Probiotic for the treatment of infertility and recurrent pregnancy loss

Abstract

The invention relates to a strain of the Lactobacillus salivarius species deposited in the Spanish Type Culture Collection (CECT) with accession number 5713 or a mutant thereof for use in the treatment and / or prevention of infertility or recurrent pregnancy loss in a female subject. Additionally, the invention relates to an in vitro monitoring method for monitoring the effect of a treatment for infertility or recurrent pregnancy loss in a female subject with a strain of the Lactobacillus salivarius species deposited in the CECT with accession number 5713 or a mutant thereof.

Description

[0001] PROBIOTIC FOR THE TREATMENT OF INFERTILITY AND RECURRENT PREGNANCY LOSS

[0002] FIELD OF INVENTION

[0003] The present invention belongs to the field of methods for the treatment of infertility and recurrent pregnancy loss.

[0004] BACKGROUND OF THE INVENTION

[0005] Currently, it is estimated that 15% of couples experience fertility problems. The cause of the problem is attributed to the woman in 33% of cases, to both partners in 25%, and to both partners in 33%, but the cause is unknown in the remaining 11% of cases.

[0006] Increasing evidence highlights the importance of the female reproductive tract microbiota in human reproduction. Under physiological conditions, and in contrast to the gut microbiota, the human vaginal microbiota is generally characterized by low bacterial diversity and an abundance and predominance of bacteria of the genus Lactobacillus.

[0007] The vaginal microbiota in most healthy women of reproductive age is composed mainly of one or a few species of Lactobacillus, which make up 90%-95% of the total bacteria.

[0008] Several factors contribute to interindividual and intraindividual changes in the vaginal microbiota, and although changes can occur between different vaginotypes, increased diversity and strict anaerobes and decreased or depleted lactobacilli are considered risk factors for bacterial vaginosis (BV).

[0009] The composition of the vaginal microbiota (and, in particular, any deviation from the low-diversity vaginal microbiome dominated by Lactobacillus) may play a key role in fertility and the outcomes of assisted reproductive technologies (ART). Although commercially available probiotics are empirically prescribed to repopulate the female reproductive tract with Lactobacillus strains (Moreno and Simón, Reprod Med Biol. 2019; 18:40-50), an adequate scientific evaluation of their actual efficacy has not been conducted. Therefore, there is a need for new treatments for infertility, as well as for recurrent pregnancy loss, based on the administration of probiotics with proven efficacy. SUMMARY OF THE INVENTION

[0010] The inventors of the present invention have surprisingly discovered that oral administration of a probiotic strain CECT 5713 of Lactobacillus salivarius to women with infertility or unexplained recurrent pregnancy loss resulted in a 66% success rate in achieving pregnancy (Table 4) and a 56% reproductive success rate (Table 4). Furthermore, the inventors of the present invention have also discovered that in both women with infertility and women with recurrent pregnancy loss, treatment with the probiotic led to a reduction in vaginal pH (Table 7) and an increase in vaginal levels of the cytokines TGFβ1, TGFβ2, and VEGF (Table 8) in those women who ultimately achieved pregnancy, but not in those who did not.

[0011] Therefore, in one aspect, the invention relates to a strain of the species Lactobacillus salivarius deposited in the Spanish Collection of Type Cultures (CECT) with accession number 5713 or a mutant thereof for use in the treatment and / or prevention of infertility or recurrent pregnancy loss in a female subject.

[0012] In another aspect, the invention relates to an in vitro method for monitoring the effect of a treatment for infertility or recurrent pregnancy loss in a female subject with a strain of the species Lactobacillus salivarius deposited in the CECT with accession number 5713 or a mutant thereof comprising:

[0013] (i) determining the level in a sample of vaginal and / or cervical mucosa from the female subject of a growth factor selected from the group consisting of VEGF, TGF-β1, and TGF-β2, and (ii) comparing this level with a baseline value obtained from the same subject before therapy, where an increase in this level relative to the baseline value indicates that the treatment is effective, and a decrease or lack of change in this level relative to the baseline value indicates that the treatment is ineffective. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1. Dominant lactobacillus species in cervicovaginal washing samples from fertile women (C), women with recurrent abortion (RA) and women with infertility of unknown origin (INF), in those cases where they could be isolated.

[0015] Figure 2. Comparison of bacterial diversity, assessed using the Shannon index (A) or the Simpson index (B), between the control, RA, and INF groups. Wilcoxon rank-sum tests with Bonferroni correction were used for pairwise comparisons between groups. Figure 3. PCoA (Principal Coordinate Analysis) plots of bacterial profiles (at the genus level) based on Bray-Curtis similarity analysis (relative abundance) (A) and the Jaccard coefficient for binary data (presence / absence) (B), from cervicovaginal wash samples. Control, circles; RA, crosses; INF, triangles. Box plots in the lower figures show the distance of the samples to the centroid in each group. The value given on each axis represents the percentage of the total variance explained by that axis. The p-value was calculated using the permutation test for homogeneity of multivariate dispersions. Figure 4.PCA (Principal Component Analysis) graphs of bacterial profiles at the genus level based on their relative abundance in cervicovaginal washing samples (Control, circles; INF, triangles; RA, squares).

[0016] Figure 5. Comparison between the RA (women with a history of recurrent spontaneous abortion) and INF (women with a history of infertility) groups of various characteristics at the time of recruitment: (A) age, weight and height; (B) pH, Nugent score and vaginal concentration of TGFβ1, TGFβ2 and VEGF and (C) lactobacillus count.

[0017] Figure 6. Changes in the characteristics of the RA group (women with a history of recurrent spontaneous abortion) after probiotic treatment, depending on the outcome (pregnancy versus no pregnancy): (A) change in pH and Nugent score; (B) change in vaginal concentration of TGFβ1, TGFβ2 and VEGF and (C) change in lactobacilli count.

[0018] Figure 7. Characteristics of the RA group (women with a history of recurrent miscarriage) before (I) and after (F) probiotic treatment, depending on the outcome (pregnancy versus no pregnancy). (A) pH and Nugent score; (B) vaginal concentration of TGFβ1, TGFβ2, and VEGF. Figure 8. Changes in the dominant species profile of the genus Lactobacillus in the RA group (women with a history of recurrent miscarriage) before (I) and after (F) probiotic treatment, depending on the outcome (pregnancy versus no pregnancy). Figure 9. Characteristics of the INF group (women with a history of infertility) before (I) and after (F) probiotic treatment, depending on the outcome (pregnancy versus no pregnancy).

[0019] Figure 10. Changes in the characteristics of the INF group (women with a history of infertility) after probiotic treatment, depending on the outcome (pregnancy versus no pregnancy): (A) change in pH and Nugent score; (B) change in vaginal concentration of TGFβ1, TGFβ2 and VEGF and (C) change in lactobacillus count.

[0020] Figure 11. Changes in the dominant species profile of the genus Lactobacillus in the INF group (women with a history of infertility) before (I) and after (F) probiotic treatment, depending on the outcome (pregnancy versus no pregnancy). Figure 12. Comparison of vaginal parameters between women in the RA and INF groups (considered together) who achieved pregnancy and those who did not: (A) change in pH and Nugent score; (B) change in vaginal concentration of TGFβ1, TGFβ2, and VEGF; and (C) change in lactobacillus count.

[0021] Figure 13. Comparison of vaginal parameters between women in the RA and INF groups (considered together) who had a pregnancy and those who did not, before (I) and after (F) probiotic treatment, depending on the outcome (pregnancy versus no pregnancy): (A) pH and Nugent score; (B) vaginal concentration of TGFβ1, TGFβ2 and VEGF.

[0022] Figure 14. Changes in lactobacillus counts (log CFU / ml) (A) and in the number of L. salivarius-specific DNA copies (copies / ml) (B) in cervicovaginal wash samples from women in groups RA and INF (considered together) who became pregnant and those who did not, before (I) and after (F) probiotic treatment, depending on the outcome (pregnancy versus non-pregnancy). Figure 15. Comparison of various characteristics of the control group with those of women in groups RA and INF who became pregnant (P) and those who did not (NP) after probiotic treatment: (A) pH and Nugent score; (B) vaginal concentration of TGFβ1, TGFβ2, and VEGF; and (C) lactobacillus count. DETAILED DESCRIPTION OF THE INVENTION

[0023] Medical uses of the invention

[0024] In one aspect, the invention relates to a strain of the species Lactobacillus salivarius deposited in the Spanish Collection of Type Cultures (CECT) with accession number 5713 or a mutant thereof for use in the treatment and / or prevention of infertility or recurrent pregnancy loss in a female subject.

[0025] Alternatively, the invention relates to the use of a strain of the species Lactobacillus salivarius deposited in the Spanish Collection of Type Cultures (CECT) with accession number 5713 or a mutant thereof for the manufacture of a medicament for the treatment and / or prevention of infertility or recurrent pregnancy loss in a female subject.

[0026] Alternatively, the invention relates to a method of treating and / or preventing infertility or recurrent pregnancy loss in a female subject comprising administering to said subject a therapeutically effective amount of a strain of the species Lactobacillus salivarius deposited in the Spanish Collection of Type Cultures (CECT) with accession number 5713 or a mutant thereof.

[0027] The term "strain", as used herein, refers to a genetic variant or subtype of a species of microorganism, preferably a bacterial species.

[0028] The terms “Lactobacilus salivarius” and “Lagilactobacillus salivarius” are used as synonyms in the context of the present invention.

[0029] In one embodiment, the invention relates to a strain of the species Lactobacillus salivarius deposited in the Spanish Collection of Type Cultures (CECT) with accession number 5713 or a mutant thereof for the treatment and / or prevention of infertility or recurrent pregnancy loss in a female subject.

[0030] In another embodiment, the invention relates to a mutant of the strain of the species Lactobacillus salivarius deposited in the Spanish Collection of Type Cultures (CECT) with accession number 5713 for the treatment and / or prevention of infertility or recurrent pregnancy loss in a female subject.

[0031] The term “mutant of the Lactobacillus salivarius strain deposited in the Spanish Type Culture Collection (CECT) with accession number 5713,” as used herein, refers to any strain obtained naturally or specifically developed from the reference strain, primarily by mutation, that retains the properties of the reference strain. The L. salivarius strain 5713 is characterized by the following properties.

[0032] (a) Antimicrobial activity against vaginal and / or cervical pathogens.

[0033] The term “vaginal and / or cervical pathogen” refers to a microorganism, primarily a fungus or bacterium, capable of colonizing the vaginal and / or cervical mucosa and potentially causing disease, such as vaginitis, vaginosis, trichomoniasis, candidiasis, or lower urinary tract infections. The term “vaginal pathogen” includes both microorganisms of external origin and microorganisms that, while they may be present in healthy vaginal mucosa, can cause disease when there is an imbalance in the vaginal flora, including a decrease in certain microorganisms that have a beneficial or protective effect, and / or an increase in the potentially pathogenic microorganism.Illustrative, non-limiting examples of vaginal pathogens include Candida albicans, Candida glabrata, Candida tropicalis, Gardnerella vaginalis, Streptococcus agalactias, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Prevotella, Peptostreptococcus, Escherichia coli, Enterococcus faecalis and Ureaplasma urealyticum. In a particular realization, the vaginal pathogen is selected from the group that consists of Gardnerella vaginalis (G. vaginalis MP14, G. vaginalis MP17, G. vaginalis MP20, G. vaginalis MP24, G. vaginalis MP29), Streptococcus agalactias (S. agalactias MP07, S. agalactias MP12, S. agalactias MP46), Candida albicans (C. albicans MP09, C. albicans MP18, C. albicans MP31), Candida glabrata (C. glabrata MP33, C. glabrata MP37), Candida parapsilosis (C. parapsilosis MP36, C. parapsilosis MP48), and Ureaplasma urealyticum ( U . urealyticum MP39, U. urealyticum MP57).

[0034] The term “antimicrobial activity,” as used here, refers to the ability to kill or inhibit the growth of a microorganism, in this case, the vaginal pathogen. Antimicrobial activity includes bactericidal activity (the ability to kill bacteria), bacteriostatic activity (the ability to inhibit bacterial growth), fungicidal activity (the ability to kill fungi), and fungistatic activity (the ability to inhibit fungal growth).

[0035] (b) Ability to co-aggregate with vaginal and cervical pathogens.

[0036] The term “vaginal and / or cervical pathogen” has been previously defined. In a particular embodiment, the vaginal pathogen is selected from the group consisting of Gardnerella vaginalis (G. vaginalis MP14, G. vaginalis MP17, G. vaginalis MP20, G. vaginalis MP24, G. vaginalis MP29), Streptococcus agalactiae (S. agalactiae MP07, S. agalactiae MP12, S. agalactiae MP46), Candida albicans (C. albicans MP09, C. albicans MP18, C. albicans MP31), Candida glabrata (C. glabrata MP33, C. glabrata MP37), Candida parapsilosis (C. parapsilosis MP36, C. parapsilosis MP48), and

[0037] Ureaplasma urealyticum (U. urealyticum MP39, U. urealyticum MP57). In a more particular embodiment, the L. salivarius strain CECT 5713 has the ability to co-aggregate with G. vaginalis, S. agalactiae, and C. albicans.

[0038] The term “co-aggregation capacity” refers to the ability of the L. salivarius CECT 5713 strain to adhere to other microorganisms of different species.

[0039] (c) High adhesion to vaginal epithelial cells.

[0040] The term “adhesion” refers to the ability of the L. salivarius strain CECT 5713 to bind to host tissues, in this case, vaginal epithelial cells. Adhesion to vaginal cells is considered high when it is at least 90% of the adhesion of the L. salivarius strain CECT 9145 when determined under the same experimental conditions; more specifically, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100%, or higher.

[0041] (d) High α-amylase activity

[0042] The term “α-amylase activity,” as used here, refers to the enzyme activity identified by EC number 3.2.1.1 capable of hydrolyzing glycosidic bonds along any point of the carbohydrate chain, breaking them down to generate maltotriose and maltose from amylose and maltose, and glucose and dextrin from amylopectin. The α-amylase activity of a bacterium is considered high when it is greater than 0.50 U / mL as determined by the method of Narita et al. (2006), Appl. Environ. Microbiol. 72: 269-275.

[0043] (e) High production of lactic acid.

[0044] The term “lactic acid production”, as used here, refers to the ability of the strain to produce lactic acid, whose official nomenclature is 2-hydroxypropanoic acid or α-hydroxypropanoic acid, in either of its two isomers L or D. The lactic acid production of a bacterium is considered high when it is greater than 8 mg / ml in the supernatants of the cultures after incubation under optimal growth conditions.

[0045] Therefore, in a particular embodiment, the mutant of the L. salivarius strain CECT 5713 retains one or more of the above properties. The term “retains,” as used herein, means that the mutant exhibits 100% or higher activity, or at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 60%, or 50% of the activity of the L. salivarius strain CECT 5713 from which it is derived, when such property is measured by appropriate quantitative assays.

[0046] In one particular embodiment, the L. salivarius strain mutant CECT 5713 maintains antimicrobial activity against vaginal pathogens of the L. salivarius strain CECT 5713. In a more particular embodiment, the mutant maintains antimicrobial activity against a vaginal pathogen selected from the group consisting of Gardnerella vaginalis (G. vaginalis MP14, G. vaginalis MP17, G. vaginalis MP20, G. vaginalis MP24, G. vaginalis MP29), Streptococcus agalactiae (S. agalactiae MP07, S. agalactiae MP12, S. agalactiae MP46), Candida albicans (C. albicans MP09, C. albicans MP18, C. albicans MP31), Candida glabrata (C. glabrata MP33, C. glabrata MP37), and Candida parapsilosis (C. parapsilosis). MP36, C. parapsilosis MP48), and

[0047] Ureaplasma urealyticum (U. urealyticum MP39, U. urealyticum MP57). In a more specific embodiment, the mutant retains antimicrobial activity against a selected vaginal pathogen from the group consisting of Gardnarella vaginalis, Streptococcus agalactiae, Candida albicans, Candida glabrata, and Ureaplasma urealyticum. Antimicrobial activity can be determined by any quantitative assay that allows the determination of the mutant's ability to kill cells of the pathogenic microorganism or inhibit their growth, for example, diffusion or agar dilution methods, as well as other methods described, for example, in Balouri et al., Journal of Pharmaceutical Analysis, 6 (2016), 71-79. In a specific embodiment, the mutant retains the antimicrobial activity of the L. salivarius strain.

[0048] CECT 5713 when this activity is determined by the superposition method described by Magnusson and Schnürer, Appl. Environ. Microbiol. 2001, 67, 1-5, which is used in the examples. In a particular embodiment, the mutant maintains 100% or more, or at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity of the L. salivarius CECT 5713 strain on the vaginal pathogen, preferably a pathogen selected from the group consisting of Gardnerella vaginalis, Streptococcus agalactiae, Candida albicans, Candida glabrata, and Ureaplasma urealyticum, when such activity is determined by the overlap assay mentioned herein.

[0049] In one particular embodiment, the L. salivarius CECT 5713 mutant retains the ability to co-aggregate with vaginal and / or cervical pathogens of the L. salivarius CECT 5713 strain. In a more specific embodiment, the mutant retains the ability to co-aggregate with a vaginal and / or cervical pathogen selected from the group consisting of G. vaginalis, S. agalactiae, and C. albicans. The ability to co-aggregate with a vaginal and / or cervical pathogen can be determined by any known co-aggregation assay, for example, the assay described by Younes et al. (PLoS One 2012, 7, e36917) used in the examples herein. In one particular embodiment, the mutant maintains the ability to co-aggregate with vaginal and / or cervical pathogens of the L salivarius CECT 5713 strain when such activity is determined by the co-aggregation assay described by Younes et al. (supra).In a particular embodiment, the mutant maintains 100% or more, or at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the co-aggregation capacity of the L. salivarius CECT 5713 strain on the vaginal and / or cervical pathogen, preferably a pathogen selected from the group consisting of G. vaginalis, S. agalactiae, and C. albicans, when such activity is determined by the aforementioned co-aggregation assay.

[0050] In one particular embodiment, the L. salivarius CECT 5713 mutant maintains the high adhesion to vaginal epithelial cells of the L. salivarius CECT 5713 strain. Adhesion to vaginal epithelial cells can be determined by any known adhesion assay, for example, the assay described by Boris et al., Infect. Immun. 1998, 66, 1985-1989, which is used in the examples herein using vaginal epithelial cells collected from premenopausal women. In one particular embodiment, the mutant maintains the high adhesion to epithelial cells of the L. salivarius CECT 5713 strain when this activity is determined by the co-aggregation assay described by Younes et al. (supra).In a particular embodiment, the mutant maintains 100% or more, or at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the adhesion of the L salivarius CECT 5713 strain to vaginal epithelial cells, when such activity is determined by the aforementioned adhesion assay. Since, as described in the examples in this document, the L salivarius strain CECT 5713 has a mean adhesion to vaginal epithelial cells obtained from premenopausal women of 329 ± 46 adherent lactobacilli in 20 random microscopic fields when adhesion is determined by the assay described by Younes et al.In one particular embodiment, the mutant of the L salivarius strain CECT 5713 exhibits similar adhesion, i.e., approximately 329 ± 46 adherent lactobacilli in 20 random microscopic fields, or higher when measured by the same assay.

[0051] In one particular embodiment, the mutant of the L. salivarius strain CECT 5713 maintains the high α-amylase activity of the L. salivarius strain CECT 5713. α-Amylase activity can be determined by any known assay, for example, by the method described by Narita et al. (Narita et al. Appl Environ Microbiol. 2006; 72(1):269-275) used in the examples, which is based on the determination of activity on the substrate 2-chloro-4-nitrophenyl 6 5 -azido-6 5- deoxy-β-maltopentaosideas. In one particular embodiment, the mutant maintains the high α-amylase activity of the L salivarius strain CECT 5713 when this activity is determined by the assay described by Narita et al. (supra). In a particular embodiment, the mutant maintains 100% or more, or at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the α-amylase activity of the L salivarius strain CECT 5713, when such activity is determined by the above-mentioned assay. Since, as described in the examples in this document, the L salivarius strain CECT 5713 has an α-amylase activity of 0.83 U / mL at 16 hours, when this activity is determined by the assay described by Narita et al.In one particular embodiment, the mutant of the L salivarius strain CECT 5713 exhibits similar α-amylase activity, i.e., approximately 0.83 U / ml at 16 hours, or higher when measured by the same assay.

[0052] In one particular embodiment, the L. salivarius CECT 5713 mutant maintains the high lactic acid production of the L. salivarius CECT 5713 strain. Specifically, the mutant maintains the high production of lactic acid, particularly L-lactic acid, of the L. salivarius CECT 5713 strain under aerobic and / or anaerobic conditions. Lactic acid production can be determined by any known assay, for example, by the method described by Martín et al. (Martín et al. 2006. Int. J. Food Microbiol. 112:35-43) used in the examples, which is based on the quantification of both lactic acid enantiomers using an enzymatic kit in the supernatant of a culture in MRS. In one particular embodiment, the mutant maintains the high lactic acid production of the L. salivarius CECT 5713 strain when this activity is determined by the assay described by Martin et al., 2006 (supra). In a particular embodiment, the mutant maintains 100% or more, or at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the lactic acid production of the L. salivarius CECT 5713 strain, when such production is determined by the above-mentioned assay. Since, as described in the examples in this document, the L. salivarius strain CECT 5713 is capable of producing 11.07 ± 0.42 mg / ml and 11.69 ± 0.58 mg / ml of L-lactic acid under aerobic and anaerobic conditions respectively when such production is determined by the assay described by Martin et al., 2006, in a particular embodiment the mutant of the L. strain.salivarius CECT 5713 is capable of producing a similar amount of L-lactic acid, namely approximately 11 mg / ml, under both aerobic and anaerobic conditions, or higher when measured by the same assay.

[0053] In one particular embodiment, the mutant of the L. salivarius strain CECT 5713 is an isogenic mutant, i.e., a strain that has a genome that differs from the L. salivarius strain CECT 5713 only by the presence of one mutation.

[0054] In one particular embodiment, the L. salivarius strain mutant CECT 5713 has a genome that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the genome of L. salivarius strain CECT 5713. In another particular embodiment, the L. salivarius strain mutant CECT 5713 is characterized by the presence of a 16S rRNA gene that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the 16S rRNA gene of L. salivarius strain CECT 5713. 5713.

[0055] In another particular embodiment, the degree of identity between a mutant and the parental strain is determined as the average nucleotide identity (ANI), which detects the conservation of core genome DNA (Konstantinidis K and Tiedje JM, 2005, Proc. Natl. Acad. Sci. USA 102: 2567-2592). In some realizations, the ANI between the variant and the original strain is approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 99.1%, approximately 99.5%, approximately 99.6%, approximately 99.7%, approximately 99.8%, approximately 99.9%, approximately 99.99%, approximately 99.999%, approximately 99.9999%, approximately 99.99999%, approximately 99.99999%, or more but less than 100%.

[0056] In another embodiment, the degree of relatedness between the mutant and the original strain is determined as the tetranucleotide signature frequency correlation coefficient, which is based on oligonucleotide frequencies (Bohlin J. et al. 2008, BMC Genomics, 9: 104). In some embodiments, the tetranucleotide signature frequency correlation coefficient between the mutant and the L. salivarius strain CECT 5713 is approximately 0.99, 0.999, 0.9999, 0.99999, 0.999999, 0.999999 or higher, but less than 1.

[0057] In another embodiment, the degree of relatedness between the mutant and the parental strain is determined as the degree of similarity obtained by analyzing the genomes of the parental and the variant strain by pulsed-field gel electrophoresis (PFGE) using one or more restriction endonucleases. The degree of similarity obtained by PFGE can be measured using the similarity coefficient of the dice. In some embodiments, the dice similarity coefficient between the mutant and the L salivarius strain CECT 5713 is approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 99.1%, approximately 99.5%, approximately 99.6%, approximately 99.7%, approximately 99.8%, approximately 99.9%, approximately 99.99%, approximately 99.999%, approximately 99.9999%, approximately 99.9999%, approximately 99.99999%, or more but less than 100%.

[0058] In another embodiment, a strain is considered a mutant of a given parental strain when both strains have the same ribotype, as obtained using any of the methods known in the art and described, for example, by Bouchet et al. (Clin. Microbiol. Rev., 2008, 21: 262-273).

[0059] In another embodiment, the degree of relatedness between the mutant and the original strain is the Pearson correlation coefficient obtained by comparing the genetic profiles of both strains obtained by repetitive PCR based on extragenic palindromic elements (REP-PCR) (see, for example, Chou and Wang, Int J Food Microbiol. 2006, 110: 135-48). In some embodiments, the Pearson correlation coefficient obtained by comparing the REP-PCR profiles of the mutant and the L. salivarius CECT 5713 strain is approximately 0.99, 0.999, 0.9999, 0.99999.

[0060] 0.999999, 0.999999 or more, but less than 1.

[0061] In another embodiment, the degree of relatedness between the mutant and the parental strain is the linkage distance obtained by comparing the genetic profiles of both strains obtained by multilocus sequence sequencing (MLST) (see, for example, Maiden, MC, 1998, Proc. Nati. Acad Sci. USA 95: 3140-3145). In some embodiments, the linkage distance obtained by MLST of the mutant and the L. salivarius strain CECT 5713 is approximately 0.99, 0.999, 0.9999, 0.99999,

[0062] 0.999999, 0.999999 or more but less than 1.

[0063] The term “treatment,” as used herein, refers to any type of therapy aimed at terminating, improving, or reducing susceptibility to a clinical condition, as described herein. Thus, “treatment,” “treat,” and their equivalent terms refer to achieving a desired effect pharmacologically or physiologically, encompassing any treatment of a pathological condition or disorder in a mammal, including humans. The effect may be prophylactic in terms of providing total or partial prevention of a disorder and / or adverse effect attributable to it.That is, "treatment" includes (1) inhibiting the disease, for example by stopping its development, (2) interrupting or ending the disorder or at least the symptoms associated with it, so that the patient would no longer suffer from the disease or its symptoms, for example by causing the regression of the disease or its symptoms by restoring or repairing a lost, absent, or defective function, or by stimulating an inefficient process, or (3) lessening, relieving, or improving the disease, or the symptoms associated with it, where lessening is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain, or immune deficiency.

[0064] The term "prevention," "prevent," or "avoid," as used herein, refers to the administration of the L. salivarius strain CECT 5713 or its mutant to a female subject who has not been diagnosed with infertility or recurrent pregnancy loss at the time of administration, but who would normally be expected to develop such a condition or be at increased risk of developing it. Prevention is intended to avoid the occurrence of such a condition. Prevention may be complete (e.g., the total absence of a condition). Prevention may also be partial, such that, for example, the occurrence of a condition in a subject is less than would have occurred without the administration of the combination or composition of the present invention. Prevention also refers to reduced susceptibility to a clinical condition. Prevention also includes reducing the risk of developing the condition.

[0065] The term “female subject”, as referred to in the present invention, applies to a female member of a mammalian animal species, and includes, but is not limited to, domestic animals, primates and humans; the subject is preferably a human being of any age or race.

[0066] For use in prevention and / or treatment according to the invention, the L. salivarius CECT 5713 strain or the mutant thereof is administered in a therapeutically effective amount.

[0067] The term "effective" or "therapeutically effective" amount of a drug or pharmacologically active agent is understood to mean a non-toxic but sufficient quantity of the drug or agent to provide the desired effect. In the therapy of the present invention, an "effective amount" of the L. salivarius strain CECT 5713, or a mutant thereof, is the amount of that microorganism that is effective in providing the desired effect.

[0068] Although individual needs vary, determining the optimum dosage ranges for effective quantities of the microorganism of the invention falls within the common expertise of those skilled in the art. In general, the dosage required to provide an effective quantity of said microorganism, which can be adjusted by a person skilled in the art, will vary depending on the patient's age, health, physical condition, sex, diet, weight, frequency of treatment, and the nature and degree of impairment or disease, medical condition, route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic profile, and toxicology of the particular compound used, whether a drug delivery system is used, and whether the compound is administered as part of a drug combination.

[0069] The term “infertility,” as used herein, refers to a woman’s inability to reproduce naturally. Specifically, it is a disease of the reproductive system defined by the inability to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse in the absence of another reason, such as breastfeeding or postpartum amenorrhea. Female infertility can be caused by a variety of factors, including:

[0070] - Menstrual cycle disorders, including anovulation, hyperprolactinemia, polycystic ovary syndrome, luteal insufficiency, premature ovarian insufficiency.

[0071] - Diseases of the fallopian tubes and uterus, including occlusion of the fallopian tubes, inflammation of the lesser pelvis (or upper genital infection), congenital malformations of the uterus and vagina.

[0072] - Changes in cervical mucus

[0073] - Endometriosis

[0074] - Infections, such as untreated gonorrhea and chlamydia, chronic infections of the cervix, and surgical treatment of cervical lesions associated with human papillomavirus (HPV) infection,

[0075] - Serious illnesses such as cancer.

[0076] - Excessive thinness or obesity.

[0077] - Endocrine disorders, such as thyroid disease, hypothalamic dysfunction, and hyperprolactinemia.

[0078] - Autoimmune disorders such as lupus, Hashimoto's disease and other types of thyroiditis and rheumatoid arthritis.

[0079] In one particular embodiment, infertility is unexplained. The term “unexplained infertility” or “idiopathic infertility,” as used herein, refers to infertility for which, after a battery of diagnostic tests performed on both the woman and the male partner, no cause is identified. In one particular embodiment, female infertility is considered unexplained when:

[0080] The physical examination shows no signs of abnormalities in the uterus, fallopian tubes, or ovaries. All ovarian reserve tests show normal levels of egg supply.

[0081] Hysterosalpingography (HSG) shows normal tubes in the uterine cavity and fallopian tubes.

[0082] The man's semen analysis shows adequate amounts of healthy sperm.

[0083] The woman's ovulation activity is normal, or any ovulation disorder (such as polycystic ovary syndrome) has been successfully treated. All blood tests for both sexes show normal results.

[0084] The term “recurrent pregnancy loss” or “recurrent miscarriage,” as used here, refers to a situation in which a woman achieves a clinical pregnancy but the pregnancy does not reach full term. Specifically, recurrent pregnancy loss is considered to exist in the following cases:

[0085] 3 consecutive pregnancy losses, including unvisualized pregnancies (European Society for Human Reproduction and Embryology, Royal College of Obstetricians and Gynaecologists)

[0086] 2 or more pregnancy losses, the pregnancy having been documented by ultrasound or histopathological examination, not necessarily consecutive.

[0087] Recurrent pregnancy loss can be due to different causes, including: Anatomical causes: malformations in the uterus, fibroids.

[0088] Lifestyle or environmental factors: smoking, drug use, excessive alcohol or caffeine consumption, obesity. Untreated illnesses such as thyroid disease or diabetes, and abnormalities of the immune system or blood clotting system.

[0089] In one particular embodiment, recurrent pregnancy loss is unexplained. The term “unexplained recurrent pregnancy loss,” as used herein, refers to recurrent pregnancy loss for which no cause is identified after a battery of diagnostic tests has been performed on both the woman and her male partner. In one particular embodiment, recurrent pregnancy loss is considered unexplained when the following tests are normal:

[0090] Uterine cavity tests, aimed at detecting abnormalities in the uterus. These may include transvaginal ultrasound, hysterosalpingography, saline infusion sonography, or magnetic resonance imaging. Genetic testing in both the woman and the man, including chromosomal analysis of aborted fetal tissue and the partner.

[0091] Hormonal tests, including thyroid studies, and analyses of prolactin, follicle-stimulating hormone, fasting glucose, and insulin.

[0092] Hematological and immunological tests.

[0093] It is possible to treat a female subject with infertility or recurrent pregnancy loss who is about to undergo assisted reproductive technology (ART) treatment with the L. salivarius strain CECT 5713, or a mutant thereof, thereby increasing the chances of success of the ART treatment. Therefore, in one particular embodiment, the L. salivarius strain CECT 5713, or a mutant thereof, is administered to a woman with infertility or recurrent pregnancy loss in combination with ART treatment.

[0094] The term “assisted reproductive technology” or “medically assisted reproductive technology,” as used here, refers to medical treatment intended to facilitate pregnancy in cases of infertility, whether male, female, or both, as well as in cases of single women or women with a female partner who wish to conceive using donor sperm. Assisted reproduction is characterized by the stimulation or induction of ovulation and may involve direct intervention on the gametes (oocytes and / or sperm) to facilitate fertilization and the transfer or placement of embryos into the uterine cavity.Examples of assisted reproduction treatments include ovulation induction, controlled ovarian stimulation, timed intercourse, intrauterine, intracervical and intravaginal artificial insemination, either with the partner's or a donor's sperm, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), sperm extraction, oocyte donation, and fertility preservation.

[0095] In one particular embodiment, the administration of the L. salivarius strain CECT 5713, or a mutant thereof, in combination with assisted reproductive treatment involves administering said strain to a female subject who has received, is receiving, or is to receive assisted reproductive treatment. In one particular embodiment, the L. salivarius strain CECT 5713, or a mutant thereof, is administered prior to treatment for a certain period of time, for example, for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 1 year prior to receiving the assisted reproductive treatment. In one particular embodiment, the L. salivarius strain CECT 5713, or a mutant thereof, is administered throughout the entire period of time that the female subject receives the assisted reproductive treatment.In one particular embodiment, the L. salivarius strain CECT 5713, or a mutant thereof, is administered after the subject has received assisted reproductive treatment, for example, for at least 1 week, at least 2 weeks, at least 3 weeks, and, if pregnancy is achieved, at least until week 4, at least until week 5, at least until week 6, at least until week 7, at least until week 8, at least until week 9, at least until week 10, at least until week 11, at least until week 12, at least until week 13, at least until week 14, at least until week 15 of pregnancy, or throughout the entire pregnancy. In a more particular embodiment, the L. salivarius strain CECT 5713, or a mutant thereof, is administered at least until week 15 of pregnancy.

[0096] The term “week of pregnancy” refers to the time elapsed since the date of the last menstrual period, assuming that the menstrual cycle lasts 28 days and that fertilization occurred on day 14 of that cycle.

[0097] The L salivarius strain CECT 5713, or a mutant thereof, can be administered vaginally or orally, preferably orally.

[0098] In one particular embodiment, the L. salivarius strain CECT 5713, or a mutant thereof, is administered in lyophilized form.

[0099] The amount of L. salivarius CECT 5713 strain, or a mutant thereof, to be administered will vary depending on the individual and the specific method of administration. Those skilled in the art will appreciate that dosages can also be determined with guidance from Goodman and Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711 and Goodman and Goldman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493. In one particular embodiment, L. salivarius CECT 5713 strain, or a mutant thereof, is administered at a daily dose between

[0100] 5.0 log 10 CFU (colony-forming units) and 13.0 log 10 UFC, for example, between 5.5 log 10 ufc and 12.5 log 10 ufc, between 6.0 log 10 ufc and 12.0 log 10 ufc, between 6.5 log 10 ufc and 11.5 log 10 ufc, between 7.0 log 10 ufc and 11.0 log 10ufc, between 7.5 log 10 ufc and 10.5 log 10 UFC, between 8.0 log UFC and 10.0 log 10 ufc, between 8.5 log 10 CFU and 9.5, approximately 9.0 log 10 CFU. Preferably, the L salivarius strain CECT 5713, or a mutant thereof, is administered at a daily dose between 8.5 log 10 ufc and 9.5 log 10 CFU, more preferably at a daily dose of approximately 9 log 10 UFC.

[0101] In one particular embodiment, the L. salivarius strain CECT 5713, or a mutant thereof, is administered for a period of time of at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, preferably at least 3 months, or until diagnosis of pregnancy, more preferably at least 6 months or until diagnosis of pregnancy.If pregnancy is diagnosed, preferably the L salivarius strain CECT 5713, or a mutant thereof, is administered at least until week 4, at least until week 5, at least until week 6, at least until week 7, at least until week 8, at least until week 9, at least until week 10, at least until week 11, at least until week 12, at least until week 13, at least until week 14, at least until week 15 of pregnancy, or throughout the entire pregnancy, more preferably, at least until week 15 of pregnancy.

[0102] Method for monitoring the invention

[0103] The authors of the present invention have detected an increase in vaginal concentrations of the growth factors VEGF, TGF-β1 and TGF-β2 induced by treatment with the L salivarius strain CECT 5713 in women who eventually achieved pregnancy compared to those who did not (Table 8), so that the concentrations of these growth factors can be a biomarker of the effectiveness of the treatment.

[0104] Therefore, in another aspect, the invention relates to an in vitro method for monitoring the effect of a treatment for infertility or recurrent pregnancy loss in a female subject with a strain of the species Lactobacillus salivarius deposited in the CECT with accession number 5713 or a mutant thereof comprising:

[0105] (i) determine the level in a sample of vaginal mucosa and / or cervical mucosa of said female subject of a growth factor selected from the group consisting of VEGF, TGF-β1 and TGF-β2 and

[0106] (ii) compare that level with a reference value obtained from the same subject before therapy, where an increase in that level relative to the reference value is indicative that the treatment is being effective and a decrease or lack of change in that level relative to the reference value is indicative that the treatment is being ineffective.

[0107] The term “in vitro”, as used here, refers to the fact that the method is not carried out on the body of a subject, human or animal, but on an isolated sample taken from that subject.

[0108] The term "monitoring the effect of a treatment" refers to the response of a patient suffering from a disease to the treatment for that disease.

[0109] The terms “infertility”, “recurrent pregnancy loss”, “female subject”, “strain of the species Lactobacillus salivarius deposited in the CECT with accession number 5713”, and “mutant thereof” have been previously defined in relation to the first inventive aspect. All particular and preferred embodiments of the first aspect in relation to these terms apply equally to the method for monitoring the treatment of the invention.

[0110] The monitoring method of the invention comprises determining the level in a sample of vaginal mucosa and / or cervical mucosa of a growth factor selected from the group consisting of VEGF, TGF-β1 and TGF-β2.

[0111] The term “vaginal and / or cervical mucosal sample,” as used herein, refers to a sample of the surface mucosa of the vagina and cervix, including small molecules, extracellular polymers, proteins and peptides, microbial cells, and host cells—in this case, from the female subject. Such a sample can be obtained by any known technique, for example, by swabbing or by washing with a fluid, such as saline solution, which is known as a “cervicovaginal” or “CVL” wash. In one particular embodiment, the vaginal and / or cervical mucosal sample is a cervicovaginal wash.

[0112] The term “VEGF, TGF-β1, or TGF-β2 level,” as used herein, refers to the amount of the corresponding proteins present in the sample. The level of a protein can be determined by any known method or technique suitable for the determination and quantification of a protein in a sample. By way of non-limiting illustration, the level of a protein can be determined by a technique involving the use of antibodies capable of specifically binding to the protein being analyzed (or to fragments thereof containing the antigenic determinants) and subsequent quantification of the resulting antigen-antibody complexes, or alternatively by a technique that does not involve the use of antibodies, such as, for example, mass spectrometry-based techniques.Antibodies can be monoclonal, polyclonal, or fragments thereof, including Fv, Fab, Fab' and F(ab')2, scFv, diabodies, tribodies, tetrabodies, and humanized antibodies. Similarly, antibodies can be labeled. Illustrative, but not limited to, examples of markers that can be used include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme cofactors or substrates, enzyme inhibitors, particles, or dyes.There is a wide variety of known tests that can be used according to the present invention, such as the combined application of unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies), Western blot or immunoblotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), two-dimensional gel electrophoresis, capillary electrophoresis, immunocytochemical and immunohistochemical techniques, immunoturbidimetry, immunofluorescence, techniques based on the use of protein biochips or microarrays that include specific antibodies, or assays based on colloidal precipitation in formats such as reagent strips and assays based on antibody-bound quantum dots. Other ways of detecting and quantifying proteins include, for example, affinity chromatography techniques or ligand-binding assays.

[0113] In one particular embodiment, the VEGF level is determined by a multiplex immunoassay. In another particular embodiment, the TGF-β1 or TGF-β2 level is determined by ELISA.

[0114] The term "VEGF" or "VEGF-A" or "Vascular Endothelial Growth Factor" refers to a member of the PDGF / VEGF growth factor family. It is a heparin-binding protein that exists as a disulfide-linked homodimer. This growth factor induces the proliferation and migration of vascular endothelial cells and is essential for both physiological and pathological angiogenesis. In humans, VEGF corresponds to the protein defined by accession number P15692 in the UniProtKB / Swiss-Prot database (version 248 of the entry, April 22, 2020; version 2 of the sequence, November 16, 2001).

[0115] The term TGF-β1, or transforming growth factor beta 1, refers to a protein belonging to the transforming growth factor beta superfamily of cytokines. It is a secretory protein involved in functions such as cell growth control, cell proliferation, differentiation processes, and apoptosis. In humans, TGF-β1 corresponds to the protein defined by accession number P01137 in the UniProtKB / Swiss-Prot database (entry version 253, April 22, 2020; sequence version 2, February 1, 1991).

[0116] The term “TGF-β2” or “transforming growth factor beta 2” refers to a protein belonging to the transforming growth factor beta superfamily of cytokines. It is a protein involved in angiogenesis and heart development. In humans, TGF-β2 corresponds to the protein defined by accession number P61812 in the UniProtKB / Swiss-Prot database (entry version 181, April 22, 2020; sequence version 1, August 1, 1988).

[0117] In one embodiment, the monitoring method of the invention comprises determining the level of VEGF in a sample of vaginal mucosa and / or cervical mucosa. In one embodiment, the monitoring method of the invention comprises determining the level of TGF-β1 in a sample of vaginal mucosa and / or cervical mucosa. In one embodiment, the monitoring method of the invention comprises determining the level of TGF-β2 in a sample of vaginal mucosa and / or cervical mucosa.

[0118] In one embodiment, the monitoring method of the invention comprises determining the level of VEGF and TGF-β1 in a sample of vaginal mucosa and / or cervical mucosa. In one embodiment, the monitoring method of the invention comprises determining the level of VEGF and TGF-β2 in a sample of vaginal mucosa and / or cervical mucosa. In one embodiment, the monitoring method of the invention comprises determining the level of TGF-β1 and TGF-β2 in a sample of vaginal mucosa and / or cervical mucosa.

[0119] In one embodiment, the monitoring method of the invention comprises determining the level in a sample of vaginal mucosa and / or cervical mucosa of VEGF, TGF-β1 and TGF-β2.

[0120] The monitoring method of the invention comprises comparing the level of VEGF, TGF-β1 and / or TGF-β2 with a reference value.

[0121] The level of VEGF, TGF-β1 and / or TGF-β2 is determined in a sample of the vaginal mucosa and / or cervical mucosa of the female subject taken after receiving treatment, specifically, after receiving administration of the L. salivarius strain CECT 5713, or a mutant thereof, for a period of at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, preferably at least 3 months, or more preferably, at least 6 months.

[0122] The term “reference value,” as used herein, refers to predetermined criteria used as a benchmark for evaluating values ​​or data obtained from samples collected from a subject. The reference value may be an absolute value, a relative value, a value with an upper or lower limit, a range of values, a mean value, a median value, a mean value, or a value compared to a particular control or baseline value. A reference value may be based on a value from an individual sample, such as, for example, a value obtained from the sample of the subject being tested, but at an earlier point in time. In one particular embodiment, the reference value is the level in a sample of vaginal and / or cervical mucosa of said growth factors before the female subject begins treatment with the L. strain.salivarius CECT 5713, or the mutant thereof, for example, 4 weeks before, 3 weeks before, 2 weeks before, 1 week before, 6 days before, 5 days before, 4 days before, 3 days before, 2 days before, 1 day before, or the day that said treatment begins.

[0123] According to the monitoring method of the invention, an increase in the level of any of VEGF, TGF-β1 and TGF-β2 with respect to their reference value is indicative that the treatment is being effective, while a decrease or lack of change in said level with respect to the reference value is indicative that the treatment is being ineffective.

[0124] The term “level increase relative to reference value” indicates that the level of the growth factor is higher than its reference value. The growth factor level is considered to be greater than its reference value when it is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200% or more greater than the reference value.

[0125] The term “decrease or lack of change in level with respect to the reference value” indicates that the level of the growth factor is less than or equal to its reference value. The growth factor level is considered to be less than or equal to its reference value when it is less than 1.5%, less than 1.0%, less than 0.5%, or equal to the reference value when it is at least 1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% less than the reference value.

[0126] In one particular instance, infertility is unexplained infertility.

[0127] In one particular embodiment, recurrent pregnancy loss is unexplained recurrent pregnancy loss.

[0128] In one particular embodiment, treatment with the L salivarius strain CECT 5713, or its mutant, is administered in combination with assisted reproductive treatment.

[0129] In one particular embodiment, the strain is administered orally.

[0130] In one particular embodiment, the strain is administered for a period of time of at least 3 months, at least 6 months, or until a pregnancy diagnosis is made.

[0131] In one particular embodiment, if pregnancy is diagnosed, administration of the strain is maintained at least until week 15 of pregnancy.

[0132] In one particular embodiment, the strain is administered at a daily dose comprising 8.5 log 10 ufc and 9.5 log 10CFU. In a more particular embodiment, the strain is administered at a daily dose of approximately 9 log 10 UFC.

[0133] The invention is described below by means of the following examples which are merely illustrative and do not limit the scope of the invention.

[0134] EXAMPLES

[0135] Materials and methods

[0136] Participants, sample, and study design

[0137] A total of 58 women, aged 28 to 45, participated in this study. The volunteers were divided into three groups. All volunteers in the AR group (n = 21) had a history of recurrent miscarriage with three or more pregnancy losses during the first 12 weeks of pregnancy. All women in the INF group (n = 23) had a history of infertility (inability to conceive) despite receiving ART at least three times, including at least one cycle of in vitro fertilization (IVF). Finally, the control group (n = 14) included women who had at least two children after uncomplicated full-term pregnancies. None of the women in the AR and INF groups received ART during the entire study period. None of the women in the AR group were diagnosed with antiphospholipid syndrome and, therefore, did not receive heparin and / or salicylic acid during the study.None of the participating women had received or consumed hormonal therapy, antibiotics, or probiotics in the 4 weeks prior to sampling. Vaginal samples were taken at least 7 days after intercourse to avoid or minimize the impact of the partner's semen on vaginal pH, microbiome, and immune profile (in the latter case, particularly regarding TGF-β1 and TGF-β2 concentrations). Women with lactose intolerance or cow's milk protein allergy were excluded due to the excipient used to administer the strain in the subsequent pilot trial (see below).

[0138] At recruitment (day 0), two samples were collected from women in all three groups within the first three days after ovulation: a vaginal swab for a Nugent score recalculation and a cervicovaginal wash (CVW) of the cervical os and vaginal walls with 10 mL of sterile normal saline (Nakra et al., J Infect Dis. 2016, 213(5):840-7) for all other analyses. Aliquots of the CVW samples were used for culture-based analysis. Subsequently, the CVW samples were clarified by low-speed centrifugation at 800 x g for 10 minutes at 4°C. Aliquots of CVW supernatant and cell pellets were stored at -80°C until immunological and metataxonomic analyses were performed.

[0139] After day 0, women in the RA and INF groups consumed (orally) one sachet daily containing ~50 mg of lyophilized probiotic (~9 µg 10⁶ CFU of L. salivarius CECT 5713) for 6 months or until pregnancy was confirmed (whichever came first). At that point, the same two samples were collected from each woman. After pregnancy was confirmed, oral administration of the strain was continued until week 15 of pregnancy. All spontaneous pregnancies occurring within the first year after day 0 were recorded in this study.

[0140] The sachets containing the probiotic were kept at 4°C throughout the study. All volunteers received questionnaires to record their adherence to the study product intake. The minimum adherence rate (% of total treatment doses) was set at 86%. All volunteers provided written informed consent to the protocol, which had been approved by the Biomedical Research Ethics Committee of the Ministry of Health and Families (Regional Government of Andalusia, Granada, Spain) (PO50 / 19, Law 11 / 19).

[0141] Vaginal pH measurement and Nugent score

[0142] During each of the two study visits, the pH of the lateral vaginal wall was measured (Whatman pH paper, pH 3.8–5.5 and pH 6.0–8.1). The Nugent score was performed as previously described (Nugent et al., J Clin Microbiol. 1991, 29(2):297–301). Briefly, the swab material was transferred to a glass slide, heat-fixed, and Gram-stained. Gram-positive, Gram-negative, and Gram-variable bacterial morphotypes were quantified. A Nugent score of 0–3 was considered normal, 4–6 was considered intermediate, and 7–10 was considered consistent with bacterial vaginosis (Nugent et al., supra).

[0143] Culture-dependent analysis

[0144] The vaginal vulva (VV) samples collected during the assay were serially diluted and plated onto Columbia nalidixic acid (CNA), Gardnerella (GAR), CHROMagar StrepB (CHR), MacConkey (MCK), Mycoplasma (MYC), and Sabouraud Dextrose Chloramphenicol (SDC) agar plates (BioMerieux, Marcy l'Etolle, France) for the selective isolation and quantification of the main non-Lactobacillus bacteria and culturable yeasts that can be found in the vagina, including the agents most frequently involved in vaginal infections. They were also spread onto MRS agar plates (Oxoid, Basingstoke, UK) supplemented with L-cysteine ​​(2.5 g / L) (MRS-C) or horse blood (5%) (MRS-B) for the isolation of lactobacilli, including L. iners (MRS-B).All plates were incubated for 48 hours at 37°C under aerobic conditions, with the exception of MRS-C and MRS-B, which were incubated anaerobically (85% nitrogen, 10% hydrogen, 5% carbon dioxide) in an anaerobic workstation (DW Scientific, Shipley, UK). After incubation, counts were recorded on each growth medium, and subsequently, at least one representative of each colony morphology was selected from the agar plates. Isolates were identified by matrix-assisted desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker, Germany). When MALDI-TOF identification was not possible at the species level (particularly in the case of lactobacilli isolates), identification was carried out by sequencing the 16S ribosomal RNA (rRNA) gene as described by Mediano et al. (J Hum Lact.2017, 33(2): 309-318) DNA extraction from samples.

[0145] Approximately 1 mL of each CVL sample was used for DNA extraction following a previously described method (Lackey et al., Front Nutr. 2019, 17; 6:45). The extracted DNA was eluted in 22 μL of nuclease-free water and stored at -20 °C until further analysis. The purity and concentration of each extracted DNA sample were estimated using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Inc., Rockland, USA). Negative (no sample) controls were added during the extraction.

[0146] Detection and quantification of Lactobacillus salivarius DNA by qPCR (quantitative real-time PCR)

[0147] The primers and conditions for quantifying DNA specifically belonging to the species L. salivarius have been previously described (Harrow et al., Appl Environ Microbiol. 2007, 73(22): 7123-7). The DNA concentration of all samples was adjusted to 5 ng / μL -1A commercial real-time PCR thermocycler (CFX96™, Biorad Laboratories, Hercules, CA, USA) was used for all experiments. Standard curves were generated using 1:10 dilutions of DNA (ranging from 2 ng to 0.2 pg) from L. salivarius CECT 5713 to estimate unknown bacterial genomic targets. Threshold cycle (Ct) values ​​between 15.29 and 20.07 were obtained for this range of L. salivarius DNA (R² = 0.9915). Measured Ct values ​​for DNA extracted from non-target species (L. reuteri MP07 and L. plantarum MP02; own collection) were ≥39.27 ± 0.64. These two control strains were selected because they belong to the species taxonomically closest to L. salivarius (Salvetti et al., 2018). All samples and standards were processed in triplicate.

[0148] Metataxonomic analysis

[0149] The hypervariable regions V3-V4 of the 16S rDNA were amplified by PCR using the universal primers SD-Bact-0341-bS-17

[0150] (CCTACGGGNGGCWGCAG, SEQ ID NO: 1) and SD-Bact-129 0785-aA-21 (GACTACHVGGGTATCTAATCC, SEQ ID NO: 2) were sequenced on the Illumina MiSeq system at the Madrid Science Park facilities (Tres Cantos, Spain) (Klindworth et al., Nucleic Acids Res. 2013, 41, e1). Barcodes attached to the 3' and 5' terminal ends of the PCR amplicons in a second PCR reaction allowed for the separation of the forward and reverse sequences. The DNA concentration in the PCR products was quantified using the 2100 Bioanalyzer system (Agilent, Santa Clara, CA, USA). After pooling the PCR products to approximately equal molar ratios, the DNA amplicons were purified using a QIAEX II gel extraction kit (Qiagen) from the excised band that was of the correct size after running on the agarose gel. The DNA concentration was then quantified using PicoGreen (BMG Labtech, Jena, Germany).The pooled, purified, and barcoded DNA amplicons were sequenced using the Illumina MiSeq end-of-pair protocol (Illumina Inc., San Diego, CA, USA) following the manufacturer's protocols. Analysis of immunological parameters.

[0151] Vascular endothelial growth factor (VEGF) was analyzed using a premixed multiplex panel (Bio-Rad, San Diego, CA). Concurrently, TGF-β1 and TGF-β2 levels were measured by ELISA using the RayBio® Human TGF-β1 and Human TGF-β2 ELISA kits, respectively (RayBlotech, Norcross, GA, USA). All determinations were performed according to the manufacturer's protocol, and standard curves were generated for each analyte.

[0152] Antimicrobial activity of L salivarius CECT5713 against vaginal pathogens

[0153] A superposition method (Magnusson and Schnürer, Appl. Environ. Microbiol. 2001, 67, 1-5) was used to determine the ability of L. salivarius CECT5713 to inhibit the growth of various bacterial and yeast species. This was performed as described by Martín et al. (2006). All strains used as indicator organisms (from our own culture collection) had been previously isolated from clinical cases of vaginal or cervical infections and included anthoses of Gardnerella vaginalis, three strains of Streptococcus agalactiae, three strains of Candida albicans, two strains of Candida glabrata, two strains of Candida parapsilosis, and two strains of Ureaplasma urealyticum. All experiments testing inhibitory activity were performed in triplicate. Production of antimicrobial compounds by L salivarius CECT5713 The production of hydrogen peroxide by the Lactobacillus strain was measured using the quantitative method of Yap and Gilliland, 2000, J.Dairy Sei.83, 628-632, modified by Martín et al. (Int. J. Food Microbiol. 2006, 112: 35-43). The production of lactic acid (L and D isomers) and acetic acid by L. salivarius CECT5713 was determined using enzyme kits (Roche Diagnostics, Mannheim, Germany), as described by Martín et al. (2006). The pH values ​​of the culture supernatants were also measured. These assays were performed in triplicate under strict aerobic and anaerobic conditions, and the values ​​are expressed as mean ± SD. Bacteriocin activity was not analyzed since previous phenotypic analyses and genome analysis of L. salivarius CECT5713 had demonstrated the inability of this strain to produce such antimicrobial compounds (Martín et al., supra; Langa et al., Appl Microbiol Biotechnol. 2012, 94(5): 1279-87). Coaggregation and adhesion assays to vaginal epithelial cell lines.

[0154] The strain's ability to aggregate with cells from the previously mentioned indicator strains was investigated following the procedure of Younes et al. (PLoS One 2012, 7, e36917). Suspensions were observed under a phase-contrast microscope after Gram staining. Adherence to vaginal epithelial cells collected from healthy premenopausal women was performed and interpreted as previously described (Boris et al., Infecí Immun. 1998, 66, 1985-1989). Adherence was measured as the number of lactobacilli adhering to vaginal cells in 20 random microscopic fields. L. salivarius CECT 9145 was used as a control strain due to its high adherence to vaginal cells (Martín et al., Nutriente. 2019, 11(4). pii: E810). The assay was performed in triplicate.

[0155] α-amylase activity of L. salivarius CECT5713

[0156] First, the α-amylase activity of L. salivarius CECT5713 was qualitatively assessed using the method described by Padmavathi et al. (J Genet Eng Biotechnol. 2018; 16(2): 357-362). Briefly, the strain was inoculated onto a modified MRS medium (0.5% peptone, 0.7% yeast extract, 0.2% NaCl, 2% starch, and 1.5% agar) supplemented with 0.25% starch. The plates were incubated at 37°C for 48 h under anaerobic conditions, and the clear zone was then observed by adding Gram-negative iodine as a detection agent. Subsequently, α-amylase activity was also measured on the surface of L salivarius CECT5713 using an α-amylase measurement kit (Kikkoman Co., Tokyo, Japan), using 2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentaosideas substrate using a protocol and conditions described above (Narita et al., 2006).One unit of activity was defined as the amount of enzyme needed to release 1 μmol of 2-chloro-4-nitrophenol from 2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentaoside per minute at 37 °C.

[0157] Bioinformatics analysis

[0158] Raw, unprocessed data were demultiplexed and filtered for quality using Illumina MiSeq Reporter analysis software. Microbiome bioinformatics was performed using QIIME 2 2019.1 (Bolyen et al., Natura Biotechnology, 2019, 37: 852-857). Disinfection was performed using DADA2 (Callahan et al., Nat Methods. 2016; 13:581-583). Taxonomy was assigned to ASVs using the q2 feature classifier (Bokulich et al. Microbiome. 2018, a; 6:90), which classifies the naive Bayes skleam taxonomy classifier against the SILVA 132 OTU reference sequences (Quast, Nuci. Acids Res. 2013, 41 (D1): D590-D596). Subsequent bioinformatics analysis was performed using R version 3.5.1 (R Core Team, 2013; https: / / www.R-project.org). A table of OTU counts per sample was generated, and bacterial taxon abundances were normalized to the total number of sequences in each sample.Alpha diversity was studied using the Shannon and Simpson diversity indices with the R Vegan package (Version: 2.5.6) (Oksanen et al., Community Ecology package, 2007, 10; 631-637). Beta diversity studies were performed using Principal Coordinate Analysis (PCoA) to map bacterial community diversity patterns using a distance matrix containing a dissimilarity value for each pairwise sample comparison. Quantitative (relative abundance) and qualitative (presence / absence) analyses were performed using the Bray-Curtis index and the Jaccard binary index, respectively. Analysis of variance of the distance matrices was performed using the Adonis non-parametric manometry test with 999 permutations to reveal statistically significant results with the R Vegan package.

[0159] Statistical analysis

[0160] Microbiological data were recorded as CFU / mL and transformed to logarithmic values ​​before statistical analysis. Quantitative data were expressed as the median and interquartile range (IQR). Differences between groups were assessed using the Kruskal-Wallis test and pairwise Wilcoxon rank-sum test to calculate comparisons between groups. Bonferroni corrections were performed. The correlation between the 20 relatively abundant genera was visualized using the R package "qgraph" (Epskamp 2012). Statistical analysis and plotting were performed in the R environment using the ggplot2 library (Wickham, 2016). Differences were considered statistically significant with a p-value <0.05. The contribution of a variable is (in percentage): (variable cos² * 100) / (total cos² of the component). Cos² = representation quality or weight of the variable in the PCA representation.

[0161] Results

[0162] Demographic data and clinical characteristics of the participants

[0163] The characteristics of the 58 women who participated in this study are presented in Table 1. The mean age (95% CI) of the women in the control group was 34.6 years (33.4–35.8 years), while those of women with recurrent miscarriages (RM) and women with unexplained infertility (UE) were 39.4 (38.5–40.4) years and 38.0 (37.1–38.9) years, respectively (Table 1). The women in the control group were approximately 4–5 years younger than the other participants (p < 0.001; one-way ANOVA), but there were no differences in mean body weight and height among the three groups of women (Table 1).

[0164] Table 1. Characteristics of the participants (n = 58), who were distributed into three groups: control group (women who had at least two children after uncomplicated full-term pregnancies), RA group (women with a history of recurrent miscarriage) and INF group (women with a history of infertility).

[0165] SD: standard deviation; Cl: confidence interval.

[0166] One-way ANOVA tests were used to assess differences in mean age, weight, height, and mean menstrual cycle length between groups. 'Freeman-Halton extension of Fisher's exact probability tests for a 2 x 3 contingency table.

[0167] Approximately 71% of women in the control group had a regular menstrual cycle, while in the other two groups (RA and INF) this percentage was 48%, although this difference was not statistically significant (p = 0.337; Freeman-Halton extension of Fisher's exact test for a 2 x 3 contingency table). No differences were observed in the mean menstrual cycle length, which was 28, 27.4, and 27.5 days for women in the control, AR, and INF groups, respectively (Table 1). Baseline vaginal health parameters

[0168] The vaginal pH in women in the control group had a mean value (95% CI) of 4.53 (4.38–4.68) (Table 2). In contrast, vaginal pH was higher in the two study groups: 5.67 (5.55–5.79) in the RA group and 5.96 (5.84–6.07) in the INF group (p = 0.000; one-way ANOVA). Significantly higher mean Nugent scores (95% CI) of 5.95 (5.54–6.37) and 6.30 (5.91–6.70) were recorded in women in the RA and INF groups, respectively, than in controls (p = 0.000; one-way ANOVA) (Table 2). There were also differences in the levels of the cytokines TGFβ1, TGFβ2, and VEGF in CVL. The CVL content of these three cytokines in women in the control group was 4.83 pg / mL (4.65-5.01) pg / mL, 3.22 pg / mL (4.65-5.01) pg / mL and 406.0 pg / mL (322.0-490.0) pg / mL for TGFβ1, TGFβ2 and VEGF, respectively, while they were reduced by approximately half in both study groups (RA and INF) (Table

[0169] 2).

[0170] Table 2. Comparison of baseline vaginal parameters (pH, Nugent score, cytokines and microbiology) of participants (n = 58) from the control group (women who had at least two children after uncomplicated term pregnancies), RA (women with a history of recurrent miscarriage) and INF (women with a history of infertility).

[0171] SD: standard deviation; Cl: confidence interval; TGFβ1, transforming growth factor β1; TGFβ2, transforming growth factor β2; VEGF, vascular endothelial growth factor.

[0172] One-way ANOVA tests were used to assess differences in means between groups. Different bold letters in the same row indicate statistically significant differences between groups.

[0173] 'Freeman-Halton extension of Fisher's exact probability tests for a 2 x 3 contingency table.

[0174] “Average in those women in whom lactobacilli could be isolated from their samples.

[0175] Lactobacilli were detected in the uterine lining (UL) of all women in the control group (n = 14), and the mean (95% CI) lactobacillus count was 7.24 log CFU / mL (6.89–7.60) log10 CFU / mL using a culture-dependent assessment. The frequency of lactobacillus detection in UL was lower in the RA and INF groups: 57% and 26%, respectively (p < 0.001, Freeman-Halton extension of Fisher's exact test for a 2 × 3 contingency table). Furthermore, lactobacillus concentrations were also 2.20 and 1.46 log10 units lower in UL samples from lactobacillus-positive women in the AR and INF groups, respectively. Another difference between these groups was related to the lactobacillus profile (Figure 1). Up to six different species of lactobacilli were identified in CVL samples from women in the control group, including L. crispatus (the dominant species), L. jensenii, L. gasseri, L. fermentum, L. salivarius, and L. vaginalis.The lactobacillus species profiles in the RA and INF study groups were narrower than in the controls, and L. fermentum, L. salivarius, and L. vaginalis were not found in any samples from the RA and INF groups. It is significant to note that L. iners was not isolated from any vaginal swab samples from the control group, but it was isolated from approximately one-third (5 out of a total of 18 lactobacillus-positive samples) of the RA and INF group samples. L. salivarius levels in vaginal swabs were also determined by species-specific qPCR. Using this technique, this bacterial species was detected in only one sample from a woman in the control group (7.29 µg10 couples / swab). This woman was the only one identified as L. salivarius positive (at a concentration of 7.3 µg10 CFU / swab) using culture techniques. The L. salivarius strain isolated from a woman in this group was genetically different from L. salivarius CECT 5713 (results not shown).

[0176] Sequencing analysis of the 16S rRNA gene from CVL samples (n = 58) yielded 4,363,364 high-quality filtered sequences, ranging from 33,160 to 139,044 per sample (median [IQR] = 73,383 [66,587–82,821]). Sequences were assigned to a total of 23 phyla and 453 genera. Analysis of alpha diversity, calculated using either Shannon or Simpson indices, revealed significant differences between the vaginal microbiota of women in the fertile control group and women with unexplained infertility (p < 0.001) (Figure 2). Beta diversity analysis, calculated according to the relative abundance of OTU (Bray-Curtis distance) and the presence / absence of OTU (Binary Jaccard distance matrix), indicated that the vaginal bacteria of the 3 groups clustered (p = 0.004 and p = 0.002, respectively) (Figure 3).Furthermore, the fertile control samples clustered closer together (shorter distance to the centroid) according to the relative abundance of OTU (Bray-Curtis distance) than those of the RA and INF groups, indicating that the bacterial profile in the CVL samples of the controls was very uniform (Figure 3).

[0177] Principal component analysis (PCA) was performed for an initial assessment of potentially dominant patterns in the bacteriological profile of vaginal samples (Figure 4). The first two PCA dimensions explained 84.9% of the total variance present in the samples, with the first dimension (principal component) accounting for 74.7% and the second dimension for 10.2% of the total variance. Overall, this analysis revealed greater similarity between samples from the fertile control group and women with a history of recurrent miscarriage than between the fertile control group and women with unexplained infertility.

[0178] The comparison of the relative abundance (% of total) of OTU at the phylum level in CVL samples from the three groups revealed statistically significant differences with respect to the 4 dominant phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes (Table 3). The most frequent (present in all samples) and abundant phylum was Firmicutes. The relative abundance of Firmicutes in samples provided by fertile controls [median (IQR) = 95.01% (99.18% - 99.8%)] was higher than in samples from women in the AR and INF groups [median (IQR) = 97.29% (72.34% - 99.35%) and 89.96% (52.46% - 98.85%), respectively] (p < 0.050 and p < 0.001; Wilcoxon tests) (Table 3). In contrast, the median (IQR) values ​​of the relative abundance of Actinobacteria, Proteobacteria, and Bacteroidetes were higher in women in the RA and INF groups than in fertile controls (p <0.012, p <0.003, and p <0.006, respectively; Kruskal-Wallis rank tests) (Table 3).

[0179] At the genus level, the only bacterial genus detected in all samples was Lactobacillus, but there were significant differences in its relative abundance in the samples from the three groups (Table 3). The median (IQR) relative abundance of Lactobacillus in samples from women in groups RA and INF [93.49% (67.18%-97.53%) and 71.95% (0.76%-94.09%), respectively] was lower than in samples from fertile control women [97.88% (96.92%-99.31%)] (p = 0.001; Kruskal-Wallis rank test) (Table 3). Other genera were present in a variable number of samples, ranging from 96% (Staphylococcus in the INF group) to 7% (Escherichia / Shigella in the control group), but the median relative abundance of any of these genera was less than <1%.The bacterial profile at the gender level in some individual samples from women in the AR and INF groups did not differ from that of the samples from women in the fertile control group, which were highly homogeneous. However, aberrant profiles with reduced content or even total absence of Lactobacillus were recorded in some samples from women in the AR and INF groups (data not shown).

[0180] Overall, the comparison of the RA and INF groups at the start of the study revealed some statistically significant differences between the two groups (Figure 6). Vaginal pH was higher in women in the INF group than in those in the RA group, but the opposite was observed for TGFβ1 and VEGF. No differences were observed with respect to other characteristics, such as age, weight, height, Nugent score, TGFβ2, and viable lactobacilli counts (Figure 5).

[0181] Main parameter of the clinical study: pregnancy success rate vs. full-term pregnancies (Reproductive outcomes / Pregnancy)

[0182] Overall, administration of probiotics of the strain to women in the RA and INF groups resulted in 29 pregnancies out of the 44 participating women. This represents a pregnancy success rate of 66% with a 95% CI of 51–78% (p = 0.017) (Table 4). Among these, there were 25 successful pregnancies and 4 miscarriages out of the 44 participating women. This represents a reproductive success rate of 56% with a 95% CI of 42–70% (p = 0.183) (Table 4). Interestingly, all successful pregnancies resulted in singleton pregnancies at term (gestational age ≥38 weeks). Gestational ages, birth weight and length, and sex of the infants are shown in Table 1.

[0183] Table 4. Main results after probiotic treatment. # Pearson Chi-square proportion.

[0184] *2 women in each group had an abortion.

[0185] Women in the AR group had the highest reproductive success rate, with 17 pregnancies (15 to term and 2 miscarriages) out of 21 participants during the study period. The rate in the INF group was lower, though still significant: 12 pregnancies (10 to term and 2 miscarriages) out of 23 participants. Comparison of pregnancy effectiveness and successful pregnancy rates (95% CI) between women in AR and INF [1.55 (0.70–3.56) and 1.64 (0.69–4.09, respectively] revealed no statistically significant differences between the two groups (Table 4). However, it should be noted that all women in these groups had previously undergone unsuccessful ART interventions in attempts to prevent miscarriage (AR group) or conceive (INF group).

[0186] Secondary response parameters associated with probiotic treatment: changes in vaginal parameters in women in the AR group

[0187] There were no differences in age, weight, or height between those women in the AR group who ended up having a successful pregnancy (n = 15) and those who did not

[0188] (n = 6).

[0189] However, differential changes were observed in this group of women (AR group) after probiotic treatment depending on the final pregnancy outcomes (Table 5). In those women who were able to complete a successful pregnancy, the mean (SD) vaginal pH value was 1.13 (0.38) units lower if they became pregnant compared to those who did not. Similar changes were observed for the Nugent score, which showed a difference of 3.33 (1.11) units between successful and unsuccessful pregnancies (p = 0.000, one-way repeated measures ANOVA) (Table 5, Figure 6).

[0190] Table 5. Differences in characteristics and comparison of changes in vaginal parameters between women who were able to complete a pregnancy to term (n = 15) and those who were not (n = 6) after intervention with L. salivarius CECT 5713 in the RA group, which included women with a history of recurrent miscarriage (n = 21). # One-way ANOVA tests were used to assess differences in means between groups, except for the presence of lactobacilli.* Fisher's exact test for a 2 x 2 contingency table. † One-way ANOVA tests were used to determine whether there were changes in each participant group.

[0191] Changes in vaginal cytokine concentrations also differed between the two groups after probiotic treatment. There were no changes in vaginal concentrations of TGFβ1, TGFβ2, and VEGF in women who did not become pregnant, but there was a significant (SD) mean increase of 1.40 (0.62) pg / mL, 1.25 (0.34) pg / mL, and 402 (252) pg / mL, respectively, in those who did (Table 5, Figure 7). Furthermore, it should be noted that differences in the concentration of these cytokines already existed between those who became pregnant and those who did not, even before treatment began (Table 5, Figure 7).

[0192] On the other hand, probiotic treatment resulted in a mean (SD) increase in lactobacillus counts of 3.52 (2.99) 10⁹ CFU / mL in women who eventually became pregnant, but not in those who did not (Table 5, Figure 6). The presence of L. salivarius [mean (SD) = 6.85 (0.61) copies / swab] was confirmed by qPCR in all women who became pregnant, but only in 50% of women with unsuccessful pregnancies, and then at a lower concentration [mean (SD) = 2.63 (1.17) copies / swab] (Table 5). The lactobacillus profile in CVL samples obtained at the beginning of probiotic treatment and after 6 months or until a pregnancy diagnosis is presented is shown in Figure 8. The most notable differences were with respect to the presence of viable L. salivarius in most women (17 / 21) after probiotic treatment. In addition, L.Iners, which was present in 3 women at the beginning of the study, was isolated at the end of treatment from only 2 women who, in particular, did not end up pregnant.

[0193] There were no differences in the metataxonomic profile at the gender level of CVL samples from women in the RA group with respect to pregnancy outcome (Table 6). Table 6. Relative frequencies, medians and interquartile range (IQR) of the most abundant bacterial phyla (shaded in gray) and genera detected in vaginal samples from women in the RA group (women with a history of recurrent miscarriage) who did or did not have a pregnancy after probiotic treatment. a n (%): Number of samples in which the phylum / genus was detected (relative detection frequency). bKruskal-Wallis rank tests with Bonferroni correction. Secondary response parameters associated with probiotic treatment: changes in vaginal parameters in women in the INF group

[0194] The women in the INF group who became pregnant after probiotic treatment and those who did not did not differ in age, weight, and height (results not shown).

[0195] CVL pH and Nugent score decreased significantly in all women in the INF group after probiotic treatment (p < 0.05; one-way repeated measures ANOVA; Figure 9), although the magnitude of change was smaller in women who did not become pregnant compared to those who did (Table 7, Figure 10). Specifically, the mean (SD) reductions in CVL pH and Nugent score in women who became pregnant were 1.32 (0.31) and 3.90 (0.74), respectively, and in women who did not become pregnant, these reductions were only 0.19 (0.18) and 0.54 (0.78), respectively (Table 7, Figure 9).

[0196] Table 7. Differences in characteristics and comparison of changes in vaginal parameters between women who were able to complete a pregnancy to term (n = 10) and those who did not (n = 13) after intervention with L. salivarius CECT 5713 in the INF group, which included women with a history of infertility of unknown origin (n = 23).

[0197] #One-way ANOVA tests were used to assess differences in means between groups.*Fisher's exact test for a 2 x 2 contingency table. **One-way ANOVA; Kruskal-Wallis, p=0.007.

[0198] The change in vaginal cytokine concentrations after probiotic treatment was similar to that described in the RA group: there was no change in vaginal concentrations of TGFβ1, TGFβ2, and VEGF in women who did not become pregnant, but there was a significant mean (SD) increase of 2.29 (0.34) pg / mL, 1.25 (0.24) pg / mL, and 462 (252) pg / mL, respectively, in those who did (Table 7, Figure 9). In this group of women, there were already differences in the concentration of TGFβ2 and VEGF, but not in that of TGFβ1, even before starting treatment between those who became pregnant and those who did not (Table 7, Figure 9).

[0199] Probiotic treatment resulted in a greater number of CVL samples where lactobacilli were detected (in all women who ultimately became pregnant) and a higher mean bacterial count (SD) [6.46 (0.51) µg10 CFU / mL] than in those women who did not (lactobacilli were detected in only 46% and mean bacterial counts (SD) were 4.95 (1.66) µg10 CFU / mL) (Table 7, Figures 9 and 10). Similarly, the presence of L. salivarius [mean (SD) = 6.85 (0.61) couplets / swab] was confirmed by qPCR in all women who became pregnant, but only in 25% of women with unsuccessful pregnancies and then at a lower concentration [mean (SD) = 3.55 (0.39) couplets / swab] (Table 7). The lactobacillus profile in CVL samples obtained at the beginning of treatment with problotics and after 6 months or until a pregnancy diagnosis is made in Figure 11, which shows that L. were detected.Viable salivarius were found in all women who became pregnant, but only in 4 of 13 of the women who did not become pregnant after probiotic treatment.

[0200] There were no differences in the metataxonomic profile at the gender level of CVL samples from women in the RA group with respect to pregnancy outcome (results not shown). Comparison of vaginal parameters between all women who became pregnant and those from both the RA and INF groups.

[0201] The mean pH value in the CVL samples was slightly more acidic in women who became pregnant [5.69 (0.48) units] than in those who did not [5.99 (0.33) units] (p = 0.024; one-way ANOVA) (Table 7). There were also differences in vaginal cytokine concentration at baseline according to pregnancy outcome, but these differences were similar to those already described separately for the RA and INF groups (Table 8; Figures 12 and 13). The only parameters that did not initially differ between the two groups were the Nugent score and the frequency of detection and count of lactobacilli (Table 8). Overall, Lactobacillus was detected in all women who became pregnant, but in only half of those who did not (p < 0.001; chi-square test). Furthermore, there was an increase of 1.There was a 6 log unit increase in Lactobacillus counts after probiotic treatment in the group of women who became pregnant, but no change in those who did not (Figure 14). Significant differences were also observed in the detection of L. salivarius in swab samples. This species was also detected, at a high concentration [mean (SD) = 6.70 (0.56) copies / swab], in all women who had a successful pregnancy compared to women who did not become pregnant (Figure 14).

[0202] Table 8. Differences in characteristics and comparison of changes in vaginal parameters between women who were able to complete a term pregnancy (n = 25) and those who did not (n = 19) after intervention with L. salivarius CECT 5713, including all women from the RA and INF groups (n=44).

[0203] One-way ANOVA tests were used to assess differences in means between groups. *Chi-square (Pearson) test for a 2 x 2 contingency table.

[0204] ** Fisher's exact probability test for a 2 x 2 contingency table.

[0205] ** Differences in the means of the positive samples.

[0206] Comparison of vaginal parameters between control women, all women who became pregnant vs those who did not in the RA and INF groups

[0207] Analysis of vaginal parameters (pH, Nugent score, TGFβ1, TGFβ2, VEGF, lactobacillus count) revealed statistically significant differences for all of them among the three groups (control group, women from the RA and INF groups who experienced a full-term pregnancy, and women from the RA and INF groups who did not become pregnant or had an abortion) (Table 9; Fig. 15).

[0208] Table 9. Comparison of vaginal parameters (pH, Nugent score, cytokines, and lactobacilli count) between participants in the control group (n=14) and participants in the RA and INF groups (combined) who had a pregnancy (n=25) or not (n=19) after probiotic treatment. SD: standard deviation; Cl: confidence interval; TGFβ1, transforming growth factor β1; TGFβ2, transforming growth factor β2; VEGF, vascular endothelial growth factor.

[0209] One-way ANOVA tests were used to assess differences in means between groups. Different bold letters in the same row indicate statistically significant differences between groups.

[0210] 'Freeman-Halton extension of Fisher's exact probability tests for a 2 x 3 contingency table.

[0211] “Average in those women in whom lactobacilli could be isolated from their samples.

[0212] Antimicrobial activity of L salivarius CECT5713 against vaginal and cervical pathogens

[0213] L salivarius CECT 5713 showed inhibitory antimicrobial activity (zone of 2 mm around the line) against all indicator organisms used in this study, under both aerobic and anaerobic conditions. To elucidate the compound(s) responsible for the antimicrobial activity, this non-bacteriogenic strain was selected for the production of hydrogen peroxide, lactate, and / or acetate. A quantitative assay showed that the mean (± SD) amount of hydrogen peroxide produced by this strain was 0.729 µg / mL (± 0.122) under aerobic conditions. No production of this compound was found when the assays were performed under anaerobic conditions. The mean (± SD) concentrations of L-lactic acid in the supernatants obtained from MRS cultures (initial pH = 6.2) of the strain were 11.07 ± 0.42 mg / mi and 11.69 ± 0.58 mg / mi under aerobic and anaerobic conditions, respectively, while the mean pH values ​​in the supernatants were 3.91 and 3.86 under aerobic and anaerobic conditions, respectively.D-Lactate was not detected in the culture supernatants. The mean (± SD) concentrations of acetic acid in the same supernatants were 0.70 mg / mL (± 0.11) and 0.46 mg / mL (± 0.14) under aerobic and anaerobic conditions, respectively.

[0214] Table 10. Antimicrobial activity and aggregation of L. salivarius CECT 5713 against various cervicovaginal pathogens. Coaggregation is defined as visible groups of bacteria and is classified as (++): large and dense groups; (+): small and sparse groups; (-): no visible groups, following the criteria of Younes et al. (2012). Full reference: Younes, JA, van der Mei, HC, van den Heuvel, E., Busscher, HJ, and Reid, G. (2012). Adhesion forces and coaggregation between vaginal staphylococci and lactobacilli. PLoS One, 7, e36917. doi: 10.1371 / ¡oumal.pone.0036917. Coaggregation of L. salivarius CECT5713 with vaginal pathogens and adhesion to vaginal cells

[0215] The strain was able to form large, well-defined coaggregates with all selected vaginal and cervical pathogens. Coaggregation was particularly intense with G. vaginalis, S. agalactiae, and C. albicans strains. In this study, the tested strain was strongly adherent to vaginal epithelial cells, with a mean of 329 ± 46 adherent lactobacilli in 20 random microscopic fields. The mean (± SD) value for L. salivarius CECT 9145, a control strain with high adherence to vaginal cells, was 336 ± 52 adherent lactobacilli.

[0216] α-amylase activity of L. salivarius CECT5713

[0217] Extracellular amylase production by L. salivarius CECT 5713 was observed by the clearing zone around the colonies (~2.0 mm) when treated with iodine solution. Later, when α-amylase activity was measured, this strain showed a high level of α-amylase activity (0.83 U / ml) at 16 h (L. salivarius CECT 5713 concentration: ~8.6 10⁹ CFU / ml), and it could be detected in supernatants at a similar level during the 48 h (when the assay was terminated).

Claims

CLAIMS 1. A strain of the species Lactobacillus salivarius deposited in the Spanish Collection of Type Cultures (CECT) with accession number 5713 or a mutant thereof for use in the treatment and / or prevention of infertility or recurrent pregnancy loss in a female subject.

2. The strain for use according to claim 1, wherein infertility is unexplained infertility.

3. The strain for use according to claim 1, wherein recurrent pregnancy loss is unexplained recurrent pregnancy loss.

4. The strain for use according to any of claims 1 to 3, wherein the strain is administered to a woman with infertility or recurrent pregnancy loss in combination with assisted reproductive treatment.

5. The strain for use according to any of claims 1 to 4, wherein the strain is administered orally.

6. The strain for use according to any of claims 1 to 5, wherein the strain is administered for a period of time of at least 3 months or until a diagnosis of pregnancy.

7. The strain for use according to claim 6, wherein the strain is administered for a period of time of at least 6 months.

8. The strain for use according to any of claims 1 to 7, wherein if pregnancy is diagnosed, administration of the strain is maintained at least until week 15 of pregnancy.

9. The strain for use according to any of claims 1 to 8, wherein the strain is administered at a daily dose comprising 8.5 log 10 UFC and 9.5 UFC lodge.

10. The strain for use according to claim 9, wherein the strain is administered at a daily dose of approximately 9 log 10 UFC.

11. An in vitro method for monitoring the effect of a treatment for infertility or recurrent pregnancy loss in a female subject with a strain of the species Lactobacillus salivarius deposited in the CECT with accession number 5713 or a mutant thereof comprising: (i) determine the level in a sample of vaginal mucosa and / or cervical mucosa of said female subject of a factor of selected growth from the group consisting of VEGF, TGF-β1 and TGF-β2 and (ii) compare that level with a reference value obtained from the same subject before therapy, where an increase in that level relative to the reference value is indicative that the treatment is being effective and a decrease or lack of change in that level relative to the reference value is indicative that the treatment is being ineffective.