alfa-tocopherol, malondialdehyde (MDA), 8-hydroxy-2'-deoxy-guanosine (8-OHdG), nitrites, creatinine, cytosine, cytidine, guanine, guanosine, total carotenoids calculated as the sum of astaxanthine, lutein, zeaxanthine, beta-apo-fra/7s-carotenal, beta-cryptoxanthine, lycopene, alfa-carotene, beta-carotene.

6) Method according to any one of the preceding claims wherein, when the first numeric value is 0 and the second numeric value is 1 , the Biomarker Score 0 to 4 and 5 to 21 respectively discriminate the fertile subjects from the infertile subjects; Biomarker scores from 5 to 12 are associated with a probability of infertility with normozoospermia higher than the probability of infertility with any of the oligo-astheno- teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia, while Biomarker Score from 13 to 21 are associated with a probability of infertility with any of the oligo-astheno- teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia higher than the infertility probability with normozoospermia.

7) Method for providing one or more control references for the in vitro diagnosis of male infertility or in vitro evaluation of the quality of cryopreserved spermatozoa, said method comprising or consisting of:

a) selecting one or more groups of subjects chosen from a group of fertile subjects and/or a group of infertile subjects and/or a group of infertile subjects with normozoospermia and/or infertile subjects with any of the oligo-astheno-teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia;

b) determining the concentration, in a biological sample of the seminal fluid or seminal plasma of each of the subjects of said one or more groups of subjects, of at least eleven or at least fifteen of the twenty-one compounds or all of the twenty selected compounds selected from ascorbic acid, beta-pseudouridine, cytidine, cytosine, creatinine, guanine, guanosine, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), inosine, hypoxanthine, malondialdehyde, nitrites, uracil, uridine, xanthine, all-trans retinol, all-trans retinoic acid, alpha-tocopherol, gamma-tocopherol, coenzyme Q₁₀, total carotenoids calculated as sum of astaxanthin, lutein, zeaxanthin, beta-apo-frans-carotenal, beta-cryptoxanthin, lycopene, alpha- carotene, beta-carotene;

c) selecting, in the group of fertile subjects, the maximum and minimum values of the concentrations cut-off of each of the said at least eleven, at least fifteen or twenty-one compounds, said maximum and minimum concentrations values of the concentrations cut-off being values that statistically discriminate concentration values of each of said at least eleven, at least fifteen or twenty-one, in fertile subjects from the concentration values of said compounds in infertile subjects such as 95% percentile of the corresponding compound in fertile subjects;

d) associating the concentration of each of said at least eleven, at least fifteen or twenty-one compounds of each subject of said one or more groups of subjects at a first numeric value, such as 0, when the concentration of the compound is equal to or comprised between the maximum and minimum concentration cut-off values and a second numerical value, different from the first one, such as 1 , when the concentration of the compound is higher or lower than the maximum and minimum concentrations cut-off values respectively;

e) calculating a Biomarker Score of each subject of said one or more groups of subjects by summing said first and second numerical values associated to the concentrations of said at least eleven, fifteen, or all twenty-one compounds of each subject;

f) calculating the posterior probability associated with each Biomarker Score of each subject in each group, said posterior probability, being the probability that each Biomarker Score belongs to said one or more groups of subjects, in order to obtain a control reference of fertile subjects and/or infertile subjects and/or infertile subjects with normozoospermia and/or infertile subjects with any of the oiigo-astheno-teratozoospermia, oligozoospermia, asthenozoospermia and teratozoospermia.

8) Method according to claim 7, wherein said at least eleven compounds are ascorbic acid, alpha-tocopherol, malondialdehyde (MDA), 8-hydroxy-2'-deoxy-guanosine (8-OHdG), nitrites, creatinine, cytosine, guanine, guanosine, total carotenoids calculated as sum of astaxanthine, lutein, zeaxanthine, beta-apo-frans-carotenal, beta-cryptoxanthine, lycopene, alpha-carotene, beta-carotene.

9) Computer program comprising code means configured in such a way that, when executed on a computer, perform the method steps of any one of claims 1-6 or 7-8.

10) Pharmaceutical composition or food supplement comprising or consisting of one or more vitamins of group B such as B1 , B2, B3, B4, B5, B6, B7, B8, B9, B10, B1 1 , B12, as active substances, in combination with one or more excipients and/or adjuvants pharmaceutically acceptable, said pharmaceutical composition or food supplement being for use in infertility therapy.

1 1) Pharmaceutical composition or food supplement according to claim 10, for the use according to claim 10, further comprising at least one or all of the compounds chosen from ascorbic acid, vitamin E (alpha-tocopherol), vitamin A {all-trans retinol) guanine, uridine, inosine, tocopherols (beta, gamma, delta tocopherol) and tocotrienols, reduced glutathione, lutein, lycopene, coenzyme Q₁₀, beta-carotene, astaxanthine, zeaxanthine, cantaxanthine, capsanthin, citranaxanthin.