IT202200022401A1 - Strains of bacteria for the treatment of urological disorders, their compositions and related uses - Google Patents

Description

Description of the Industrial Invention entitled:

?Bacteria strains for the treatment of urological disorders, their compositions and related uses?

The present invention relates to selected bacterial strains, belonging to the species Lactobacillus and Bifidobacterium and to their mixtures, compositions and their use in therapy, especially in the field of male urology for the treatment of male fertility. More specifically, the invention relates to the use of specific bacterial strains in the curative or prophylactic treatment of benign prostatic hyperplasia and myometrial dysfunctions. Furthermore, the present invention relates to selected bacterial strains, belonging to the species Lactobacillus and Bifidobacterium, and to their mixtures and compositions, for use in a method of treatment, preferably a preventive or curative treatment method, of male urinary pathologies, preferably benign prostatic hyperplasia (BPH). Finally, the present invention relates to selected bacterial strains, belonging to the species Lactobacillus and Bifidobacterium, and to their mixtures and compositions, for use in a method of treatment, preferably a preventive or curative treatment method, of gynecological pathologies, preferably pathologies associated with alterations of the trophism of the myometrium and endocrine and metabolic disorders such as polycystic ovary syndrome (PCOS), endometrial hyperplasia and endometriosis.

Urological disorders.

One of the most commonly diagnosed urological diseases in men over the age of 50 is benign prostatic hyperplasia (BPH).

Among the most significant manifestations of BPH are lower urinary tract symptoms (LUTS) and benign prostatic enlargement (BPE); LUTS usually arise due to bladder outlet obstruction (BOO), which leads to benign prostatic obstruction (BPO). The development of BPH is often associated with other pre-existing pathologies; in fact, a relationship between metabolic syndrome (MetS) and the risk of LUTS and BPH has already been demonstrated.

In the 1950s, it was discovered that there is a link between BPH and prostate cancer (PCa): both arise due to chronic inflammation, are dependent on the presence of androgens, and their development can be associated with metabolic factors.

Considering that short-chain fatty acids (SCFA) have a very high immunomodulatory potential, more and more research is currently being conducted on their effect not only in the intestinal microenvironment, but also in cells and tissues of other organs.

Short-chain fatty acids (SCFA) have less than 6 carbon atoms in their chain (C1-C6); the most common SCFAs, generated by particular gut microbiomes, are acetate (C2), propionate (C3) and butyrate (C4). SCFAs are known to play a role in regulating host health or disease mainly through two mechanisms: the regulation of target cell epigenetics after SCFA entry into the cell and signal transduction through membrane receptors; in fact, one of the main characteristics of SCFAs is their activity as endogenous HDAC inhibitors (HDACi), especially propionate and butyrate.

These acids are present in humans in certain quantities, and the concentrations depend on the ratio of bacteria inhabiting the intestine and disturbances in the intestinal microflora (dysbiosis), which affects the amount of SCFA produced. Thus, the levels of SCFA vary depending on the composition of the intestinal microflora and food intake; however, differences in analytical methods and methodology of preparing material for research on the correlation between SCFA and disorders such as BPH, cause difficulties in interpreting the results obtained.

The influence of SCFAs on the development of BPH has not been fully studied. Only a few publications on the impact of the intestinal microflora on the prostate can be found in the literature. They mainly concern the influence of intestinal bacteria on the synthesis of metabolites and androgens, which may be associated with the development of prostate cancer in men. There are also reports on the impact of inflammatory bowel disease (IBD) on the risk of prostate cancer. So far, differences in the composition of the intestinal microflora have only been confirmed in one pilot study, in which the composition of the intestinal microflora in patients with PCa and BPH was analyzed. It has been observed that chronic inflammation of the prostate predisposes to BPH and PCa; this inflammation can be caused by bacterial infection, but can also be associated with low-grade systemic inflammation. It seems very likely, however, that the disturbed intestinal microflora does not directly affect the prostate gland, but contributes to the development of chronic systemic inflammation. Cells and inflammatory factors (including cytokines) from the intestinal environment, together with the circulation, can enter the gland and cause "local" inflammation and stimulate growth factors of the prostatic stroma, which in turn can lead to prostatic hyperplasia. The influence of the intestinal microflora and its metabolites entering the systemic circulation has been confirmed by studies of the urinary, nervous and respiratory systems, as well as autoimmune diseases.

In BPH patients, stool analysis showed increased levels of some SCFAs, mainly isobutyric acid (C4:0i), isovaleric acid (C5:0i), and isocaproic acid (C6:0i); this suggests that they are most likely to influence factors that predispose men to BPH. Specifically, isobutyric acid was significantly elevated in men with BPH compared to healthy controls (%SCFA - mean: 4,695, median: 4,702 vs. 3,814, 3,726; p = 0.008). Isovaleric acid levels were also increased (%SCFA - mean: 9,499, median: 9,221 vs. 6,911, 6,730; p < 0.001). Finally, the predominant acid among the acids isolated from the feces of healthy controls was isocaproic acid (% SCFAs - mean: 0.359, median: 0.174 vs. 0.186, 0.142; p = 0.038). SCFAs produced by the gut microbiota, mainly acetate, propionate and butyrate, play a key role in maintaining homeostasis in humans. These three most common acids account for 95% of total SCFAs. In the large intestine and stool samples, SCFAs are present in an approximate molar ratio of acetic acid:propionic acid:butyric acid of 60:20:20. SCFA levels in the gut range from 20 to 140 mM and depend on the composition of the gut microflora, SCFA absorption from the gut and dietary fiber content. Acetic, propionic and butyric acids are produced as a result of saccharolytic fermentation and have health benefits. SCFAs are considered mediators in the communication between the gut microbiome and the immune system. The signal they produce is transferred, among other things, into immune cells via free fatty acid receptors (FFARs), which belong to the family of G protein-coupled receptors (GPCRs). Considering their immunomodulatory potential, they can be useful in the prevention of chronic but persistent low-grade inflammation. It is also worth noting that the concentrations of SCFAs and BCFAs (branched chain fatty acids) fluctuate in a healthy population throughout life, from newborns to the elderly. These values are influenced by the composition of the gut microflora, the age and health of the subjects. Many studies have confirmed that the gut microflora changes with the aging of the body. Yes? It has been observed that the positive impact of the microflora (characterized by a decrease in the taxonomic diversity of the intestinal microbiota) on the human body decreases with age. These changes are also reflected in the physiology of the host organism and manifest themselves, among other things, as increased inflammation. These differences are noted between adult men (average age 42 years) and elderly men (average age 77 years). In young people, the bacteria that predominate in the composition of the intestinal microflora are those with immunomodulatory potential, such as Clostridiales and Bifidobacterium. In elderly people, however, bacterial communities are enriched with pathogens, e.g. Enterobacteriaceae. Butyric acid (C4:0) is an important factor influencing metabolic processes, which has been confirmed in both animal models and humans, but the exact mechanism of its action still requires a more detailed explanation. Butyric acid supplementation has been shown to prevent obesity, hyperinsulinemia, and hypertriglyceridemia, and may also reduce appetite and activate brown adipose tissue (BAT) via vagal nerve signaling. Butyric and valeric acids have also been shown to be inhibitors of class I histone deacetylases.

Isobutyric acid (C4:0i) is a short-chain branched fatty acid (BCFA), the geometric isomer of butyric acid, which results in its different physical but not chemical properties. In the human intestine, the fermentation of branched-chain amino acids is carried out mainly by the genera Bacteroides and Clostridium. Among the bacterial populations that participate in the fermentation of peptides and amino acids, there are also those bacteria responsible for the production of BCFA: 0.6% of the population is involved in the synthesis of isovaleric acid and up to 40% of the bacteria in the synthesis of isobutyric acid. The highest levels of BCFA are found in the distal parts of the large intestine. BCFA concentrations in feces, as well as SCFA concentrations, can be modified depending on the food consumed. Little is known about the effects of BCFA on the host organism. However, there is evidence that these acids can be oxidized if the amount of BCFA is too high. of butyric acid is insufficient and can therefore be an energy source for colonocytes. Isobutyric acid (C4:0i), isovaleric acid (C5:0i) and isocaproic acid (C6:0i) have been shown to be the most likely to influence the factors that predispose men to BPH. In addition, during proteolytic fermentation, along with the increase in BCFA, harmful metabolites such as: ammonia, phenols and hydrogen sulfide are produced. The resulting components can contribute to the initiation of inflammation and excessive proliferation of colonocytes and thus influence local pathological states. In addition to inducing epithelial cell proliferation, inflammation can also affect tight junctions (TJs). Damage to tight junctions causes the leakiness and dysfunction of cellular barriers and is the cause of "leaky states" and chronic inflammation, which are observed in digestive tract cancers. Impaired tight junction function in the digestive tract (mainly in the intestine) can also be caused by the state of intestinal dysbiosis and reduced amount of SCFA, resulting, among other things, from the use of antibiotics. SCFA, mainly butyric acid, strengthen the intestinal barrier by regulating the transcription of claudin-1 which is a protein that builds tight junctions. Leakage of the intestinal barrier causes bacterial particles and factors, pro-inflammatory cytokines, immune cells, toxins and antigens to enter the bloodstream and move from the digestive tract to distant locations, including the prostate. Many studies have confirmed that SCFA are involved in the pathophysiology of IBD and can be a prognostic marker of the disease state.

Research has also confirmed the role of SCFAs in the pathogenesis of IBD. Their analysis showed that in people with inflammatory bowel disease, the levels of the main fatty acids - acetic, butyric and propionic - decrease dramatically compared to healthy people (162.0; 86.9; 65.6 ?mol/g versus 209.7; 176.0; 93.3 ?mol/g). In addition, the effect of L. plantarum in alleviating the symptoms of diarrhea-predominant IBS (IBS-D) was investigated: this study demonstrated how L. plantarum significantly restores the composition and diversity of the intestinal microbiota, in particular its benefits may be related to the increase in bacteria capable of producing butyric acid.

Changes in fecal short-chain fatty acids have also been observed in morbidly obese patients after surgery. When they lost weight and changed their diet, the total amount of SCFA decreased. A similar effect was obtained for the relative amounts of straight-chain SCFA, namely acetic, propionic and butyric acids. At the same time, the levels of BCFA (isobutyric, isovaleric and isocaproic acid) increased. These changes suggest a predominant proteolytic fermentation that may have adverse effects on health. One therapy may consist of changing a high-protein diet to a diet rich in carbohydrates, fibre and polysaccharides in order to increase saccharolytic fermentation and the level of the main simple SCFA (acetate, propionate and butyrate), which have health-promoting properties. Butyrate levels are also altered in patients with prostate cancer; in one study it was found that the levels of butyrate were altered. It has been observed that several pathways leading to butyrate production are activated in greater abundance than in benign controls. In addition, sodium butyrate (the sodium salt of butyric acid) has been shown to decrease androgen receptor expression in some postpartum cancer cell lines.

Herbal remedies, pharmacotherapy, and surgery are currently available for BPH. European and non-European guidelines focusing on the therapeutic options for the disease also include pharmacotherapy, lifestyle recommendations, surgical options, and phytotherapy.

Additionally, researchers are focusing on new herbal alternatives for the treatment of BPH, especially traditional Chinese medicine.

Drugs based on the pharmacological actions of active compounds include

? ?1-adrenergic antagonists

? 5?-reductase inhibitors

? muscarinic receptor antagonists

? phosphodiesterase type 5 inhibitors and vasopressin analogues.

Gynecological disorders.

Currently, there is a lot of evidence showing that many of the bacteria that colonize the human body have established beneficial relationships with their host, actively participating in the formation and maintenance of normal physiological balance. On the contrary, dysbiosis promotes the development of various diseases. In fact, the microbial colonies that live in the human body play an important role in the state of eubiosis of each individual; in particular, the cervicovaginal microbiome plays a fundamental role in the reproductive health of women, interacting with estrogens, androgens, insulin and other hormones. It has been shown that an alteration of the natural state of the microbiota contributes to endocrine and metabolic disorders such as polycystic ovary syndrome (PCOS), endometrial hyperplasia and endometriosis, and several comorbidities such as diabetes. Unfortunately, the data available in the scientific literature are scarce, especially regarding the mechanisms.

The gut microbiome modulates many biological mechanisms; for example, commensal bacteria can produce and secrete hormones, and communication between microbes and hormones can influence host metabolism, immunity, and behavior. In addition, sex hormones, such as progesterone, estradiol, and testosterone, also participate in communication between microorganisms and their hosts and play several important physiological roles in reproduction, differentiation, cell proliferation, apoptosis, inflammation, metabolism, homeostasis, and brain function. In this context, several studies have highlighted how perturbation of the vaginal microbiome of women of reproductive age affects all stages of a woman's reproductive life. More precisely, the human microbiome influences every stage and level of female reproduction, including follicle and oocyte maturation in the ovary, fertilization and embryo migration, implantation, and the entire pregnancy, including childbirth. Alterations in the microbiome, particularly in the gut, have a specific impact on the reproductive endocrine system, and correcting abnormal microbiomes can lead to improved reproductive outcomes.

The Human Microbiome Project has shown that the microbiome of a normal vagina is populated by several indigenous lactobacilli species and that, compared to other body sites, the microbial diversity within the reproductive tract is relatively low. In this context, many studies have shown that dietary supplementation or direct intervention with probiotics can alleviate reproductive dysfunction and have positive therapeutic effects on associated diseases; furthermore, there is considerable evidence that probiotics can alter the biological activity of microbes, thus exerting their effects by directly regulating the composition of the host microbiota and also influencing its metabolism and health. In particular, probiotics can improve the host's reproductive function by regulating its metabolism, since metabolic health is linked to reproductive function.

In view of the above, it is evident that it would be useful to identify new probiotic-based treatments for the urological and gynecological disorders described above as an alternative to conventional phytotherapeutic and pharmacological treatments.

The Applicant, following an intense and prolonged research and development activity, has surprisingly found that specific bacterial strains of the genus Bifidobacterium are able to exert an important beneficial activity in the case of male urological disorders.

The Applicant has also found that specific bacterial strains of the genus Bifidobacterium and Lactobacillus show interesting and unexpected activities at the gynecological level.

In particular, the Applicant surprisingly found that specific bacterial strains of the genus Bifidobacterium, in particular the strains:

- Bifidobacterium psychraerophilum ?Q5?, filed with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 33121, since July 7, 2019, and

- Bifidobacterium longum subsp. longum ?novaBLG1?, deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with deposit number DSM 34338, dated July 26, 2022, and mixtures thereof, are able to carry out an important beneficial activity against urological disorders in men, in particular, but not only, in the curative or prophylactic treatment of benign prostatic hyperplasia (BPH).

Furthermore, the Applicant surprisingly found that specific bacterial strains of the genus Bifidobacterium and Lactobacillus, in particular the strains:

- Bifidobacterium bifidum ?novaBBF9? filed with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34337, as of July 26, 2022,

- Lactobacillus crispatus ?novaLCR6? deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 34348, dated 26 July 2022, and

- Lactobacillus fermentum (Limosilactobacillus fermentum) ?novaLF58? deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with deposit number DSM 34340, dated July 26, 2022, and their mixtures, are able to perform an important beneficial activity against gynecological disorders, in particular, but not only, by activating mechanisms compromised in conditions of myometrial alteration and improving myometrial activity through the modulation of cell cycle progression.

All the above-mentioned bacterial strains have been deposited in accordance with the provisions of the Budapest Treaty; the Applicant of said bacterial strains described and claimed in this patent application and the Applicant express their consent to make the strains available for the duration of the patent.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1 to 12 relate to the first aspect of the invention, Figures 13 to 21 relate to the second aspect of the invention. In Figures 13 to 21 the association A2 is defined as MIX.

Figure 1 shows a schematic example of a 3D model of intestinal epithelium connected to the prostate.

Figure 2 shows 3D spheroids in culture monitored during growth.

Figure 3 shows the intestinal passage of metabolites produced by the A1 combination.

Figures 4, 5 and 6 show the effects of the A1 association on the activity of prostate cells. Figures 7A and 7B show the effects of the A1 association on the reduction of oxidative stress (markers: ROS, SOD).

Figure 8 shows the effects of the A1 combination on reducing inflammation.

Figure 9 shows the effects of the A1 combination on androgen receptor (AR) activity. Figure 10 shows the effects of the A1 combination on testosterone levels.

Figure 11 shows the effects of the A1 combination on serotonin levels.

Figure 12 shows the effects of the A1 combination on prostate-specific antigen (PSA).

Figure 13 shows the cell viability of PHM1-41 cells incubated with all single agents for 24 h as measured by the MTT assay; data are the mean ± SD of five independent experiments performed in triplicate versus control (0% line). *p<0.05 versus control; #p<0.05 versus other concentrations.

Figure 14 shows mitochondrial metabolism and TEER analysis. In panel A, mitochondrial metabolism assessed by MTT at 6 hours. In B, TEER during 6 hours. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs single agents; ! p<0.05 vs Association A2+DCI 150mg; y p<0.05 vs DCI 150mg. Figure 15 shows absorption analysis. In panel A, fluorescein rate at 6 hours. In B, butyric acid quantification measured at 6 hours. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs single agents; ! p<0.05 vs Association A2+DCI 150mg; y p<0.05 vs DCI 150mg.

Figure 16 shows the proliferation analysis. In panel A, mitochondrial metabolism assessed by MTT assay. In panel B, proliferation analysis by crystal violet staining. In C, cyclin D1 activity assessed by ELISA kit. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs single agents; ! p<0.05 vs A2+DCI 150mg Association; y p<0.05 vs DCI 150mg. Figure 17 shows myometrial damage. In panel A, ROS production measured by CytoC reduction. In panel B, TNFα activity analyzed by ELISA kit. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs single agents; ! p<0.05 vs Association A2+DCI 150mg; y p<0.05 vs DCI 150mg.

Figure 18 shows the contractile activity. Oxytocin concentration (A) and MAGT1 activity (B) measured by ELISA kit. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs single agents; ! p<0.05 vs Association A2+DCI 150mg; y p<0.05 vs DCI 150mg.

Figure 19 shows the intracellular pathway. ERK/MAPK (A) and PI3K/AKT (B) activities assessed by ELISA kit. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs single agents; ! p<0.05 vs Association A2+DCI 150mg; y p<0.05 vs DCI 150mg.

Figure 20 shows the intracellular pathways. ERb (A), PAX8 (B) and PAK1 (C) activities. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs single agents; ! p<0.05 vs A2+DCI 150mg Association; y p<0.05 vs DCI 150mg.

Figure 21 shows the hormonal activity. LH (A) and FSH (B) concentration measured by ELISA kit. Data are expressed as mean?SD (%) of 5 independent experiments normalized to control. *p<0.05 vs control; #p<0.05 vs single agents; ! p<0.05 vs Association A2+DCI 150mg; y p<0.05 vs DCI 150mg.

DESCRIPTION OF THE INVENTION

According to a first aspect, the invention has as its object a bacterial strain chosen from:

- Bifidobacterium psychraerophilum ?Q5?, filed with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 33121, from July 7, 2019;

- Bifidobacterium longum subsp. longum ?novaBLG1?, filed with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34338, from 26 July 2022;

and their blends.

The first aspect of the invention also relates to a pharmaceutical or nutraceutical association or combination or food supplement consisting of the two strains of the invention (association A1) Bifidobacterium psychraerophilum ?Q5? DSM 33121 and Bifidobacterium longum subsp. longum ?novaBLG1? DSM 34338, preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example 1:3 to 3:1, or 1:2 to 2:1, or 1:1, preferably all the strains of bacteria are in freeze-dried form. The first aspect of the invention also relates to a pharmaceutical or nutraceutical composition or food supplement comprising only one of the strains described above or their association or combination (A1), optionally together with at least one physiologically and/or pharmaceutically acceptable or food grade excipient and/or vehicle.

The first aspect of the invention also relates to the use of said bacterial strains and/or mixtures thereof in therapy and, more specifically, in the curative or prophylactic treatment of male urinary pathologies, preferably benign prostatic hyperplasia (BPH).

The first aspect of the invention also includes the use of the compositions of the invention in therapy and, more specifically, in the curative or prophylactic treatment of male urinary pathologies, preferably benign prostatic hyperplasia (BPH).

Preferably, the first aspect of the invention relates to a composition comprising 1 to 100 mg of B. Longum novaBLG1 and 1 to 100 mg of B. Psychaerophilum Q5, both in lyophilized form, preferably 5 mg to 50 mg of B. Longum novaBLG1 and 5 mg to 50 mg of B. Psychaerophilum Q5, even more preferably 10 mg to 40 mg of B. Longum novaBLG1, for example 20 mg or 30 mg of B. Longum novaBLG1, and 10 mg to 40 mg of B. Psychaerophilum Q5, for example 20 mg or 30 mg of B. Psychaerophilum Q5, preferably all strains of bacteria are in lyophilized form.

Preferably, a composition may comprise 5 to 20 mg of B. Longum novaBLG1 and 5 to 20 mg of B. Psychaerophilum Q5, both in lyophilized form, preferably 10 mg of B. Longum novaBLG1 and 10 mg of B. Psychaerophilum Q5, preferably all strains of bacteria are in lyophilized form.

Preferably, the first aspect of the invention relates to a composition comprising 10 mg of B. Longum novaBLG1 and 20 mg of B. Psychaerophilum Q5, both in freeze-dried form. Preferably, the compositions of the first aspect of the invention may comprise one or more physiologically and/or pharmacologically acceptable or food-grade excipients and/or vehicles.

Preferably, each of the two strains of the first aspect of the invention is administered in daily doses ranging from 1.0x10<6>CFU/day to 1.0x10<12>CFU/day, preferably from 1.0x10<8>CFU/day to 1.0x10<10>CFU/day, for example approximately 1.0x10<9>CFU/day (CFU = Colony Forming Units).

The compositions can be administered once or more times a day, preferably once a day.

The strains of the first aspect of the invention may be present in the composition of the first aspect of the invention as live and viable bacteria, dead bacteria or their cellular components, cellular extracts, lysates or tyndallized.

Preferably, the compositions of the first aspect of the invention may contain at least one physiologically and/or pharmacologically acceptable or food grade excipient having prebiotic characteristics or properties. Preferably, said at least one physiologically and/or pharmacologically acceptable or food grade excipient may include a monosaccharide, a disaccharide, a polysaccharide, soluble fibers and/or insoluble fibers, GOS, FOS, inulin, maltodextrin and mixtures thereof.

Preferably, said at least one physiologically and/or pharmacologically acceptable or food grade excipient may include partially hydrolyzed guar gum (PHGG).

Preferably, said at least one physiologically and/or pharmacologically acceptable or food grade excipient may be selected from the group comprising or, alternatively, consisting of: inulin, a fructooligosaccharide (FOS), maltodextrin and mixtures thereof.

The compositions according to the first aspect of the invention are preferably compositions for oral use, advantageously in the form of capsules. Other pharmaceutical forms may however be used, such as, for example, powders, tablets or dispersions.

The two strains of the first aspect of the invention have been the subject of numerous experimental tests, reported below.

During these tests, the Applicant observed that in particular when associated or combined, the probiotic strains of the invention, Q5 and novaBLG1 (combination A1), improve the function of the androgen receptor, the level of testosterone and serotonin, restoring their balance and improving the BPH situation (markers evaluated: Ki67, androgen receptor activity, testosterone level, 5Htr1a receptor, serotonin levels, PSA).

All these important effects are believed to be due to the presence of SCFAs secreted by probiotics, such as butyric acid, which are able to act as second messengers and activate various mechanisms impaired in BPH.

Furthermore, the tested strains did not induce any damage to the intestinal epithelia, confirming their non-toxicity and their ability to enhance antioxidant mechanisms (markers: ROS, SOD) and their anti-inflammatory activity (markers: TNF-a, IL-6 and IL-10), also acting on the prostate serotoninergic pathway and decreasing proliferative activity.

The tests conducted and the results obtained with the A1 association of the first aspect of the invention will be set out below, with reference to the attached Figures.

Experimental Section - BPH

Legend

Longum = B. Longum novaBLG1

Psy = B. Psychaerophilum Q5

Association = Longum Psy

TRANSWELL SYSTEM

The product metabolized by intestinal cells in a Transwell system was directly in contact with prostatic cells seeded in a basolateral environment as reported in the literature, in order to reproduce in vitro what happens in the human body (Figure 1).

Prostatic organoids were pretreated with 0.5?M testosterone to mimic prostatic hyperplasia in vitro (Figure 2).

ANALYSIS OF THE INTESTINAL PHASE

These data demonstrate that the chosen formulation is able to stimulate the beneficial effect on mitochondrial metabolism after intestinal passage.

In particular, the association Longum 10mg+Psy 10mg and 20mg does not produce any damage at intestinal level compared to the single agents, suggesting that these two formulations (Figure 3 A). The evaluation of transepithelial electrical resistance (TEER) allows to evaluate the integrity of the cell monolayer. The TEER values of 500 ? 52.9 ? *cm 2 for intestinal CaCo 2 cells are in agreement with the data available in the literature and suggest that the cells maintain an intact monolayer after treatments (Figure 3B).

Intestinal passage analysis demonstrated that all selected agents are able to produce butyric acid (probiotic metabolite) as a second messenger. The association increases the intestinal passage of metabolites suggesting that the probiotic formulation is able to directly reach the target organ (Figure 3C).

PROSTATIC ANALYSIS AFTER INTESTINAL METABOLISM

Evaluation of prostate cell activity

Prostatic analysis after intestinal passage reveals that all tested probiotics are able to counteract the proliferation rate that leads to hyperplasia. In particular, the association between Longum 10mg+Psy 10mg and Longum 10mg+Psy. 20 mg reduced cell volume and, consequently, proliferation activity was reduced compared to the single agents (p<0.05) (Figures 4 and 5).

Hyperplasia aggravated prostate functions, significantly increased the activity of Ki67, a marker of proliferation, leading to cellular suffering. Probiotics are able to slow down this process (p<0.05) and the positive effects exerted by Longum and Psychaerophilum are amplified thanks to their cooperative activity on the intestinal epithelium (p<0.05) (Figure 6). Reduction of oxidative stress

All probiotics alone or combined are able to significantly reduce oxidative stress that leads to tissue damage (p<0.05) causing the condition of hyperplasia confirming the absence of side effects (safety) and the ability to modulate disease progression (Figure 7). Reduction of inflammation

Inflammation analyses demonstrate that all probiotics alone or combined are able to significantly reduce the inflammatory condition (p<0.05) of hyperplasia confirming the absence of side effects (safety) and the ability to directly modulate the progression of the disease (Figure 8).

Androgen receptor (AR) activity

AR hyperactivity is a marker of prostatic hyperplasia; many therapeutic strategies have involved inhibiting AR activity to reduce the size of the prostate gland. The association of probiotics maintains normal AR activity by inducing its reduction during hyperplasia (Figure 9).

Testosterone Level

During hyperplasia, testosterone levels are lower due to AR hyperactivity. The combination of probiotics improves testosterone levels while maintaining lower AR activity. These data confirm the ability of probiotics to improve prostate condition without side effects (Figure 10).

Serotonin Level and Receptor Activity

Serotonin is a strong negative regulator of prostate growth. The association of probiotics decreases the level of serotonin while keeping the activity of its receptor (5-Htr1a) low. These data confirm the ability of probiotics to improve the prostate condition by inhibiting prostate growth (Figure 11).

PSA Level

Prostate specific antigen (PSA) is a sensitive and specific marker for prostate diseases, in which it is increased. PSA is produced during the hyperplasia condition (p<0.05), but all the single agents are able to reduce this production suggesting their positive role during BPH. In particular, the association between Longum and Psy is able to amplify the positive role of probiotics with the maximum effect exerted by Longum 10mg+Psy 10mg (Figure 12). The results of the tests conducted have shown that the association of the first aspect of the invention is able to reduce the proliferation of prostate cells, reduces oxidative stress and the inflammatory network reducing the negative consequences of BPH.

In addition, the combination improves the function of androgen receptor, testosterone level and serotonin restoring the balance between them to reduce the pathology.

All these important effects are believed to be due to the SCFAs secreted by probiotics, such as butyric acid, which are able to act as second messengers and activate various mechanisms that are altered during BPH.

It is important to note that the tested strains did not induce any damage on the epithelia, confirming their safety properties by enhancing the antioxidant mechanisms and the anti-inflammatory activity during BPH also acting on the serotoninergic pathway of the prostate and decreasing the proliferative activity.

In a second aspect, the invention has as its object at least one bacterial strain selected from the group comprising or, alternatively, consisting of:

- Bifidobacterium bifidum ?novaBBF9? deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 34337, dated 26 July 2022 dated 26 July 2022,

- Lactobacillus crispatus ?novaLCR6? deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under deposit number DSM 34348, dated 26 July 2022, and

- Lactobacillus fermentum ?novaLF58? filed with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) with filing number DSM 34340, as of July 26, 2022;

and their blends.

The second aspect of the invention also includes a pharmaceutical or nutraceutical association or combination or food supplement which comprises or, alternatively, consists of two strains of the invention (Association A2):

- Bifidobacterium bifidum ?novaBBF9? DSM 34337 and Lactobacillus crispatus ?novaLCR6? DSM 34348; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example 1:3 to 3:1; or 1:2 to 2:1; or 1:1, preferably all strains of bacteria are in freeze-dried form.

- Bifidobacterium bifidum ?novaBBF9? DSM 34337 and Lactobacillus fermentum ?novaLF58? DSM 34340; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example 1:3 to 3:1; or 1:2 to 2:1; or 1:1, preferably all strains of bacteria are in freeze-dried form.

- Lactobacillus crispatus ?novaLCR6? DSM 34348 and Lactobacillus fermentum ?novaLF58? DSM 34340; preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example 1:3 to 3:1; or 1:2 to 2:1; or 1:1, preferably all strains of bacteria are in freeze-dried form.

or, a pharmaceutical or nutraceutical association or combination or food supplement comprising or, alternatively, consisting of the three strains of the invention: - Bifidobacterium bifidum ?novaBBF9? DSM 34337, Lactobacillus crispatus ?novaLCR6? DSM 34348 and Lactobacillus fermentum ?novaLF58? DSM 34340; preferably in a weight ratio of 1:1:2, or 1:2:1, or 2:1:1, or 1:1:1, preferably all strains of bacteria are in freeze-dried form.

The second aspect of the invention also concerns a pharmaceutical composition or

nutraceutical or food supplement that includes one of the strains described above or their

association (A2), optionally together with at least one excipient and/or physiologically compatible vehicle

and/or pharmaceutically acceptable.

The second aspect of the invention also concerns the use of the said bacterial strains and/or the

their mixtures in therapy and, more specifically, in the curative or prophylactic treatment of pathologies

gynecological, preferably of pathologies associated with alterations of myometrial trophism

and endocrine and metabolic disorders such as polycystic ovary syndrome (PCOS), thyroid hyperplasia

endometrial and endometriosis.

The second aspect of the invention also concerns the use of the compositions

of the invention in therapy and, more specifically, in the curative or prophylactic treatment of

gynecological pathologies, preferably pathologies associated with alterations in the trophism of the

myometrium and endocrine and metabolic disorders such as polycystic ovary syndrome (PCOS),

endometrial hyperplasia and endometriosis.

Preferably, the second aspect refers to a composition comprising 1 to 100 mg

of Bifidobacterium bifidum novaBBF9, from 1 to 100 mg of Lactobacillus crispatus novaLCR6 and from

0.1 to 100 mg of Lactobacillus fermentum novaLF58, preferably 5 mg to 50 mg of

Bifidobacterium bifidum novaBBF9, 5 mg to 50 mg of Lactobacillus crispatus novaLCR6 and

1 mg to 50 mg of Lactobacillus fermentum novaLF58, even more? preferably from 10 mg to 40 mg

of Bifidobacterium bifidum novaBBF9, for example 20 mg or 30 mg of Bifidobacterium bifidum

novaBBF9, 10 mg to 40 mg of Lactobacillus crispatus novaLCR6, e.g. 20 mg or 30 mg

of Lactobacillus crispatus novaLCR6 and from 5 mg to 40 mg of Lactobacillus fermentum novaLF58,

for example 20 mg or 30 mg of Lactobacillus fermentum novaLF58, preferably all strains of

bacteria are in freeze-dried form.

Preferably, a composition may comprise from 5 mg to 20 mg of Bifidobacterium bifidum

novaBBF9, 5 mg to 20 mg of Lactobacillus crispatus novaLCR6 and 3 mg to 10 mg of

Lactobacillus fermentum novaLF58; preferably all strains of bacteria are in the form

freeze-dried.

Preferably, the second aspect of the invention refers to a composition comprising 10 mg of Bifidobacterium bifidum novaBBF9, 10 mg of Lactobacillus crispatus novaLCR6 and 5 mg of Lactobacillus fermentum novaLF58, preferably all strains of bacteria are in freeze-dried form.

The compositions of the second aspect of the invention may comprise one or more pharmaceutically acceptable excipients and/or carriers.

Preferably, the strains Bifidobacterium bifidum novaBBF9 and Lactobacillus crispatus novaLCR6 are administered in daily doses ranging from 1.0x10<6>CFU/day to 1.0x10<12>CFU/day, preferably from 1.0x10<8>CFU/day to 1.0x10<10>CFU/day, for example approximately 1.0x10<9>CFU/day, while the strain Lactobacillus fermentum novaLF58 is administered in daily doses ranging from 0.5x10<6>CFU/day to 0.5x10<12>CFU/day, preferably from 0.5x10<8>CFU/day to 0.5x10<10>CFU/day, for example approximately 0.5x10<9>CFU/day (CFU = Colony Forming Units).

The compositions can be administered once or more times a day, preferably once a day.

The strains of the second aspect of the invention may be present in the composition of the second aspect of the invention as live and viable bacteria, dead bacteria or their cellular components, cellular extracts, lysates or tyndallized.

According to one embodiment, the at least one physiologically and/or pharmacologically acceptable excipient may include prebiotics. According to one embodiment, the at least one physiologically and/or pharmacologically acceptable excipient may include a monosaccharide, a disaccharide, a polysaccharide, soluble fiber and/or insoluble fiber, GOS, FOS, inulin, maltodextrin, and mixtures thereof.

According to one embodiment, the at least one physiologically and/or pharmacologically acceptable excipient may include partially hydrolyzed guar gum (PHGG). According to various embodiments, the physiologically and/or pharmacologically acceptable excipient may be selected from the group consisting of: inulin, a fructooligosaccharide (FOS), maltodextrin and mixtures thereof.

The compositions according to the first aspect of the invention are preferably compositions for oral use, advantageously in the form of capsules. Other pharmaceutical forms may however be used.

The strains of the second aspect of the invention have been the subject of numerous experimental tests, reported below.

During these tests, the Applicant observed that in particular when associated, the A2 association is able to stimulate the intestine/myometrium axis, maintaining myometrial function. Furthermore, the association is able to improve myometrial activity by modulating cell cycle progression.

The tests conducted and the results obtained with the A2 association of the second aspect of the invention will be presented below, with reference to the attached Figures.

Experimental section - myometrial activity

Legend

L. Crispastus = Lactobacillus crispatus novaLCR6

L. Fermentum = Lactobacillus fermentum novaLF58

B Bifidum = Bifidobacterium bifidum novaBBF9

UFC = colony forming units

A dose-response study was conducted in an in vitro model using PHM1-41 cells to evaluate the safety of different probiotics and substances, as follows:

or Lactobacillus crispatus novaLCR6 in a range from 10 mg/ml to 50 mg/ml (corresponding to 1x10<9>-5x10<9 >CFU)

or Lactobacillus fermentum novaLF58 in a range from 10 mg/ml to 50 mg/ml (corresponding to 1x10<9>-5x10<9 >CFU)

or Bifidobacterium bifidum novaBBF9 in a range from 10 mg/ml to 50 mg/ml (corresponding to 1x10<9>-5x10<9 >CFU)

or a Lactobacillus Gasseri in a range from 5 mg/ml to 50 mg/ml (corresponding to 1x10<9>-1x10<8 >CFU)

- di-chiro-inositol (DCI) in a range from 150 mg/ml to 300 mg/ml

or inositol in a range from 18 ?g/ml to 1000 mg/ml.

All substances were prepared directly in the stimulation medium.

The best concentration of each probiotic (based on conversion from CFU to mg/ml per day) was maintained for all subsequent experiments.

Based on the data obtained, the best concentration of each substance was tested for the intestinal barrier or for the modulation of mediators released by probiotics. In this context, a gut/myometrium co-culture model was established by seeding intestinal CaCo-2 cells in the apical part of the Transwell? system and stimulating the cells with L. Crispastus 10 mg/ml, L. Fermentum 5 mg/ml, B. Bifidum 10 mg/ml, alone and in combination. Samples metabolized by intestinal cells were used to stimulate myometrial epithelium (PHM1-41 cells) placed in the basolateral compartment for 48 hours, in order to analyze mitochondrial metabolism. Further experiments were then performed to evaluate myometrial cell proliferation (Crystal Violet), ROS and oxytocin production, TNF-a activity and to evaluate the influence of ROS activity on the myometrial cell proliferation. ovarian by analyzing the level of FSH and LH, which are mainly involved in the fertility program.

Materials and methods

Preparation of solutions

Freeze-dried L. Crispatum, L. Fermentum and B. Bifidum were dissolved directly in PBS1x to prepare different concentrations for testing. L. Crispatum and L. Fermentum were tested in the range of 50 mg/mL to 10 mg/mL, and B.Bifidum from 200 μg/mL to 5 mg/mL. Concentrations of each probiotic correspond to oral human use as reported in Table 1.

Table 1: Sample concentration range

Inositol and di-chiroinositol (DCI) were dissolved in PBS1x in a range normally used in commercial formulations (from 150 mg/mL to 1000 mg/mL).

Cell culture

Human intestinal epithelial cells, CaCo-2, purchased from the American Type Culture Collection (ATCC), were cultured in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham's (DMEM-F12, Merck Life Science, Rome, Italy) containing 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin and maintained in a 37°C, 5% CO2 incubator. Cells for experiments were used at passage numbers ranging from 26 to 32 to preserve the integrity of the permeability and paracellular transport properties while maintaining similarity to the intestinal absorption mechanism following oral intake in humans.

The PHM1-41 cell line was derived from gravid human myometrium and maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% FBS, 2mM L-glutamine and 1% penicillin/streptomycin and incubated at 37°C at 5% CO2 and 95% humidity.

CHO-K1 (Chinese Hamster Ovary) cells, a widely used model to evaluate the role of probiotics in the fertility program, were cultured in Dulbecco's modified Eagle's medium: Nutrient Mixture F-12 supplemented with 10% fetal bovine serum (FBS, Sigma, Milan, Italy), 2 mM glutamine and 1% penicillin/streptomycin (Sigma, Milan, Italy) and incubated at 37 °C, 5% CO2 and 95% humidity. MTT assay

After each stimulation, cells were washed with PBS1? and incubated with DMEM without phenol red and FBS containing 1% MTT dye (MTT-Based In Vitro Toxicology Assay Kit; Sigma-Aldrich) for 2 h at 37?C and 5% CO2. Cell viability was determined by measuring absorbance with a spectrophotometer (Infinite 200 Pro MPlex, Tecan) at 570 nm with correction at 690 nm and calculated by comparing the results with control cells (100% viable). Crystal Violet

Cell proliferation was studied by Cristal Violet staining as reported in the literature. After treatment, cells were washed with PBS1x and fixed with 1% glutaraldehyde in PBS1x (% v/v) for 15 minutes at room temperature. 100 ?l of 0.1% Cristal Violet in aqueous solution (% v/v) was added to each well for 20 minutes at room temperature. Cells were washed again and 100 ?l of 10% acetic acid in PBS1x (% v/v) was added to solubilize them, then absorbance was detected with a spectrophotometer (Infinite 200 Pro MPlex, Tecan) at 595 nm. Cell number was calculated by comparing the data with control cells and normalized to untreated cells examined at time zero.

Intestine/myometrium co-culture model

CaCo-2 cells were seeded, as previously described, into Transwell? inserts at a density of 20,000 cells/cm2 and cultured for 21 days in complete medium, as previously reported. Then, 3-5 days before expiration, PHM1-41 cells were added to the basolateral side of the insert system at a concentration of 50,000 cells/600 μL and the co-culture was maintained in complete medium for three days. The medium was changed starting from the apical side of the wells and incubated for up to 2 hours, then treatments were performed. L. crispatum 10mg/ml,

L. Fermentum 5 mg/ml and B. Bifidum 10 mg/ml, alone and in combination, were added to the apical part from 2h to 6h, mimicking intestinal absorption. At the end of the time, the basolateral medium was collected to measure the probiotics metabolization by the butyric acid assay kit. At 6 hours after stimulation, the transwell insert was removed to perform the subsequent tests on PHM1-4, such as the analysis of proliferation, oxidative stress and inflammatory processes, hormonal and molecular pathways involved.

Intestinal/ovarian co-culture model

CaCo-2 cells were seeded, as previously described, into Transwell? inserts at a density of 20,000 cells/cm2 and cultured for 21 days in complete medium, as previously reported. Then, 3-5 days before expiration, CHO-K1 cells were added to the basolateral side of the insert system at a concentration of 60,000 cells/600 μL and the co-culture was maintained in complete medium for three days. The medium was changed starting from the apical side of the wells and incubated for up to 2 hours, then treatments were performed. L. crispatum 10mg/ml,

L. Fermentum 5 mg/ml and B. Bifidum 10 mg/ml, alone and in combination, were added to the apical medium for 2h to 6h, simulating intestinal absorption. At 6 hours after stimulation, the Transwell? insert was removed to perform the subsequent FSH and LH production assay.

Absorption analysis

Cells were stimulated with the selected agents added to the apical part of the Transwell? system in a time-dependent study (2h to 6h), to verify at each time point the intestinal absorption or bioavailability using the marker dye fluorescein 0.04% of transepithelial transport. Fluorescence was detected with a fluorescence spectrophotometer (Infinite 200 Pro MPlex, Tecan, Cernusco sul Naviglio, MI, Italy) at excitation/emission wavelengths of 490/514 nm.

Quantification of butyric acid

To quantify probiotic metabolites, butyric acid production was analyzed using the ELISA kit (Cloud-Clone, Wuhan) according to the manufacturer's instructions. The absorbance of each sample was measured after the addition of the stop solution at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan) and the OD was interpolated with a standard curve (10,000 pg/mL to pg/mL), expressing the data as mean (pg/mL) versus control. ROS production

The rate of superoxide anion release was measured using a standard protocol based on the reduction of cytochrome C. Both treated and untreated cells were added with 100 μL of cytochrome C and, in another sample, 100 μL of superoxide dismutase for 30 minutes in an incubator (all substances were supplied by Sigma-Aldrich). The absorbance in the culture supernatants was measured at 550 nm with a spectrometer (Infinite 200 Pro MPlex, Tecan) and O2 was expressed as the mean ± SD (%) of nanomoles of reduced cytochrome C per microgram of protein compared to the control.

TNF-ELISA Kit?

The concentration of TNF-? in the basolateral medium was determined using the TNF-? ELISA kit (Merck Life Science, Rome, Italy) according to the manufacturer's instructions. The absorbance of each well was measured after the addition of the stop solution at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan).

LH ELISA Kit

Luteinizing hormone (LH) concentration was determined with the Human LH (Luteinizing Hormone) ELISA kit (FineTest) according to the instructions. Briefly, 100uL of each sample was added to each well and the plate was incubated at 37?C for 90 minutes. At the end of the incubation, the material in each well was removed and the wells were washed twice with wash buffer. 100uL of biotin-labeled antibody working solution was added to the wells and the plate was incubated at 37?C for 60 minutes. At the end of the incubation, the solution in each well was removed and the wells were washed three times with wash buffer. Then, 100 ?L of SABC working solution was added to each well and the plate was incubated at 37?C for 30 minutes. Finally, the wells were washed five times and 90 uL of TMB substrate was added to each well. After 10-20 minutes, 50 uL of stop solution was added to each well and the plate was immediately stained at 450nm using a plate reader (Infinite 200 Pro MPlex, Tecan). A standard curve relating the color intensity to the concentration of the standard was plotted (range 0.938 to 60 mIU/mL).

FSH ELISA Kit

Follicle-stimulating hormone (FSH) concentration was determined using the Human FSH (Follicle-Stimulating Hormone) ELISA kit (FineTest) according to the instructions. Briefly, 100 µL of each sample was added to each well and the plate was incubated at 37?C for 90 minutes. At the end of the incubation time, the material in each well was removed and the wells were washed twice with wash buffer. 100 µL of biotin-labeled antibody working solution was added to the wells and the plate was incubated at 37?C for 60 minutes. At the end of the incubation, the solution in each well was removed and the wells were washed three times with wash buffer. Then, 100 µL of SABC working solution was added to each well and the plate was incubated at 37?C for 60 minutes. was incubated at 37?C for 30 minutes. Finally, the wells were washed five times and 90?L of TMB substrate was added to each well. After 10-20 minutes, 50?L of stop solution was added to each well and the plate was immediately stained at 450 nm using a plate reader (Infinite 200 Pro MPlex, Tecan). A standard curve relating the color intensity to the concentration of the standard (range 1,563 to 100 mIU/mL) was plotted.

Oxytocin ELISA Kit

Oxytocin (OT) concentration was determined using the Oxytocin ELISA Kit (Cayman Chemical Company; Ann Arbor, MI, USA) according to the instructions. This assay is based on the competition between oxytocin and acetylcholinesterase (AChE)-conjugated oxytocin (oxytocin tracer) for a limited amount of oxytocin polyclonal antiserum. Since the concentration of oxytocin tracer is held constant while the concentration of oxytocin varies, the amount of oxytocin tracer that can bind to the oxytocin polyclonal antiserum will be inversely proportional to the concentration of oxytocin in the well. The plate was washed to remove unbound reagents and then Ellman's Reagent was added to the wells. The product of the enzymatic reaction is ? was reddened at a wavelength between 405 and 420 nm. A standard curve is plotted relating the intensity of the color to the concentration of the standard (range 15,625 to 1000 pg/mL).

AKT/ERK ELISA Kit

AKT/ERK activation was measured by the InstantOneTM ELISA method (Thermo Fisher, Milan, Italy) on chondrocyte lysates as reported in the literature. The strips were measured by a spectrometer at 450 nm (Infinite 200 Pro MPlex, Tecan). The results were expressed as mean absorbance (%) compared to the control.

Cyclin D1 ELISA Kit

Cyclin D1 production was determined using the ELISA kit (MyBiosource, San Diego, CA, USA) that analyzes cyclin D1 in cell lysates, according to the manufacturer's instructions. Briefly, 100 μL of each sample was added to the wells and the plate was incubated for 80 min at 37°C; then the wells were washed 3 times with 200 μL of washing solution and 100 μL of biotinylated antibody was added before incubating the plate for 50 min at 37°C. Then the wells were washed again 3 times with 200 μL of washing solution before adding 100 μL of Streptavidin-HRP working solution for 50 min at 37°C. Before adding 90 μL of TMB substrate solution to each well, the wells were washed again 5 times. Finally, after 50 minutes of incubation at 37?C, 50?L of Stop reagent was added to each well. Samples were analyzed with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. Concentration is expressed as ng/mL relative to a standard curve (range 0.32 to 20 ng/mL) and results are expressed as a percentage (%) relative to the control (line 0).

Annexin V ELISA Kit

Annexin V production was determined using the ELISA kit (Abcam, Cambridge, UK) that analyzes Annexin V in cell supernatants, according to the manufacturer's instructions. Briefly, 50 μl of standards or samples were added to each well with 50 μl of antibody cocktail before incubating the plate for 1 h with shaking. Subsequently, each well was washed 3 times with 350 μl of 1x wash buffer before incubating the plate for 10 min in the dark with 100 μl of TMB developer solution in each well. Finally, 100 μl of stop solution was added to each well and the samples were analyzed with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. The concentration was expressed in ng/mL against a standard curve (range 46.9 pg/mL - 3000 pg/mL) and results are expressed as a percentage (%) compared to the control (line 0).

ELISA MAGT1 Kit

MAGT1 production was determined using the ELISA kit (MyBioSource, Vancouver, Canada) that analyzes MAGT1 in cell lysates, according to the manufacturer's instructions. Samples were analyzed with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. Concentration is expressed as ng/mL relative to a standard curve (range 0.25 ng/mL-8 ng/mL) and results are expressed as a percentage (%) relative to the control (line 0).

PAX8 ELISA Kit

PAX8 production was determined using the ELISA kit (MyBiosource, San Diego, CA, USA) that analyzes PAX8 in cell lysates, according to the manufacturer's instructions. Samples were analyzed with a spectrometer (Infinite 200 Pro MPlex, Tecan) at 450 nm. Briefly, 100 μL of each sample or standard was added to each well and the plate was incubated for 1 h at 37°C. Then the wells were washed 3 times with wash buffer and 100 μL of HRP-Streptavidin conjugate was added before incubating the plate for 30 min at 37°C. After 5 washes with wash buffer, 90 μL of TMB substrate was added to each well and the plate was incubated for 15–30 min at 37°C. Finally, 50 ?L of stop solution was added to each well and the absorbance of the samples was read at 450 nm with a spectrometer (Infinite 200 Pro MPlex, Tecan). The concentration is expressed in ng/mL against a standard curve (range 15.6 to 1000 pg/mL) and the results are expressed as a percentage (%) compared to the control (line 0).

Statistical analysis

Results are expressed as mean ? SD of at least 5 biological replicates for each experimental protocol, and each replicate was repeated 3 times for each experimental protocol. Statistical comparisons between groups were performed using one-way ANOVA with Bonferroni post hoc test or Mann-Whitney U test, as appropriate, using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). p<0.05 was considered statistically significant. All densitometric analysis data were normalized to control values (defined as 1). All other data from each experimental protocol were normalized to control values in percent (defined as 0%).

Results

PHM1 cells were used to analyze the interaction between the microbiome and myometrium using different probiotics, combined in a novel formulation, able to improve female infertility. A dose-response study was then performed for 24 hours in order to evaluate the best concentration to be used in the final formulation. The concentration range was tested based on the literature. Since different treatments for PCOS were formulated using inositol and di-chiroinositol (DCI), these compounds were also tested in a range normally used in commercial formulations. As reported in figure 13, all probiotics were able to improve cell viability compared to the control (p<0.05); in particular, L. Crispatus, L. Fermentum and B. Bifidum were able to maintain cell health compared to all tested agents (p<0.05). As for Inositol and DCI, none of the tested substances induce the desired results, but only DCI 150 mg, which reported less harmful results and for this reason was included in the combination. Based on the results obtained, the hypothesized concentrations are L. Crispatus 10 mg/ml L. Fermentum 5 mg/ml+ B. Bifidum 10 mg/ml (combination A2) and L. Crispatus 10 mg/ml L. Fermentum 5 mg/ml+ B. Bifidum 10 mg/ml DCI 150 mg (combination A2+DCI 150 mg) (Figure 13).

Before conducting further analyses, investigations were conducted to gain more insight into physiological absorption in an EMA and FDA validated intestinal barrier model. To this end, both mitochondrial metabolism and absorption were analyzed by testing B.Bifidum 10 mg/ml, L. Crispatus 10 mg/ml, L. Fermentum 5 mg/ml and DCI 150 mg/ml, alone and in combination, over 6h (maximum intestinal absorption time) to demonstrate that all substances are able to exert a beneficial effect without altering intestinal cells, excluding any irritability to confirm the safety of the A2 association. As shown in Figure 2, cell viability shows an increase in mitochondrial metabolism for all tested agents compared to the control, although the greatest effect (p<0.05) was observed with the combination of all probiotics. In contrast, the A2+DCI150 mg combination dramatically reduced the cell viability of intestinal cells (about four-fold, p<0.05), suggesting that the addition of DCI could alter the activity of probiotics. Consequently, TEER analyses were performed to confirm the integrity of the intestinal monolayer. The reported TEER values are 500 ? 52.9 !*cm2 for CaCo-2 intestinal cells according to the literature suggesting that the cells form an intact monolayer induced by the treatments (Figure 14).

Further analyses were conducted using the same model, analyzing two main aspects of probiotic bioavailability: permeability and metabolites produced. As shown in Figure 3, the intestinal passage analysis demonstrated that all the selected agents are able to cross the intestinal epithelium, confirming the safety data already obtained. In particular, the chosen combination increases the intestinal passage, suggesting that the probiotic formulation is able to directly reach the target organ more efficiently than the single agents and, above all, compared to the combination A2+DCI150 mg (p<0.05). The same results were also obtained by analyzing the probiotic metabolite (butyric acid), demonstrating that the ability of probiotics to release short-chain fatty acids (SCFA) and to influence the secretion of sexual hormones is controlled by the A2 association which amplified the effects exerted by the single agents (p<0.05). Also in this case, the A2+DCI150 mg association does not contribute to correct intestinal passage, suggesting that DCI should not be relevant for myometrial homeostasis (p<0.05).

All these data on intestinal absorption are important to understand the behavior of the formulation within the intestinal compartment: in fact, the A2 association stimulates the correct passage of the intestinal barrier, improving the functions of the intestinal barrier and reaching the plasma environment, also thanks to the synergistic activity exerted by the chosen probiotics, enhancing their bioavailability (Figure 15).

Given these premises, it becomes important to consider the direct gut-myometrium effect in a 24-hour co-culture model of myometrial alteration. Therefore, as shown in figure 16, analyses of the myometrium after intestinal passage reveal that all the tested probiotics are able to improve the biological activity of the myometrium during myometrial alteration. Our results demonstrated that the combination L. Crispatus 10 mg/ml L. Fermentum 5 mg/ml + B. Bifidum 10 mg/ml (called association A2) improved mitochondrial metabolism and, consequently, proliferation activity, amplifying the effects exerted by the single agents (p<0.05). In particular, the analysis of the mitochondrial metabolism of myometrial cells, in contact with intestinal cells, confirms the hypothesis that the chosen combination is able to increase the viability of the myometrium. compared to the single agents (about 1.25 times more than L. Crispatus; 3 times more than L. Fermentum and 1 time more than B. Bifidum), confirming that it is more effective in increasing cellular metabolism, suggesting that it may also play an important role in cell proliferation. In fact, as shown in panel B, proliferation activity is improved after treatment with the single agents (p<0.05), but the synergistic effect, obtained when all probiotics are combined, produces an increase in proliferative activity (about 1.66 times more than L. Crispatus; 1 time more than L. Fermentum and 2 times more than B. Bifidum) also confirmed by the analysis of cyclin D1, a cell cycle regulatory protein that is necessary for proliferative activity. On the contrary, the association A2+DCI 150 mg does not produce any significant effect with regards to the proliferation analysis (p<0.05), confirming the minor role of the DCI in the formulation (Figure 16).

To demonstrate that the increase in proliferative capacity is not due to an alteration of myometrial homeostasis, further experiments were performed during myometrial alteration, analyzing ROS production and TNFα activity. As reported in Figure 5, the analysis of oxidative stress (panel A) and inflammation (panel B) demonstrates that all the tested concentrations did not induce ROS production and did not determine any inflammatory phenomenon. All the combined probiotics exerted the maximum effect, confirming the absence of side effects. On the contrary, the association A2+DCI 150 mg did not alter ROS production (p<0.05) but induced a slight increase in inflammatory levels, which remained below the normal physiological ranges, confirming the controversial role of DCI (Figure 17).

Since all these results on myometrial epithelia revealed very encouraging data, further experiments were performed to evaluate the hormonal activity that regulates myometrial function. In particular, several results suggest that oxytocin may be involved in a dual role: in the induction of myometrial contractility and in the control of the secretion of some hormones of the anterior pituitary gland. More specifically, regarding the contractile role of oxytocin, it plays an essential role in the mechanisms of myometrial contractility through the interaction between myosin and actin, independently of the variations in intracellular calcium and magnesium concentration in smooth muscle tissue. Therefore, the hypercontraction activity of the myometrium may cause several physiological changes such as hypertrophy, hyperplasia, contraction and apoptosis, which are essential for fertility activity. Therefore, to exclude myometrial hypercontraction, oxytocin level and MAGT1, a magnesium transporter implicated in maintaining intracellular magnesium levels, were observed. Furthermore, as reported in Figure 18, the results demonstrated that the combination of L. Crispatus 10 mg/ml L. Fermentum 5 mg/ml+ B. Bifidum 10 mg/ml maintained oxytocin and MAGT1 expression at basal level better than the DCI formulation (p<0.05), confirming the active role of the A2 association in myometrial relaxation. These results are important because the A2 association is able to maintain the correct balance between calcium and magnesium movement by inhibiting spontaneous myometrial contractility (Figure 18).

To clarify the mechanisms underlying the observed results, selective biomarkers involved in the maintenance of myometrial homeostasis were analyzed by ELISA kits (Figure 7A). ERK/MAPK activity induced by all probiotics combined was higher than that of the single agents (p<0.05), supporting better results regarding myometrial viability and proliferation capacity. Furthermore, AKT expressions (Figure 7B) confirmed the role of the new formulation in myometrial activity compared to the control (p<0.05) and compared to the single agents (about 4-fold more than L. Crispatus; 60% compared to L. Fermentum and 40% compared to B. Bifidum, p<0.05), confirming its greater influence. Also in this case, ? the role of DCI was investigated and, as expected, the A2+150 mg combination was not able to maintain the balance of both kinases supporting its controversial effects (p<0.05) (Figure 19).

Furthermore, the main intracellular pathways involved in myometrial dysfunction were analyzed by an ELISA kit to confirm the beneficial effects of the new formulation. Accordingly, Er?, which could be clinically useful to predict hormonal imbalances and treat inflammatory conditions associated with menopause, infertility or myometrial dysfunction, PAX8, which is frequently upregulated and functionally essential in a major subset of ovarian tumors leading to infertility, and PAK-1, which is a key member of the CDC42/PAK1 signaling pathway involved in the regulation of cell cycle proliferation and apoptosis during PCOS, were analyzed. As reported in Figure 20, all tested agents were able to modulate the selected markers (p<0.05), in particular the A2 association amplified the effects exerted by the single agents confirming the data already reported. obtained on the improvement, safety and maintenance of myometrial function (p<0.05). However, the addition of DCI in the formulation slightly attenuated the alteration observed during myometrial dysfunctions without inducing any significant improvement, confirming once again that this molecule does not induce beneficial effects on the myometrium and does not interact directly with probiotics (Figure 20).

Finally, since LH and FSH play complementary roles in follicular development and ovulation through a complex interaction in the hypothalamus, anterior pituitary, reproductive organs and oocytes, additional experiments were conducted to investigate the effects of the A2 combination and the A2+DCI 150 mg combination on ovarian cells. Impaired gonadotropin production or action results in a relative or absolute deficiency of LH and FSH that impairs gametogenesis and gonadal steroid production, thereby reducing fertility. In women, LH and FSH deficiency is a spectrum of conditions with different functional or organic causes, characterized by low or normal gonadotropin levels and low estradiol levels; the determinants of reduced FSH and LH action are associated with a reduced response to ovarian stimulation. Therefore, the analysis of these two hormones on ovarian cells supported the importance of the gut/brain axis on the regulation of fertility homeostasis; as reported in Figure 9, the association between probiotics can improve the related fertility hormones (FSH and LH) (p<0.05) compared to the control. The evidence showed that the combination between the chosen probiotics improved hormone secretion better than the single agent (p<0.05) and then the association A2+DCI 150 mg (about 6 times more than LH and about 3 times more than FSH), demonstrating once again that the presence of DCI does not improve the activity of probiotics (p<0.05). Furthermore, these data support the results reported in the literature on the importance of recombinant LH and FSH supplementation to improve hormone levels during fertility programs, since These cells naturally produce these hormones after stimulation with butyric acid (Figure 21).

Claims (10)

1. A pharmaceutical or nutraceutical composition or food supplement comprising at least one strain of bacteria selected from the group comprising or, alternatively, consisting of: - Bifidobacterium psychraerophilum ?Q5?, DSM deposit number 33121; - Bifidobacterium longum subsp. longum ?novaBLG1?, DSM deposit number 34338; and mixtures thereof, and, optionally, at least one physiologically and/or pharmaceutically acceptable or food grade excipient and/or carrier. 2. The composition according to claim 1, wherein said composition comprises or, alternatively, consists of Bifidobacterium psychraerophilum ?Q5? DSM 33121 and Bifidobacterium longum subsp. longum ?novaBLG1? DSM 34338, preferably in a weight ratio of 1:10 to 10:1, even more preferably in a weight ratio of 1:5 to 5:1, for example 1:3 to 3:1, or 1:2 to 2:1, or 1:1. 3. The composition according to claim 1 or 2, wherein said composition comprises from 1 to 100 mg of B. Longum novaBLG1 and from 1 to 100 mg of B. Psychaerophilum Q5, both in lyophilized form, preferably from 5 mg to 50 mg of B. Longum novaBLG1 and from 5 mg to 50 mg of B. Psychaerophilum Q5, even more preferably from 10 mg to 40 mg of B. Longum novaBLG1, for example 20 mg or 30 mg of B. Longum novaBLG1, and from 10 mg to 40 mg of B. Psychaerophilum Q5, for example 20 mg or 30 mg of B. Psychaerophilum Q5. 4. The composition according to any of claims 1-3, wherein said composition comprises from 5 to 20 mg of B. Longum novaBLG1 and from 5 to 20 mg of B. Psychaerophilum Q5, both in lyophilized form, preferably 10 mg of B. Longum novaBLG1 and 10 mg of B. Psychaerophilum Q5. 5. The composition according to any of claims 1-4, wherein said composition is administered in daily doses ranging from 1.0x10<6>CFU/day to 1.0x10<12>CFU/day, preferably from 1.0x10<8>CFU/day to 1.0x10<10>CFU/day, for example approximately 1.0x10<9>CFU/day. 6. The composition according to any of claims 1-5, wherein said composition is for use as a medicament, preferably for use in the treatment of male fertility. 7. The composition for use according to claim 6, wherein said composition is for use in a method of treatment, preferably in a method of preventive or curative treatment, of male urinary disorders, preferably benign prostatic hyperplasia (BPH). 8. A strain of bacteria selected from the group comprising or, alternatively, consisting of: - Bifidobacterium psychraerophilum ?Q5?, DSM deposit number 33121; - Bifidobacterium longum subsp. longum ?novaBLG1?, DSM filing number 34338; and their blends. 9. The bacterial strain according to claim 8, wherein said bacterial strain is for use as a medicament, preferably for use in the treatment of male fertility. 10. The bacterial strain for use according to claim 9, wherein said bacterial strain is for use in a method of treatment, preferably in a method of preventive or curative treatment, of male urinary pathologies, preferably benign prostatic hyperplasia (BPH).