Probiotic for the treatment of infertility and recurrent miscarriage

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of methods for treating infertility and recurrent miscarriage. BACKGROUND

[0002] Today, it is estimated that 15% of couples face fertility problems. In 33% of cases, the problem is in the woman; 25% in the man; 33% in both the man and the woman; but in the remaining 11% of cases, the cause is unknown.

[0003] There is increasing evidence that the female reproductive tract microbiota is involved in human reproduction. Under physiological conditions, the human vaginal microbiota is typically characterized by low bacterial diversity, high numbers of and predominance of Lactobacillus bacteria, compared to the gut microbiota.

[0004] The vaginal microbiota of most healthy women of fertile age is composed mainly of one or several Lactobacillus species, which represent 90-95% of the total number of bacteria.

[0005] Multiple factors contribute to inter- and intra-individual variation in the vaginal microbiota, although changes between different vaginal types can occur, an increase in diversity and obligate anaerobes and a decrease or depletion of Lactobacillus are considered risk factors for bacterial vaginosis (BV).

[0006] The composition of the vaginal microbiota, especially any deviation from a Lactobacillus-dominated low-diversity vaginal microbiota, can play a key role in the outcome of fertility and assisted reproductive treatments (ART). However, there are some probiotics on the market that are prescribed on an empirical basis to attempt to recolonize the female reproductive tract with Lactobacillus strains (Moreno and Simon, Reprod Med Biol. 2019; 18:40-50), although their actual use has not been properly scientifically evaluated.

[0007] Therefore, there is a need to provide new therapeutic methods for infertility and recurrent miscarriage, on the basis of the administration of effective probiotics. SUMMARY

[0008] The inventors have found, surprisingly, that oral administration of the probiotic Lactobacillus salivarius strain CECT 5713 to women with unexplained infertility or recurrent miscarriage achieves a 66% pregnancy rate (Table 4) and a 56% successful pregnancy (Table 4). Furthermore, the inventors have also found that in infertile women and women with recurrent miscarriage, probiotic treatment leads to a decrease in vaginal pH (Table 7) and an increase in vaginal TGFpi, TGFp2 and VEGF cytokine levels (Table 8) in women who eventually become pregnant, but not in those who do not.

[0009] Thus, in one aspect, the present application relates to a Lactobacillus salivarius strain deposited at the Spanish Type Culture Collection (CECT) under deposit number 5713, or a mutant thereof, for use in the treatment and / or prevention of infertility or recurrent miscarriage in a female subject.

[0010] In another aspect, the present application relates to an in vitro monitoring method for monitoring the therapeutic effect of a treatment of infertility or recurrent miscarriage in a female subject with a Lactobacillus salivarius strain deposited at the CECT under deposit number 5713, or a mutant thereof, said method comprising:

[0011] (i) determining the level of a growth factor selected from the group consisting of VEGF, TGF-β1 and TGF-β2 in a vaginal mucus sample and / or a cervical mucus sample of said female subject, and

[0012] (ii) comparing said level to a reference value obtained from the same subject prior to treatment,

[0013] wherein an increase of said level relative to said reference value is indicative of said treatment being effective, and a decrease or no change of said level relative to the reference value is indicative of the treatment being ineffective. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 : Main Lactobacillus species in cervical-vaginal lavage samples of women of fertile age (C), women with recurrent miscarriage (RM) and women with unexplained infertility (INF) in isolatable cases.

[0015] Figure 2 : Comparison of bacterial diversity assessed using the Shannon index (A) or the Simpson index (B) between the control group, the RM group and the INF group. Pairwise comparisons between groups were performed using the Wilcoxon rank-sum test with Bonferroni correction.

[0016] Figure 3 : PCoA (Principal Coordinates Analysis) plot of bacterial distribution (at genus level) of cervical-vaginal lavage samples based on Bray-Curtis similarity analysis (relative abundance) (A) and binary data (presence / absence) of Jaccard coefficient (B). Control, circles; RM, crosses; INF, triangles. The boxplot in the bottom panel shows the distance of the samples to the centroid of each group. The values given on each axis represent the percentage of the total variance explained by the axis. P-values were calculated by a permutation test of multivariate dispersion homogeneity.

[0017] Figure 4 : PCA (Principal Component Analysis) plot of bacterial distribution at genus level (control, circles; INF, triangles; RM, crosses) according to the relative abundance of bacteria in cervical-vaginal lavage samples.

[0018] Figure 5 Comparison of various characteristics of the RM group (women with a history of recurrent spontaneous abortion) and the INF group (women with a history of infertility) at recruitment: (A) age, weight and height; (B) pH, Nugent score, concentration of TGFβ1, TGFβ2 and VEGF in the vagina and (C) Lactobacillus count.

[0019] Figure 6 Changes in characteristics of the RM group (women with a history of recurrent spontaneous abortion) after probiotic treatment according to outcome (pregnancy vs. non-pregnancy): (A) change in pH and Nugent score; (B) change in concentration of TGFβ1, TGFβ2 and VEGF in the vagina; and (C) change in Lactobacillus count.

[0020] Figure 7 Characteristics of the RM group (women with a history of recurrent spontaneous abortion) before (I) and after (F) probiotic treatment according to outcome (pregnancy vs. non-pregnancy). (A) pH and Nugent score; (B) concentration of TGFβ1, TGFβ2 and VEGF in the vagina.

[0021] Figure 8 Change in distribution of dominant species of Lactobacillus in the RM group (women with a history of recurrent spontaneous abortion) before (I) and after (F) probiotic treatment according to outcome (pregnancy vs. non-pregnancy).

[0022] Figure 9 Characteristics of the INF group (women with a history of infertility) before (I) and after (F) probiotic treatment according to outcome (pregnancy vs. non-pregnancy).

[0023] Figure 10 Changes in characteristics of the INF group (women with a history of infertility) after probiotic treatment according to outcome (pregnancy vs. non-pregnancy): (A) change in pH and Nugent score; (B) change in concentration of TGFβ1, TGFβ2 and VEGF in the vagina; and (C) change in Lactobacillus count.

[0024] Figure 11 Change in distribution of dominant species of Lactobacillus in the INF group (women with a history of infertility) before (I) and after (F) probiotic treatment according to outcome (pregnancy vs. non-pregnancy).

[0025] Figure 12 Comparison of vaginal parameters of pregnant and non-pregnant women in the RM and INF groups (as a whole): (A) change in pH and Nugent score; (B) change in concentration of TGFβ1, TGFβ2 and VEGF in the vagina; and (C) change in Lactobacillus count.

[0026] Figure 13 : Comparison of vaginal parameters (A) pH and Nugent score; (B) concentrations of TGFpi, TGFp2 and VEGF in the vagina, in pregnant women vs. non-pregnant women, in the RM group and in the INF group (as a whole), before (I) and after (F) probiotic treatment, according to the outcome (pregnancy vs. non-pregnancy).

[0027] Figure 14 : Changes in the counts of Lactobacillus (log cfu / ml) (A) and in the number of copies of DNA specific for Lactobacillus salivarius (copies / ml) (B) in the cervical-vaginal lavage samples, in pregnant women vs. non-pregnant women, in the RM group and in the INF group (as a whole), before (I) and after (F) probiotic treatment, according to the outcome (pregnancy vs. non-pregnancy). 10

[0028] Figure 15 : Comparison of various characteristics in the control group vs. characteristics in pregnant (P) and non-pregnant (NP) women in the RM group and in the INF group, after probiotic treatment: (A) pH and Nugent score; (B) concentrations of TGFpi, TGFp2 and VEGF in the vagina; and (C) counts of Lactobacillus. DETAILED DESCRIPTION

[0029] MEDICAL USES OF THE INVENTION

[0030] In one aspect, the present application relates to a Lactobacillus salivarius strain deposited at the Spanish Type Culture Collection (CECT) under accession number 5713, or a mutant thereof, for use in the treatment and / or prevention of infertility or recurrent miscarriage in a female subject.

[0031] Alternatively, the present application relates to the use of a Lactobacillus salivarius strain deposited at the Spanish Type Culture Collection (CECT) under accession number 5713, or a mutant thereof, for the manufacture of a pharmaceutical product for the treatment and / or prevention of infertility or recurrent miscarriage in a female subject.

[0032] Alternatively, the present application relates to a method for the treatment and / or prevention of infertility or recurrent miscarriage in a female subject, said method comprising administering to said subject an effective therapeutic amount of a Lactobacillus salivarius strain deposited at the Spanish Type Culture Collection (CECT) under accession number 5713, or a mutant thereof.

[0033] As used herein, the term "strain" refers to a genetic variant or subtype of a species of microorganism, preferably a bacterial species.

[0034] ​The terms "Lactobacillus salivarius" and "Lagilactobacillus salivarius" are used as synonyms in the context of the present application.

[0035] In one embodiment, the present application relates to the Lactobacillus salivarius strain deposited at the Spanish Type Culture Collection (CECT) under the accession number 5713 for use in the treatment and / or prevention of infertility or recurrent miscarriage in a female subject.

[0036] In another embodiment, the present application relates to a mutant of the Lactobacillus salivarius strain deposited at the Spanish Type Culture Collection (CECT) under the accession number 5713 for use in the treatment and / or prevention of infertility or recurrent miscarriage in a female subject.

[0037] As used herein, the term "a mutant of the Lactobacillus salivarius strain deposited at the Spanish Type Culture Collection (CECT) under the accession number 5713" refers to any strain that is naturally obtained or developed in properties from the reference strain mainly by mutation and that maintains the properties of the reference strain, the strain being deposited at the address of the University of Valencia Research Building, Valencia, Spain, on the date of 11 June 2002. Lactobacillus salivarius 5713 is characterized by the presence of the following properties.

[0038] (a) Antimicrobial activity against vaginal and / or cervical pathogens.

[0039] The term "vaginal and / or cervical pathogen" refers to a microorganism, primarily a fungus or a bacterium, capable of colonizing the mucosa of the vagina and / or cervix, which can lead to disease, such as vaginitis, vaginosis, trichomoniasis, candidiasis, or lower urinary tract infection. For example, the term "vaginal pathogen" includes microorganisms of external origin and microorganisms that, while possibly present in the healthy vaginal mucosa, can cause disease when the vaginal bacterial flora is unbalanced, including a decrease in certain microorganisms with beneficial or protective effects and / or an increase in potentially pathogenic microorganisms. Non-limiting illustrative examples of vaginal pathogens include Candida albicans, Candida glabrata, Candida tropicalis, Gardnerella vaginalis, Streptococcus agalactiae, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Prevotella, Peptostreptococcus, Escherichia coli, Enterococcus faecalis, and Ureaplasma urealyticum. In particular embodiments, the vaginal pathogen is selected from Gardnerella vaginalis (Gardnerella vaginalis MP14, Gardnerella vaginalis MP17, Gardnerella vaginalis MP20, Gardnerella vaginalis MP24, Gardnerella vaginalis MP29), Streptococcus agalactiae (Streptococcus agalactiae MP07, Streptococcus agalactiae MP12, Streptococcus agalactiae MP46), Candida albicans (Candida albicans MP09, Candida albicans MP18, Candida albicans MP31), Candida glabrata (Candida glabrata MP33, Candida glabrata MP37), Candida parapsilosis (Candida parapsilosis MP36, Candida parapsilosis MP48), and Ureaplasma urealyticum (Ureaplasma urealyticum MP39, Ureaplasma urealyticum MP57).

[0040] As used herein, the term "antimicrobial activity" refers to the ability to kill or inhibit the growth of a microorganism (herein a pathogenic vaginal microorganism). Antimicrobial activity includes bactericidal activity (the ability to kill bacteria), bacteriostatic activity (the ability to inhibit the growth of bacteria), fungicidal activity (the ability to kill fungi), and fungistatic activity (the ability to inhibit the growth of fungi).

[0041] (b) the ability to co-aggregate with vaginal and cervical pathogens.

[0042] The term "vaginal and / or cervical pathogen" has been defined hereinabove. In particular embodiments, the vaginal pathogen is selected from the group consisting of Gardnerella vaginalis (Gardnerella vaginalis MP14, Gardnerella vaginalis MP17, Gardnerella vaginalis MP20, Gardnerella vaginalis MP24, Gardnerella vaginalis MP29), Streptococcus agalactiae (Streptococcus agalactiae MP07, Streptococcus agalactiae MP12, Streptococcus agalactiae MP46), Candida albicans (Candida albicans MP09, Candida albicans MP18, Candida albicans MP31), Candida glabrata (Candida glabrata MP33, Candida glabrata MP37), Candida parapsilosis (Candida parapsilosis MP36, Candida parapsilosis MP48) and Ureaplasma urealyticum (Ureaplasma urealyticum MP39, Ureaplasma urealyticum MP57). In more particular embodiments, Lactobacillus salivarius strain CECT 5713 has the ability to co-aggregate with Gardnerella vaginalis, Streptococcus agalactiae and Candida albicans.

[0043] The term "co-aggregation ability" refers to the ability of Lactobacillus salivarius strain CECT 5713 to adhere to other microorganisms of different species.

[0044] (c) High adhesion to vaginal epithelial cells.

[0045] The term "adhesion" refers to the ability of Lactobacillus salivarius strain CECT 5713 to bind to host tissues, in this case vaginal epithelial cells. Lactobacillus salivarius strain CECT 5713 is considered to have a higher adhesion to vaginal cells when it is at least 90% of the adhesion to vaginal cells of Lactobacillus salivarius strain CECT 9145, more particularly 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 more, when measured under the same experimental conditions.

[0046] (d) High alpha-amylase activity

[0047] As used herein, the term "alpha-amylase activity" refers to the enzymatic activity determined by the enzyme numbered EC 3.2.1.1, which is capable of hydrolyzing glycosidic linkages anywhere along the carbohydrate chain, breaking these bonds, producing maltotriose and maltose from amylase and maltose, and glucose and dextrin from amylopectin. A bacterium is considered to have a high alpha-amylase activity when its alpha-amylase activity is greater than 0.50 U / mL, as determined by the method described by Narita et al. (2006), Appl. Environ. Microbiol. 72: 269-275.

[0048] (e) High lactic acid production.

[0049] As used herein, the term "lactate production" refers to the ability of a strain to produce lactic acid, officially named 2-hydroxypropanoic acid or a-hydroxypropanoic acid, which is either of the two L or D isomers. A bacterium is considered to have a high lactate production when the lactate production in the culture supernatant after incubation under optimal growth conditions is greater than 8 mg / ml.

[0050] Thus, in a particular embodiment, the mutants of Lactobacillus salivarius strain CECT 5713 maintain one or more of the aforementioned properties. As used herein, the term "maintain" refers to an activity that, when measured by a suitable quantitative assay, shows 100% or more activity of the activity of the Lactobacillus salivarius strain CECT 5713 from which it was derived, 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 Lactobacillus salivarius strain CECT 5713 from which it was derived.

[0051] In a particular embodiment, the mutants of Lactobacillus salivarius strain CECT 5713 retain the antimicrobial activity of Lactobacillus salivarius strain CECT 5713 against vaginal pathogens, said mutants retaining the antimicrobial activity against a vaginal pathogen selected from the group consisting of Gardnerella vaginalis (Gardnerella vaginalis MP14, Gardnerella vaginalis MP17, Gardnerella vaginalis MP20, Gardnerella vaginalis MP24, Gardnerella vaginalis MP29), Streptococcus agalactiae (Streptococcus agalactiae MP07, Streptococcus agalactiae MP12, Streptococcus agalactiae MP46), Candida albicans (Candida albicans MP09, Candida albicans MP18, Candida albicans MP31), Candida glabrata (Candida glabrata MP33, Candida glabrata MP37), Candida parapsilosis (Candida parapsilosis MP36, Candida parapsilosis MP48) and Mycoplasma urealyticum (Mycoplasma urealyticum MP39, Mycoplasma urealyticum MP57). In a more particular embodiment, said mutants retain the antimicrobial activity against a vaginal pathogen selected from the group consisting of Gardnerella vaginalis, Streptococcus agalactiae, Candida albicans, Candida glabrata and Mycoplasma urealyticum. Said antimicrobial activity can be determined by any quantitative assay method that allows determining the ability of the mutant to kill or inhibit the growth of the pathogenic microorganism cells, such as agar dilution or diffusion methods and other methods described, for example, by Balouri et al., in Journal of Pharmaceutical Analysis, 6 (2016), 71-79. In a particular embodiment, said mutants retain the antimicrobial activity of Lactobacillus salivarius strain CECT 5713 when the activity is determined by the overlay method described by Magnusson and Schnürer, Appl. Environ. Microbiol. 2001, 67, 1-5, used in the examples. In a particular embodiment, said mutants retain 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 Lactobacillus salivarius strain CECT 5713 against vaginal pathogens, preferably selected from the group consisting of Gardnerella vaginalis, Streptococcus agalactiae, Candida albicans, Candida glabrata and Mycoplasma urealyticum, when said activity is determined by the overlay assay mentioned herein.

[0052] In a particular embodiment, the mutants of Lactobacillus salivarius strain CECT 5713 retain the ability of Lactobacillus salivarius strain to co-aggregate with vaginal and / or cervical pathogens. In a more particular embodiment, said mutants retain the ability to co-aggregate with vaginal and / or cervical pathogens selected from the group consisting of Gardnerella vaginalis, Streptococcus agalactiae and Candida albicans. The ability to co-aggregate with vaginal and / or cervical pathogens can be determined by any known co-aggregation assay method, such as the one described by Younes et al. (PLoS One 2012, 7, e36917) used in the examples herein. In a particular embodiment, the mutants retain the ability of Lactobacillus salivarius strain CECT 5713 to co-aggregate with vaginal and / or cervical pathogens when said activity is determined by the co-aggregation assay method described by Younes et al. (supra). In a particular embodiment, said mutants retain 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 ability of Lactobacillus salivarius strain CECT 5713 to co-aggregate with vaginal and / or cervical pathogens, preferably selected from the group consisting of Gardnerella vaginalis, Streptococcus agalactiae and Candida albicans, when said activity is determined by the co-aggregation assay method described above.

[0053] In a particular embodiment, the mutants of Lactobacillus salivarius strain CECT 5713 maintain the high adhesion to vaginal epithelial cells of Lactobacillus salivarius strain CECT 5713. The adhesion to vaginal epithelial cells can be determined by any known adhesion assay method, for example by the method described by Boris et al. in Infect. Immun. 1998, 66, 1985-1989, used in the examples, wherein vaginal epithelial cells collected from premenopausal women are used. In a particular embodiment, the mutants maintain the high adhesion to epithelial cells of Lactobacillus salivarius strain CECT 5713 when the activity is determined by the co-aggregation assay described by Younes et al. (see above). In particular embodiments, the mutants maintain 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 to vaginal epithelial cells of Lactobacillus salivarius strain CECT 5713 when the activity is determined by the adhesion assay described above. In this regard, Lactobacillus salivarius strain CECT 5713 has an average adhesion to vaginal epithelial cells obtained from premenopausal women of 329 ± 46 adherent lactobacilli in 20 random microscopic fields when the adhesion is determined by the assay method described by Younes et al. as described in the examples herein, and the mutants of Lactobacillus salivarius strain CECT 5713 have similar adhesion, i.e. about 329 ± 46 adherent lactobacilli or more in 20 random microscopic fields when determined by the same assay method.

[0054] In a particular embodiment, the mutants of Lactobacillus salivarius strain CECT 5713 maintain the high alpha-amylase activity of Lactobacillus salivarius strain CECT 5713. The alpha-amylase activity can be determined by any known assay method, for example by the method described by Narita et al. (Appl Environ Microbiol. 2006; 72(1): 269-275) used in the examples, which is based on the determination of the substrate 2-chloro-4-nitrophenyl 6 5 - azido-6 5- deoxy-beta-maltosidase activity. In a particular embodiment, the mutant strain maintains the high alpha-amylase activity of Lactobacillus salivarius strain CECT 5713 when said activity is determined by the assay described by Narita et al. (see above). In particular embodiments, 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 alpha-amylase activity of Lactobacillus salivarius strain CECT 5713 when said activity is determined using the assay described above. In this regard, Lactobacillus salivarius strain CECT 5713 has an alpha-amylase activity of 0.83 U / mL after 16 hours when said activity is determined by the assay described by Narita et al. as described in the examples herein, and in particular embodiments, the Lactobacillus salivarius strain CECT 5713 mutants have a similar alpha-amylase activity, i.e. around 0.83 U / mL or more after 16 hours when measured by the same assay.

[0055] In a particular embodiment, the mutant of Lactobacillus salivarius strain CECT 5713 maintains the high lactic acid production of Lactobacillus salivarius strain CECT 5713. In a particular embodiment, the mutant of Lactobacillus salivarius strain CECT 5713 maintains the high lactic acid production, particularly L-lactic acid production, of Lactobacillus salivarius strain CECT 5713 under aerobic and / or anaerobic conditions. Said 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 the two enantiomers of lactic acid by an enzymatic kit in the MRS culture supernatant. In a particular embodiment, the mutant maintains the high lactic acid production of Lactobacillus salivarius strain CECT 5713 when said activity is determined by the assay described by Martín et al. 2006 (see above). In particular embodiments, 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 Lactobacillus salivarius strain CECT 5713 when said production is determined by the assay described above. In this regard, Lactobacillus salivarius strain CECT 5713 is able to produce 11.07 ± 0.42 mg / ml and 11.69 ± 0.58 mg / ml of L-lactic acid under aerobic and anaerobic conditions, respectively, when said production is determined by the assay described by Martín et al. (2006) as described in the examples herein. In a particular embodiment, the mutant of Lactobacillus salivarius strain CECT 5713 is able to produce similar amounts of L-lactic acid under aerobic and anaerobic conditions, i.e. about 11 mg / ml or more, when measured by the same assay.

[0056] In a particular embodiment, the mutant of Lactobacillus salivarius strain CECT 5713 is an isogenic mutant, i.e. a strain having a genome that differs from that of Lactobacillus salivarius strain CECT 5713 only by the presence of the mutation.

[0057] In another particular embodiment, the mutant of Lactobacillus salivarius strain CECT 5713 has a genome with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the genome of Lactobacillus salivarius strain CECT 5713.

[0058] In another particular embodiment, the mutants of Lactobacillus salivarius strain CECT 5713 are characterized by the presence of a 16S rRNA gene having 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 Lactobacillus salivarius strain CECT 5713.

[0059] In another particular embodiment, the degree of identity between the mutant and the parent strain is determined as the average nucleotide identity (ANI) that detects the conservation of core genomic DNA (Konstantinidis K and Tiedje JM, 2005, Proc. Natl. Acad. Sci. USA 102:2567-2592). In some embodiments, the ANI between the variant and the original strain is about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.99%, about 99.999%, about 99.9999%, about 99.99999%, about 99.999999% or more, but less than 100%.

[0060] In another embodiment, the degree of relatedness between the mutant and the parent strain is determined as the tetranucleotide signature frequency correlation coefficient based on oligonucleotide frequency (Bohlin J. et al., 2008, BMC Genomics, 9: 104). In some embodiments, the tetranucleotide signature frequency correlation coefficient between the mutant and Lactobacillus salivarius strain CECT 5713 is about 0.99, 0.999, 0.9999, 0.99999, 0.999999, 0.999999 or more, but less than 1.

[0061] In another embodiment, the degree of relatedness between the mutant and the parent strain is determined as the degree of similarity obtained when the genomes of the parent and variant strains are analyzed by the method of pulsed field gel electrophoresis (PFGE) using one or more restriction enzymes. The degree of similarity obtained by the method of PFGE can be measured by the Dice similarity coefficient. In some embodiments, the Dice similarity coefficient between the mutant and Lactobacillus salivarius strain CECT 5713 is about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.99%, about 99.999%, about 99.9999%, about 99.99999%, about 99.999999% or more, but less than 100%.

[0062] In another embodiment, when two strains have the same ribotype (as obtained using any method known in the art and described by Bouchet et al. (Clin. Microbiol. Rev., 2008, 21 :262-273), the strains are considered to be mutants of a given parent strain.

[0063] In another embodiment, the degree of relatedness between a mutant and an original strain is the Pearson correlation coefficient obtained by comparing the genetic profiles of the two strains obtained based on repetitive extragenic palindromic polymerase chain reaction (REP-PCR) (see, e.g., Chou and Wang, Int J Food Microbiol. 2006 110: 135-48). In some embodiments, the Pearson correlation coefficient obtained when comparing the REP-PCR profiles of a mutant and Lactobacillus salivarius strain CECT 5713 is about 0.99, 0.999, 0.9999, 0.99999, 0.999999, 0.999999, or more, but less than 1.

[0064] In another embodiment, the degree of relatedness between a mutant and a parent strain is the linkage distance obtained by comparing the genetic profiles of the two strains obtained by multilocus sequence typing (MLST) (see, e.g., Maiden, MC, 1998, Proc. Natl. Acad. Sci. USA 95:3140-3145). In some embodiments, the linkage distance obtained by MLST between a mutant and Lactobacillus salivarius strain CECT 5713 is about 0.99, 0.999, 0.9999, 0.99999, 0.999999, 0.999999, or more, but less than 1.

[0065] As used herein, the term "treatment" refers to any type of treatment, with the aim of stopping, improving or reducing the susceptibility of suffering from a clinical condition, as described herein. Thus, "treatment", "treat" and equivalents thereof mean obtaining a pharmacological or physiologic effect, including any treatment of a condition or disease in a mammal, including humans. The effect can be prophylactic in terms of providing a complete or partial prevention of a disease and / or adverse effect attributable to the disease. In other words, "treatment" includes (1) inhibiting the disease, e.g., arresting its development, (2) interrupting or terminating the disease or at least symptoms associated therewith, such that the patient no longer suffers from the disease or its symptoms, e.g., by restoring or repairing a lost, missing or deficient function, or stimulating an ineffective process, leading to the regression of the disease or its symptoms, or (3) slowing, reducing or ameliorating the disease or symptoms associated therewith, wherein slowing is used in a broad sense to mean at least reducing the magnitude of a parameter, e.g., inflammation, pain or immune deficiency.

[0066] As used herein, the term "prevention", "preventing" or "prevent" refers to the administration of Lactobacillus salivarius strain CECT 5713 or a mutant thereof to a female subject who has not been diagnosed with infertility or recurrent miscarriage, but who is generally expected to suffer from or be at risk of said disease. Prevention aims to stop the occurrence of the disease described above. Prevention can be complete (e.g., complete non-suffering from the disease). Prevention can also be partial, such that the degree of onset of the disease in the subject is less than that which would occur, for example, in the absence of administration of the combination or composition of the application. Prevention also refers to a reduction in the susceptibility to a clinical condition. Prevention also includes reducing the risk of suffering from a disease.

[0067] The term "female subject" as described herein refers to a female member of a mammalian species, including but not limited to domestic animals, primates and humans; the subject is preferably a human of any age or ethnicity.

[0068] According to the present application, an effective therapeutic amount of Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered for prevention and / or treatment.

[0069] The term "effective" amount or "effective therapeutic amount" of a pharmaceutical or pharmacologically active agent is understood as a non-toxic amount of the drug or agent, but which is sufficient to provide the desired effect. In the treatment of the present application, an "effective amount" of Lactobacillus salivarius strain CECT 5713 or a mutant thereof is the amount of the microorganism effective to provide the desired effect.

[0070] Although individual needs can vary, determination of the optimal range of effective amounts of the microorganisms of the present application is within the skill of those in the art. Generally, the dosage required to provide the effective amount of the microorganisms (which can be adjusted by those skilled in the art) will depend on the age, health, physical condition, sex, diet, body weight, frequency of treatment, the nature and extent of the malaise or disease, the patient's medical history, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetics and toxicology profile of the particular compound used, whether systemic drug delivery is used, and whether the compound is administered as part of a drug combination.

[0071] As used herein, the term "infertility" refers to the inability of a woman to conceive by natural means. In particular, it is a reproductive system disorder defined as the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected intercourse in the absence of other causes (such as breastfeeding or lactational amenorrhea). Female infertility can be due to different causes, including:

[0072] - changes in the menstrual cycle, including anovulation, hyperprolactinemia, polycystic ovary syndrome, luteal phase defects, premature ovarian failure.

[0073] - tubal and uterine diseases, including tubal obstruction, small pelvis inflammation (or upper genital tract infection), congenital uterine and vaginal malformations.

[0074] - changes in cervical mucus

[0075] - endometriosis

[0076] - infections, such as untreated gonorrhea and chlamydia, chronic infection of the cervix, and surgical treatment of cervical lesions associated with human papillomavirus (HPV) infection,

[0077] - serious diseases such as cancer.

[0078] - extreme thinness or obesity.

[0079] - endocrine disorders, such as thyroid disease, hypothalamic dysfunction, and hyperprolactinemia.

[0080] - autoimmune diseases, such as lupus, Hashimoto's disease, other types of thyroiditis, and rheumatoid arthritis.

[0081] In a particular embodiment, infertility is unexplained infertility. As used herein, the term "unexplained infertility" or "idiopathic infertility" refers to infertility that cannot be determined after a series of diagnostic tests on the woman and her male partner. In a particular embodiment, female infertility is considered unexplained infertility in the following cases:

[0082] - physical examination does not show signs of uterine, tubal or ovarian abnormalities.

[0083] - All ovarian reserve tests show a normal level of follicle supply.

[0084] - Hysterosalpingography (HSG) shows normal uterine cavity and tubal patency.

[0085] - Analysis of semen obtained from the man shows an appropriate number of healthy spermatozoa.

[0086] - The woman has a normal ovulatory activity or any ovulatory disorder (such as polycystic ovary syndrome) has been successfully treated.

[0087] - Blood tests are normal for both the man and the woman.

[0088] As used herein, the term “recurrent miscarriage” or “recurrent spontaneous miscarriage” refers to the situation where a female subject obtains a clinical pregnancy but said pregnancy does not reach its term. In particular, recurrent miscarriage is considered to be present in the following cases:

[0089] - 3 consecutive miscarriages, including non-visualized miscarriages (ESHRE, RCOG)

[0090] - 2 or more miscarriages, not necessarily consecutive, and with the pregnancy recorded by means of ultrasound or histopathological examination.

[0091] Recurrent miscarriage can be caused by different reasons, including:

[0092] - Anatomical causes: uterine malformation, fibroids.

[0093] - Lifestyle or environment-related causes: smoking, drug abuse, excessive alcohol or caffeine, obesity.

[0094] - Untreated diseases, such as thyroid or diabetes disease, abnormalities of the immune or coagulation system.

[0095] In particular embodiments, the recurrent miscarriage is unexplained recurrent miscarriage. As used herein, the term “unexplained recurrent miscarriage” refers to a recurrent miscarriage for which no cause is identified after a series of diagnostic tests performed on the woman and her male partner. In a particular embodiment, the recurrent miscarriage is considered unexplained when the following tests are normal:

[0096] - Uterine cavity tests aimed at detecting uterine abnormalities. These examinations can include transvaginal ultrasound, hysterosalpingography, saline infusion sonography or magnetic resonance.

[0097] - Genetic testing on the woman and the man, including testing of the miscarried fetal tissue and of the couple’s tissues for chromosomal abnormalities.

[0098] - Hormone tests include thyroid studies and prolactin, follicle-stimulating hormone, fasting blood sugar, and insulin analysis.

[0099] - Hematology and immunology tests.

[0100] Female subjects with infertility or recurrent miscarriage who are undergoing assisted reproductive treatment can be treated with Lactobacillus salivarius strain CECT 5713 or a mutant thereof, thereby increasing the chances of success of the assisted reproductive treatment. Thus, in a particular embodiment, Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered in combination with assisted reproductive treatment to a female suffering from infertility or recurrent miscarriage.

[0101] As used herein, the term "assisted reproductive treatment" or "medical assisted reproductive treatment" refers to a medical treatment aimed at favoring pregnancy in the presence of infertility problems, whether male, female or male and female together infertility problems, and in the case of a woman without a partner or with a female partner who wishes to become pregnant with donor sperm. The feature of assisted reproduction is the stimulation or induction of ovulation, which can include direct action on the gametes (oocytes and / or spermatocytes) to favor fertilization and embryo transfer or deposition in the uterine cavity. Examples of assisted reproductive treatment include ovulation induction, controlled ovarian stimulation, planned intercourse, intrauterine, intra-cervical and intra-vaginal insemination, whether using the partner's or donor's sperm, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), sperm extraction, egg donation, fertility preservation.

[0102] In a particular embodiment, the administration of Lactobacillus salivarius strain CECT 5713 or a mutant thereof in combination with assisted reproductive treatment means that the strain is administered to a female subject who has received, is receiving or will receive assisted reproductive treatment. In a particular embodiment, Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered for a certain period of time before the treatment, for example, 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, at least 1 year before the assisted reproductive treatment. In a particular embodiment, Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered throughout the period of time in which the female subject receives assisted reproductive treatment. In a particular embodiment, Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered to a subject after she has received assisted reproductive treatment, for example, for at least 1 week, at least 2 weeks, at least 3 weeks, and in case a pregnancy has been achieved, at least up to the 4thweek, at least up to the 5thweek, at least up to the 6thweek, at least up to the 7thweek, at least up to the 8thweek, at least up to the 9thweek, at least up to the 10thweek, at least up to the 11thweek, at least up to the 12thweek, at least up to the 13thweek, at least up to the 14thweek, at least up to the 15thweek, or throughout the pregnancy. In a more particular embodiment, Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered at least up to the 15thweek of pregnancy.

[0103] The term "week of pregnancy" refers to the time elapsed from the last menstrual period, assuming a menstrual cycle of 28 days, with fertilization occurring at the 14thday of the cycle.

[0104] Lactobacillus salivarius strain CECT 5713 or a mutant thereof can be administered vaginally or orally, preferably orally.

[0105] In a particular embodiment, Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered in the form of a lyophilisate.

[0106] The amount of administration of Lactobacillus salivarius strain CECT 5713 or a mutant thereof will vary depending on the subject and the particular mode of administration. The skilled person will understand that the dose can also be determined according to the guidelines of Goodman and Goldman, The Pharmacological Basis of Therapeutics, 9th edition (1996), Appendix II, pages 1707-1711 and Goodman and Goldman, The Pharmacological Basis of Therapeutics, 10th edition (2001), Appendix II, pages 475-493, with a daily dose of Lactobacillus salivarius strain CECT 5713 or a mutant thereof of 5.0 log 10 cfu (colony forming units) to 13.0 log 10 cfu, for example 5.5 log 10 cfu to 12.5 log 10 cfu, 6.0 log 10 cfu to 12.0 log 10 cfu, 6.5 log 10 cfu to 11.5 log 10 cfu, 7.0 log 10 cfu to 11 log 10 cfu, 7.5 log 10 cfu to 10.5 log 10 cfu, 8.0 log 10 cfu to 10.0 log 10 cfu, 8.5 log 10 cfu to 9.5 log 10 cfu, about 9.0 log 10 cfu. Preferably, Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered in a daily dose of 8.5 log 10 cfu to 9.5 log 10 cfu, more preferably in a daily dose of about 9 log 10 cfu.

[0107] In a particular embodiment, the Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered for 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 diagnosis of pregnancy is made, preferably, the Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered at least until the 4th week of pregnancy, at least until the 5th week of pregnancy, at least until the 6th week, at least until the 7th week of pregnancy, at least until the 8th week of pregnancy, at least until the 9th week of pregnancy, at least until the 10th week of pregnancy, at least until the 11th week of pregnancy, at least until the 12th week of pregnancy, at least until the 13th week of pregnancy, at least until the 14th week of pregnancy, until the 15th week of pregnancy, or throughout the pregnancy, more preferably at least until the 15th week of pregnancy.

[0108] Monitoring method of the application

[0109] The inventors have detected that, in women who finally achieve pregnancy, the concentration of the growth factors VEGF, TGF-β1 and TGF-β2 in the vagina increases after treatment with the Lactobacillus salivarius strain CECT 5713 compared to women who do not achieve pregnancy (Table 8), so that the concentration of these growth factors can be used as a biomarker of the effectiveness of the treatment.

[0110] Therefore, in another aspect, the present application relates to an in vitro monitoring method for monitoring the effectiveness of the treatment of infertility or recurrent miscarriage in a female subject treated with the Lactobacillus salivarius strain deposited in the CECT with accession number 5713 or a mutant thereof, said method comprising:

[0111] (i) determining the level of a growth factor selected from the group consisting of VEGF, TGF-β1 and TGF-β2 in a vaginal mucus sample and / or a cervical mucus sample of said female subject, and

[0112] (ii) comparing said level with a reference value obtained from the same subject before treatment,

[0113] wherein an increase in said level with respect to said reference value indicates that said treatment is effective, and

[0114] a decrease or no change in said level with respect to the reference value indicates that the treatment is not effective.

[0115] As used herein, the term "in vitro" refers to a method performed not on the body of a human or animal subject, but on a sample isolated from said subject.

[0116] The term "monitoring the effect of a treatment" refers to the response of a patient suffering from a disease to a treatment that treats said disease.

[0117] The terms "infertility", "recurrent miscarriage", "female subject", "Lactobacillus salivarius strain preserved at CECT with accession number 5713", "mutants thereof" have been defined above in relation to the first aspect of the invention. All specific and preferred embodiments of the first aspect in relation to said terms are also applicable to the monitoring method of the effect of the treatment of the invention.

[0118] The monitoring method of the invention comprises determining the level of a growth factor selected from the group consisting of VEGF, TGF-β1 and TGF-β2 in a vaginal mucus sample and / or a cervical mucus sample.

[0119] As used herein, the term "vaginal mucus sample and / or cervical mucus sample" refers to a sample from the vaginal and cervical mucosal surface, including small molecules, extracellular polymers, proteins and peptides, microbial cells and host cells (in this case, cells of the female subject). Said sample can be obtained by any known technique, for example, by swabbing or by a method of washing with a liquid, such as saline, which is known as "cervicovaginal" lavage or "CVL". In a particular embodiment, the vaginal mucus sample and / or cervical mucus sample is a cervicovaginal lavage.

[0120] As used herein, the term "level of VEGF, TGF-β1 or TGF-β2" refers to the amount of the corresponding protein present in the sample. The level of protein can be determined by any method known in the art suitable for determining and quantifying proteins in a sample. By way of non-limiting illustration, the level of protein can be determined by a technique comprising the use of an antibody having the ability to specifically bind the protein under analysis (or a fragment thereof containing an antigenic determinant), followed by quantification of the resulting antigen-antibody complex, or by a technique not comprising the use of an antibody, for example by a method based on mass spectrometry techniques. The antibody can be monoclonal, polyclonal or fragments thereof, Fv, Fab, Fab' and F(ab')2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies. Similarly, the antibody can be labeled. Non-limiting illustrative examples of labels that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, co-factors or enzyme substrates, enzyme inhibitors, particles or dyes. According to the present application, a variety of known tests can be used, for example the combined use of an unlabelled antibody (primary antibody) and a labelled antibody (secondary antibody), Western blot or immunoblot, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), two-dimensional gel electrophoresis, capillary electrophoresis, immunocytochemistry and immunohistochemistry techniques, immunoturbidimetry, immunofluorescence, techniques based on the use of biochips or protein microarrays, including specific antibodies or analyses based on colloidal precipitation, such as in the form of reaction bands and analyses based on antibody-bound quantum dots. Other methods of detecting and quantifying proteins include, for example, affinity chromatography techniques or ligand binding assays.

[0121] In particular embodiments, the level of VEGF is determined by a multiplex immunoassay. In particular embodiments, the level of TGF-β1 or TGF-β2 is determined by an ELISA method.

[0122] 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. The growth factor induces vascular endothelial cell proliferation and migration and is essential for physiological and pathological angiogenesis. In humans, VEGF corresponds to the protein defined by accession number P15692 in the UniProtKB / Swiss-Prot database (entry version 248 dated 22 April 2020; sequence version 2 dated 16 November 2001).

[0123] The term "TGF-β1" or "transforming growth factor beta 1" refers to a protein belonging to the superfamily of cytokines transforming growth factor-betas. It is a secreted protein involved in functions such as control of cell growth, 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).

[0124] The term "TGF-β2" or "transforming growth factor beta 2" refers to a protein belonging to the superfamily of cytokines transforming growth factor-betas. It is a protein involved in functions such as 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).

[0125] In one embodiment, the monitoring method of the application comprises determining the level of VEGF in a vaginal mucus sample and / or a cervical mucus sample. In one embodiment, the monitoring method of the application comprises determining the level of TGF-β1 in a vaginal mucus sample and / or a cervical mucus sample. In one embodiment, the monitoring method of the application comprises determining the level of TGF-β2 in a vaginal mucus sample and / or a cervical mucus sample.

[0126] In one embodiment, the monitoring method of the application comprises determining the level of VEGF and TGF-β1 in a vaginal mucus sample and / or a cervical mucus sample. In one embodiment, the monitoring method of the application comprises determining the level of VEGF and TGF-β2 in a vaginal mucus sample and / or a cervical mucus sample. In one embodiment, the monitoring method of the application comprises determining the level of TGF-β1 and TGF-β2 in a vaginal mucus sample and / or a cervical mucus sample.

[0127] In one embodiment, the monitoring method of the application comprises determining the level of VEGF, TGF-β1 and TGF-β2 in a vaginal mucus sample and / or a cervical mucus sample.

[0128] The monitoring method of the application comprises comparing the level of VEGF, TGF-β1 and / or TGF-β2 to a reference value.

[0129] The levels of VEGF, TGF-β1 and / or TGF-β2 in the vaginal mucus sample and / or the cervical mucus sample of the female subject are determined after the treatment has been received, in particular after 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 of administration of Lactobacillus salivarius strain CECT 5713 or a mutant thereof.

[0130] As used herein, the term "reference value" refers to a predetermined criterion used as a reference to evaluate a value or data obtained from a sample collected from a subject. The reference value can 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, an average value, or a value compared to a particular control or baseline value. The reference value can be based on a value of a single sample, e.g., obtained from a sample of the subject being analyzed, e.g., at an earlier time. In a particular embodiment, the reference value is the level of the growth factor in a vaginal mucus sample and / or a cervical mucus sample prior to the female subject starting treatment with Lactobacillus salivarius strain CECT 5713 or a mutant thereof, e.g., 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, or the actual date of the start of said treatment.

[0131] According to the monitoring method of the present application, an increase in the level of any of VEGF, TGF-β1 and TGF-β2 relative to its reference value indicates that the treatment is effective, while a decrease or no change in said level relative to the reference value indicates that the treatment is not effective.

[0132] The term "increase in the level relative to the reference value" means that the level of the growth factor is higher than its reference value. The level of the growth factor is considered higher 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 than its reference value.

[0133] The term "decrease or no change in level with respect to a reference value" means that the growth factor level is lower than or equal to its reference value. A growth factor level is considered lower than its reference value when it is at least 1.5% lower, at least 1.0% lower, at least 0.5% lower or equal to the reference value, when it is lower than 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% of its reference value.

[0134] In a particular embodiment, the infertility is unexplained infertility.

[0135] In a particular embodiment, the recurrent miscarriage is unexplained recurrent miscarriage.

[0136] In a particular embodiment, the treatment with Lactobacillus salivarius strain CECT 5713 or a mutant thereof is administered in combination with assisted reproductive treatment.

[0137] In a particular embodiment, the strain is administered orally.

[0138] In a particular embodiment, the strain is administered for a period of at least 3 months, at least 6 months, or until pregnancy is diagnosed.

[0139] In a particular embodiment, if pregnancy is diagnosed, the administration of the strain is maintained at least until the 15th week of pregnancy.

[0140] In a particular embodiment, the strain is administered at a daily dose of 8.5 log 10 cfu to 9.5 log 10 cfu. In a more particular embodiment, the strain is administered at a daily dose of about 9 log 10 cfu.

[0141] **************

[0142] The present application is described below by means of purely illustrative and non-limiting examples, which do not limit the scope of the application.

[0143] Example

[0144] Materials and methods

[0145] Design of the study

[0146] A total of 58 women aged 28 to 45 years participated in this study. Volunteers were divided into three groups. All volunteers in the RM group (n=21) had a history of recurrent spontaneous abortion within the first 12 weeks of pregnancy, with three or more miscarriages. All women in the INF group (n=23) had a history of infertility (inability to conceive), despite having undergone at least three ART procedures, including at least one in vitro fertilization (IVF) cycle. Finally, the control group (n=14) consisted of women who had at least two children at term without complications. Throughout the study, neither the RM nor INF groups received ART. No women in the RM group were diagnosed with antiphospholipid syndrome and therefore did not receive heparin and / or salicylates during the study. All female participants did not receive or undergo hormone therapy, antibiotics, or probiotics for four weeks prior to sampling. Vaginal samples were collected at least seven days after intercourse to prevent or minimize the influence of partner semen on vaginal pH, microbiota, and immune status (especially in the latter case, related to TGF-β1 and TGF-β2 concentrations). Women who were lactose intolerant or allergic to milk protein were excluded because excipients were used to administer the strain in subsequent preliminary trials (see below).

[0147] During recruitment (day 0), two samples were collected from three groups of women within the first three days after ovulation: one vaginal swab sample was used to re-determine the Nugent score, and the other was obtained by cervical-vaginal irrigation (CVL) of the cervix and vaginal wall with 10 ml of sterile saline (Nakra et al., J Infect Dis. 2016, 213(5):840-7), used for all other analyses. Aliquots of the CVL samples were used for culture-based analyses. The CVL samples were then centrifuged at 800×g for 10 min at 4 °C to clarify them. The aliquots of the CVL supernatant and cell pallets were stored at -80 °C until immunological and metabolomics analysis was performed.

[0148] After day 0, women in the RM and INF groups (orally) took one sachet of approximately 50mg of freeze-dried probiotics (approximately 9 log) daily. 10 Lactobacillus salivarius CECT 5713 (CFU) was administered orally for 6 months or until pregnancy was diagnosed (whichever came first). Two identical samples were collected from each woman at this time. After pregnancy was diagnosed, the strain was administered orally until week 15 of gestation. This study recorded all spontaneous pregnancies occurring within the first year from day 0.

[0149] The sachets containing probiotics were kept at 4°C throughout the study. All volunteers received a questionnaire to record the intake of the product of the study. The minimum compliance (% of total treatment dose) was 86%. All volunteers gave written informed consent for the protocol, which was approved by the Bioethics Committee of Biomedical Research of the Ministry of Health and Family (Government of Andalusia, Granada, Spain) (P050 / 19, Law 11 / 19).

[0150] Determination of vaginal pH and Nugent score

[0151] The pH of the vaginal lateral wall was measured at two study visits (Whatman pH paper, pH 3.8-5.5 and pH 6.0-8.1). Nugent score was calculated as previously described (Nugent et al., J Clin Microbiol. 1991, 29(2):297-301). Briefly, the material of the swab was transferred to a glass slide, heat fixed, and Gram-stained. Quantitative Gram-positive, Gram-negative, and Gram-variable bacterial morphotype types were quantified. Nugent scores 0-3 were considered normal, 4-6 intermediate, and 7-10 consistent with bacterial vaginosis (Nugent et al., supra).

[0152] Culture-dependent analysis

[0153] The CVL samples collected during the assay were serially diluted and plated on Columbia Nalidixic Acid (CNA) agar, Gardnerella (GAR) agar, CHROMagar StrepB (CHR) agar, MacConkey (MCK) agar, Mycoplasma (MYC) agar and Sabouraud Dextrose Chloramphenicol (SDC) agar (BioMerieux, Marcy L’Etoile, France) for the selective isolation and quantification of the main cultivable non-lactobacilli and yeasts found in the vagina, including the most common pathogens of vaginal infections. Samples were also plated on MRS agar (Oxoid, Basingstoke, UK) plates 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, except MRS-C and MRS-B, were incubated for 48 hours at 37°C in aerobic conditions, while MRS-C and MRS-B were incubated in anaerobic conditions (85% nitrogen, 10% hydrogen, 5% carbon dioxide) in an anaerobic workstation (DW Scientific, Shipley, UK). After incubation, counts were recorded in each growth medium and then at least one representative cell of each colony morphology was selected from the agar plates. Isolates were identified by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker, Germany). When identification at the species level was not possible by MALDI-TOF (especially in the case of lactobacilli isolates), identification was performed by sequencing of the 16S ribosomal RNA (rRNA) gene as described by Mediano et al. (J Hum Lact. 2017, 33(2): 309-318).

[0154] DNA extraction from samples

[0155] Approximately 1 ml of each CVL sample was used for DNA extraction following the method previously described (Lackey et al., Front Nutr. 2019, 17; 6:45). Extracted DNA was eluted in 22 μΐ of nuclease-free water and stored at -20°C until subsequent analysis. The purity and concentration of each extracted DNA were estimated using a NanoDrop 1000 spectrophotometer (NanoDrot Technologies Inc., Rockland, USA). A negative control (no sample) was added during the extraction process.

[0156] Detection and quantification of Lactobacillus salivarius DNA by qPCR (quantitative real-time PCR)

[0157] Primers and conditions for quantification of DNA specific for the species Lactobacillus salivarius have been previously described (Harrow et al., Appl Environ Microbiol. 2007, 73(22):7123-7). DNA concentration of all samples was adjusted to 5 ng pL -1 All experiments were performed using a commercially available real-time PCR thermocycler (CFX96 TM , Biorad Laboratories, Hercules, CA, USA). A dilution series of 1 : 10 (from 2 ng to 0.2 pg) of Lactobacillus salivarius CECT 5713 DNA was used as a standard curve to calculate unknown bacterial genomic targets. Ct values between 15.29 and 20.07 (R2= 0.9915) were obtained for this range of Lactobacillus salivarius DNA. Ct values of DNA extracted from non-target species (Lactobacillus reuteri MP07 and Lactobacillus plantarum MP02; collected by the Applicant) were > 39.27 ± 0.64. These two control strains were chosen because they are taxonomically the closest species to Lactobacillus salivarius (Salvetti et al., 2018). All samples and standards were processed in triplicate.

[0158] Metabolomic analysis

[0159] The variable region V3-V4 of 16S rDNA was amplified by PCR using the universal primers SD-Bact-0341-bS-17 (CCTACGGGNGGCWGCAG, SEQ ID NO: 1) and SD-Bact-129 0785-aA-21 (GACTACHVGGGTATCTAATCC, SEQ ID NO: 2) and sequenced in an Illumina MiSeq system at the Madrid Science Park facilities (Tres Cantos, Spain) (Klindworth et al., Nucleic Acids Res. 2013, 41, el). In a second PCR reaction, barcodes added to the 3' and 5' ends of the PCR amplicons allowed the separation of forward and reverse sequences. DNA concentration in the PCR products was quantified by the 2100 Bioanalyzer System (Agilent, Santa Clara, CA, USA). After grouping the PCR products in approximately the same molar proportions, DNA amplicons on the removed bands, which had the correct size after running on an agarose gel, were purified using the QIAEX II Gel Extraction Kit (Qiagen). DNA concentration was then quantified with PicoGreen (BMG Labtech, Jena, Germany). Sequencing of the grouped and purified DNA amplicons containing barcodes was performed using the Illumina MiSeq paired-end protocol (Illuminia Inc., San Diego, CA, USA) according to the manufacturer's protocol.

[0160] Immunological parameters analysis

[0161] Vascular endothelial growth factor (VEGF) was analyzed using a pre-mixed multiplex panel (Bio-Rad, San Diego, CA). Human VEGF-A, VEGF-B, and VEGF-C levels were measured in parallel by ELISA using the Human VEGF-A, VEGF-B, and VEGF-C ELISA kits (RayBiotech, Norcross, GA, USA), respectively. All assays were performed following the manufacturer's protocol and standard curves were plotted for each analyte.

[0162] Antimicrobial activity of Lactobacillus salivarius CECT 5713 against vaginal pathogens Production of antimicrobial compounds by Lactobacillus salivarius CECT 5713

[0163] ​The ability of L. salivarius CECT 5713 to inhibit the growth of various bacteria and yeasts was determined using the checkerboard method (Magnusson and Schnürer, Appl. Environ. Microbiol. 2001, 67, 1-5). The method was performed as described by Martín et al. (2006). All the strains used as indicator organisms (cultures collected by the Applicant) had been previously isolated from clinical cases of vaginal or cervical infections, including five strains of G. vaginalis, three strains of S. agalactiae, three strains of C. albicans, two strains of C. glabrata, two strains of C. parapsilosis and two strains of U. urealyticum. Inhibition activity was tested in triplicate in all experiments.

[0164] Co-aggregation and vaginal epithelial cell line adhesion assays

[0165] The production of hydrogen peroxide by the lactobacilli strains was measured using the quantitative method modified by Martín et al. (Int. J. Food Microbiol. 2006, 112:35-43) using Yap and Gilliland (2000, J. Dairy Sci. 83, 628-632). The production of lactic acid (L and D isomers) and acetic acid by L. salivarius CECT 5713 was determined using an enzymatic kit (Roche Diagnostics, Mannheim, Germany) as described by Martín et al. (2006). The pH of the culture supernatant was also measured. These determinations were performed in triplicate under strict aerobic and anaerobic conditions and the values are expressed as mean ± SD. Bacteriocin activity was not analyzed because previous phenotypic analysis and analysis of the genome of L. salivarius CECT 5713 had already shown that this strain is not able to produce such antimicrobial compounds (Martín et al., supra; Langa et al., Appl Microbiol Biotechnol. 2012, 94(5): 1279-87).

[0166] Alpha-amylase activity of Lactobacillus salivarius CECT 5713

[0167] The ability of the strains to form aggregates with the cells of the above indicated strains was studied following the method of Younes et al. (PLoS One 2012, 7, e36917). Suspensions were observed under phase contrast microscopy after Gram staining. Adhesion was performed on vaginal epithelial cells collected from healthy premenopausal women and interpreted as described above (Boris et al., Infect. Immun. 1998, 66, 1985-1989). Adhesion was the number of lactobacilli adhered to vaginal cells in 20 random microscopic fields. Lactobacillus salivarius CECT 9145 was used as control strain due to its high adhesion to vaginal cells (Martin et al., Nutrients. 2019, 11(4). pii: E810). Assays were performed in triplicate.

[0168] Bioinformatic analysis Statistical analysis

[0169] On the other hand, alpha-amylase activity of Lactobacillus salivarius CECT 5713 was qualitatively evaluated using the method described by Padmavathi et al. (J Genet Eng Biotechnol. 2018; 16(2): 357-362). Briefly, strains were inoculated in modified MRS medium (0.5% peptone, 0.7% yeast extract, 0.2% NaCl, 2% starch and 1.5% agar) added with 0.25% starch. Plates were incubated at 37°C in anaerobic bacteria for 48 hours and then clearing zones were observed by adding Gram's iodine as detection agent. Next, alpha-amylase activity on the surface of Lactobacillus salivarius CECT 5713 was also measured using an alpha-amylase assay kit (Kikkoman Co., Tokyo, Japan) using 2-chloro-4-nitrophenyl 6 5 -azido-6 5 -deoxy-beta-maltoside, using the protocol and conditions previously described (Narita et al., 2006). One unit of activity was defined as the amount of enzyme required to release 1 pmol of 2-chloro-4-nitrophenol per minute at 37°C. 5 -azido-6 5 -deoxy-beta-maltoside, using the protocol and conditions previously described (Narita et al., 2006). One unit of activity was defined as the amount of enzyme required to release 1 pmol of 2-chloro-4-nitrophenol per minute at 37°C.

[0170] Demographic data and clinical characteristics of participants

[0171] Raw, unprocessed data was parsed and quality filtered using Illumina MiSeq Reporter analysis software. Microbiome bioinformatics analysis was performed using QIME 2 2019.1 (Bolyen et al., Nature Biotechnology, 2019, 37: 852-857). Denoising 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 Sklearn naive Bayes classifier with SILVA 132 OTU reference sequences (Quast, Nucl. Acids Res. 2013, 41 (D1): D590-D596). Subsequent bioinformatics analysis was performed using version R 3.5.1 (R Core Team, 2013; https: / / www.R-project.org). OTU count tables were generated for each sample and abundances of bacterial taxonomic units were normalized by the total number of sequences in each sample. Alpha and diversity were investigated using Shannon and Simpson diversity indices using the R Vegan package (version: 2.5.6) (Oksanen et al., Community Ecology package, 2007, 10; 631-637). Beta diversity investigation was performed using Principal Coordinate Analysis (PCoA) by plotting bacterial community diversity patterns through distance matrices comparing each pair of samples by distance matrices containing different values. Quantitative (relative abundance) and qualitative (presence / absence) analyses were performed using Bray-Curtis index and Jaccard binary index, respectively. Variance of distance matrices was subjected to 999 permutations analysis using Adonis “non-parametric MANOVA test” to reveal statistical significance of RVegan package.

[0172] Baseline vaginal health parameters

[0173] Microbial data were recorded as CFU / ml and converted to log values prior to statistical analysis. Quantitative data are expressed as median and interquartile range (IQR). Differences between groups were assessed by Kruskal-Wallis test and paired Wilcoxon rank-sum test to calculate comparisons between groups. Bonferroni correction test was performed. Correlations between the 20 relatively abundant genera were visualized using the R "qgraph" package (Epskamp 2012). Statistical analysis and plotting were performed in the R environment using the ggplot2 library (Wickham, 2016). Differences with p-value < 0.05 were considered statistically significant. The contribution of a variable was (in percentage form): (cos2 variable*100) / (total cos2 of the component). Cos2 = quality of representation or weight of the variable in the PCA representation.

[0174] Results

[0175] Figure 1

[0176] The characteristics of the 58 women participating in this study are presented in Table 1. The mean age (CI 95%) of the control women was 34.6 (33.4-35.8) years, while the mean age of the women with recurrent miscarriage (RM) and unexplained infertility (INF) was 39.4 (38.5-40.4) years and 38.0 (37.1-38.9) years, respectively (Table 1). The women of the control group were about 4-5 years younger than the other participants (p<0.001; one-way ANOVA), but there were no differences in the mean values of weight and height between the three groups of women (Table 1).

[0177]

[0178]

[0179] Table 1. Characteristics of the participants (n=58) divided into three groups: control group (women with at least two children after full-term pregnancy and without complications), RM group (women with a history of recurrent miscarriage) and INF group (women with a history of infertility).

[0180] SD: standard deviation; CI: confidence interval.

[0181] One-way ANOVA test for assessing differences in mean age, mean weight, mean height and mean duration of menstrual cycle between groups.

[0182] * Freeman Halton extension of the Fisher's exact probability test for 2 x 3 contingency tables.

[0183] Approximately 71% of the women in the control group had regular menstrual cycles, while in the other two groups (RM and INF) this proportion was 48%, although this difference was not statistically significant (p=0.337; Fisher's exact probability test of the Freeman Halton extension of the 2x3 contingency table). The average menstrual cycle of the women in the control group, RM group and INF group was 28, 27.4 and 27.5 days, respectively, and no differences were observed (Table 1).

[0184] Figure 2

[0185] The average vaginal pH value (CI 95%) of the women in the control group was 4.53 (4.38-4.68) (Table 2). In contrast, the vaginal pH value was higher in the two groups studied: 5.67 (5.55-5.79) in the RM group and 5.96 (5.84-6.07) in the INF group (p=0.000; one-way ANOVA). The average Nugent score of the women in the RM group and INF group was 5.95 (5.54-6.37) and 6.30 (5.91-6.70), respectively, with greater significance (CI 95%) compared to the control group (p=0.000; one-way ANOVA) (Table 2). There were also differences in the levels of the cytokines TGFβ1, TFGβ2 and VEGF in the CVL. The content of these three cytokines in the CLV of the 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 (TGFβ1, TFGβ2 and VEGF), respectively, while in the two groups studied (RM and INF) it was reduced to approximately half (Table 2).

[0186]

[0187]

[0188] Table 2. Comparison of the baseline vaginal parameters (pH value, Nugent score, cytokines and microbiology) of the participants (n=58) in the control group (women with at least two children after a full-term pregnancy and without complications), the RM group (women with a history of recurrent miscarriages) and the INF group (women with a history of infertility).

[0189] SD: standard deviation; CI: confidence interval, TGFβ1, transforming growth factor beta 1; TGFβ2, transforming growth factor beta 2; VEGF, vascular endothelial growth factor.

[0190] #One-way ANOVA was used to assess differences in means between groups. Different letters in bold in the same row indicate statistically significant differences between groups.

[0191] Freeman-Halton extension of Fisher's exact probability test for 2×3 contingency tables.

[0192] **The average number of women from whom lactobacilli can be isolated from the sample.

[0193] In the control group (n=14), lactobacilli were detected in the CVL of all women. Using culture-dependent assessment, the mean lactobacillus count (CI 95%) was 7.24 log cfu / ml (range 6.89–7.60 log cfu / ml). 10 The detection frequency of Lactobacillus in CVL samples from Lactobacillus-positive women was lower in the RM 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, the concentration of Lactobacillus in CVL samples from Lactobacillus-positive women was also 2.20 and 1.46 log lower, respectively, in the RM and INF groups. 10 Units. Another difference between these groups relates to the distribution of lactobacilli ( Figure 3 Up to six different lactobacilli were identified in CVL samples from women in the control group, including *Lactobacillus crispatus* (the dominant species), *Lactobacillus jensenii*, *Lactobacillus gasseri*, *Lactobacillus fermentum*, *Lactobacillus salivarius*, and *Lactobacillus vaginalis*. The lactobacilli-specific distribution in the study groups (i.e., the RM and INF groups) was narrower than in the control group; *Lactobacillus fermentum*, *Lactobacillus salivarius*, and *Lactobacillus vaginalis* were not found in any samples from either the RM or INF groups. Interestingly, *Lactobacillus inertes* was not isolated from any CVL samples from the control group, while it was isolated from approximately one-third of the samples in the RM and INF groups (5 out of a total of 18 lactobacilli-positive samples). The level of *Lactobacillus salivarius* in vaginal swabs was also determined by species-specific qPCR. Using this technique, this bacterium was detected in a sample from only one woman in the control group (7.29 log...). 10 (Copy / swab). This woman was the only one identified as positive for *Lactobacillus salivarius* (concentration 7.3 log) via culture. 10 A female (CFU / swab) was isolated from one of the women in this group. The *Lactobacillus salivarius* strain isolated from this female was genetically different from *Lactobacillus salivarius* CECT 5713 (results not shown).

[0194] 16S rRNA gene sequencing analysis of CVL samples (n=58) resulted in 4,363,364 high-quality filtered sequences, from 33,160 to 139,044 per sample (median [IQR]=73,383 [66,587-82,821]). A total of 23 phyla and 453 genera were identified. Alpha diversity analysis (calculated by Shannon index or Simpson index) showed statistically significant differences in the vaginal microbiota of control women of reproductive age and women with unexplained infertility (p<0.001) Figure 3 ) Beta diversity analysis calculated from the relative abundance of OTUs (Bray-Curtis distance) and the presence / absence of OTUs (Jaccard binary distance matrix) showed that the vaginal bacteria of the 3 groups were clustered (p=0.004 and p=0.002, respectively) Figure 4 ) In addition, according to the relative abundance of OTUs (Bray-Curtis distance), the samples of the fertile control group clustered more closely (shorter distance from the centroid) compared to the RM and INF groups, indicating a high degree of consistency in the bacterial distribution in the control CVL samples Figure 6

[0195] PCA analysis was performed to preliminarily assess potential dominant patterns in the bacterial distribution of the vaginal samples Figure 5 ) The first two PCA dimensions explained 84.9% of the total variation in the samples, the first dimension (principal component) represented 74.7% of the total variation and the second dimension represented 10.2% of the total variation. Overall, this analysis showed that the similarity between the samples of the fertile control group and women with a history of recurrent miscarriage was greater than the similarity between the fertile control group and women with unexplained infertility.

[0196] Comparison of the relative abundance of OTUs (in % of total abundance) at the phylum level in the three groups of CVL samples showed statistically significant differences compared to the 4 dominant phyla (Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes) (Table 3). Firmicutes was the most abundant and most frequent (present in all samples) phylum. The relative abundance of Firmicutes [median (IQR)=95.01% (91.48-97.48)] in the samples provided by the fertile control group was statistically significantly higher than in the samples provided by the women with unexplained infertility [median (IQR)=89.48% (81.48-94.48)] (p=0.001) and in the samples provided by the women with a history of recurrent miscarriage [median (IQR)=89.48% (81.48-94.48)] (p=0.001).

[0197] ​The median (IQR) of relative abundance of Firmicutes in female samples of the RM group and of the INF group [93.49% (67.18%-97.53%) and 71.95% (0.76%-94.09%), respectively] was lower than in the fertile control group [97.88% (96.92%-99.31%)] (p=0.001 ; Kruskal-Wallis rank test) (Table 3). Other genera were present in different numbers of samples, ranging from 96% (Staphylococcus genus in the INF group) to 7% (Escherichia / Shigella genus in the control group), but the median of relative abundance of these genera was less than 1%. At the genus level, the bacterial profile in some individual samples of the RM group and of the INF group females was not different from the microbial profile in the fertile control group females, the two samples being very similar. However, an abnormal profile with a decrease or even an absence of Lactobacillus content was recorded in some samples of the RM group and of the INF group females (data not shown).

[0198]

[0199]

[0200] Table 3. Relative frequency, median and interquartile range (IQR) of the detected phyla (highlighted in grey) and most abundant bacterial genera in vaginal samples of the control group (women with at least two children after full-term pregnancy and without complications), of the RM group (women with a history of recurrent miscarriage), of the INF group (women with a history of infertility). a n (%): number of phyla / genera detected in the sample (relative detection frequency). Mann-Whitney test with Bonferroni correction b Kruskal-Wallis rank test.

[0201] At the genus level, the only bacterial genus detected in all samples was Lactobacillus, but its relative abundance was significantly different in the three groups of samples (Table 3). The median (IQR) of relative abundance of Lactobacillus in female samples of the RM group and of the INF group [93.49% (67.18%-97.53%) and 71.95% (0.76%-94.09%), respectively] was lower than the median of relative content of Lactobacillus in the fertile control female samples (97.88% (96.92%-99.31%)] (p=0.001 ; Kruskal-Wallis rank test) (Table 3). Other genera were present in different numbers of samples, ranging from 96% (Staphylococcus genus in the INF group) to 7% (Escherichia / Shigella genus in the control group), but the median of relative abundance of these genera was less than 1%. At the genus level, the bacterial profile in some individual samples of the RM group and of the INF group females was not different from the microbial profile in the fertile control group females, the two samples being very similar. However, an abnormal profile with a decrease or even an absence of Lactobacillus content was recorded in some samples of the RM group and of the INF group females (data not shown).

[0202] In general, the comparison between the RM and INF groups at the beginning of the study revealed statistically significant differences between the two groups Main parameters of the clinical study: pregnancy and term pregnancy effect (reproduction / pregnancy outcome) ). The vaginal pH of the women in the INF group was higher than in the RM group, but the opposite was true for TGFβ1 and VEGF. No differences were observed in other characteristics such as age, weight, height, Nugent score, TGFβ2 and live lactobacilli count Secondary response parameters related to probiotic treatment: changes in vaginal parameters of RM group women

[0203] Figure 6

[0204] In general, the administration of the probiotic to the women in the RM and INF groups with the strain described made 29 of the 44 female participants pregnant. This means that the pregnancy efficiency was 66%, with a CI 95% of 51-78% (p = 0.017) (Table 4). Of these pregnancies, 25 of the 44 female participants were successful and 4 were miscarriages. This means that the reproductive success rate was 56%, with a CI 95% of 42-70% (p = 0.183) (Table 4). Interestingly, all the successful pregnancies resulted in term singleton pregnancies (gestational age > 38 weeks). The gestational age, birth weight and height values and the sex of the babies are shown in Table 1.

[0205]

[0206]

[0207] Table 4. Main results after probiotic treatment.

[0208] # Pearson's chi-squared test.

[0209] * 2 women miscarried in each group.

[0210] The women in the RM group had the highest reproductive success rate, since 17 of the 21 participants were pregnant (15 term pregnancies and 2 miscarriages) during the study. The rate was lower but still noteworthy in the INF group: 12 of the 23 participants were pregnant (10 term pregnancies and 2 miscarriages). The comparison of the pregnancy efficiency and the rate of successful pregnancies (CI 95%) of the women in the RM and INF groups [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 the women in these groups had previously received ART intervention, which had not been successful, in an attempt to prevent spontaneous miscarriage (RM group) or pregnancy (INF group).

[0211] Figure 7

[0212] There were no differences in age, weight or height between the women who successfully conceived (n = 15) and those who did not (n = 6) in the RM group.​

[0213] However, different changes were observed in this group of women (RM group) after probiotic treatment, depending on the final pregnancy outcome (Table 5). In those women who were able to successfully conceive, if they got pregnant, the mean (SD) of vaginal pH was 1.13 (0.38) units lower than in those who did not get pregnant. Similar changes were observed in Nugent score, recording a difference of 3.33 (1.11) units between successful and failed pregnancies (p=0.000, one-way repeated measures ANOVA) (Table 5, Figure 7 ).

[0214]

[0215]

[0216]

[0217] Table 5. Comparison of the characteristics and changes in vaginal parameters between women who were able to complete a term pregnancy (n=15) and those who did not (n=6) after the intervention with Lactobacillus salivarius CECT 5713 in the RM group (women with a history of recurrent spontaneous abortions (n=21).

[0218] # Differences in the mean values between groups were assessed using one-way ANOVA test, except for the presence of lactobacilli. * Fisher's exact probability test for 2x2 contingency tables. One-way ANOVA test was used to determine whether there were changes in each group of participants.

[0219] Changes in the concentration of vaginal cytokines were also different between the two groups after probiotic treatment. There were no changes in the vaginal concentration of TGFβ1, TGFβ2 and VEGF in non-pregnant women, but the mean (SD) concentration in pregnant women was 1.40 (0.62) pg / mL, 1.25 (0.34) pg / mL and 402 (252) pg / mL, respectively (Table 5, Figure 6 ). In addition, it should be taken into account that there were already differences in the concentration of these cytokines between pregnant and non-pregnant women even before starting the treatment (Table 5, Figure 8 ).

[0220] In addition, probiotic treatment caused an average (SD) increase of 3.52 (2.99) log 10 cfu / ml in the count of lactobacilli in women who finally got pregnant, but not in those who did not (Table 5, Secondary response parameters related to probiotic treatment: changes in vaginal parameters of INF group women). In all pregnant women, the presence of L. salivarius was confirmed by qPCR [mean (SD) = 6.85 (0.61) copies / swab] but only in 50% of the women with pregnancy loss and at lower concentrations [mean (SD) = 2.63 (1.17) copies / swab] (Table 5). The distribution of lactobacilli in CVL samples obtained at the beginning of probiotic treatment and after 6 months or until diagnosis of pregnancy is shown in Figure 9 . The most significant difference was the presence of live L. salivarius in the majority of women (17 / 21) after probiotic treatment. Moreover, at the beginning of the study, the presence of L. iners in 3 women, at the end of the treatment, was isolated, in particular, only in 2 women who did not have a pregnancy termination.

[0221] The metabolic profile of the CVL samples of the RM group women, at genus level, did not differ in comparison with the pregnancy outcome (Table 6).

[0222]

[0223] Table 6. Relative frequency, median and interquartile range (IQR) of bacterial phyla (highlighted in grey) and most abundant bacterial genera detected in vaginal samples of RM group women (women with a history of recurrent miscarriage) pregnant or not after probiotic treatment.

[0224] an (%): number of samples in which the phylum / genus was detected (relative detection frequency).

[0225] With Bonferroni correction b Kruskal-Wallis test for ordinal data.

[0226] Figure 10

[0227] Women pregnant and not pregnant in the INF group after probiotic treatment did not differ in age, weight and height (results not shown).

[0228] CVL pH values and Nugent scores of all women in the INF group significantly decreased after probiotic treatment (p < 0.05; one-way repeated measures ANOVA; Figure 9 ), but the magnitude of the change was smaller in women not pregnant compared to women pregnant (Table 7, Figure 9 ). Specifically, the mean decrease (SD) in CVL pH values and Nugent scores was 1.32 (0.31) and 3.90 (0.74), respectively, in women pregnant and 0.19 (0.18) and 0.54 (0.78), respectively, in women not pregnant (Table 7, Figure 9 ).

[0229]

[0230]

[0231] Table 7. Differences in characteristics and changes in vaginal parameters between women who completed a term pregnancy (n=10) and those who did not (n=13) in the INF group (women with a history of unexplained infertility, n=23) after receiving L. salivarius CECT5713 intervention.

[0232] One-way ANOVA test was used to assess differences in means between groups. Fisher's exact test for 2x2 contingency tables. **One-way ANOVA; Kruskal Wallis, p=0.007.

[0233] Changes in vaginal cytokine concentrations after probiotic treatment were similar to those described in the RM group: vaginal concentrations of TGFpi, TGFp2 and VEGF did not change in non-pregnant women, but they significantly increased in pregnant women by an average of (SD) 2.29 (0.34) pg / mL, 1.25 (0.24) pg / mL and 462 (252) pg / mL, respectively (Table 7, Figure 9 ) In this group of women, even before starting treatment, concentrations of TGFp2 and VEGF were already different between pregnant and non-pregnant women, but not TGFpi (Table 7, Figure 10

[0234] Probiotic treatment resulted in higher numbers of lactobacilli detected in CVL samples (in all women who eventually became pregnant) and in average bacterial counts (SD) [(6.46 (0.51) log 10 cfu / ml)] than in non-pregnant women (lactobacilli were detected in only 46% of them, with an average bacterial count (SD) of 4.95 (1.66) log 10 cfu / ml) (Table 7, Figure 11 and Comparison of vaginal parameters in all pregnant and non-pregnant women in the RM and INF groups ) Similarly, the presence of L. salivarius was confirmed by qPCR in all pregnant women [average (SD) = 6.85 (0.61) copies / swab], but only in 25% of the unsuccessfully pregnant women, and at lower concentrations [average (SD) = 3.55 (0.39) copies / swab] (Table 7). The distribution of lactobacilli in CVL samples obtained at the start of probiotic treatment and 6 months after starting or until diagnosis of pregnancy is shown in Figure 12 , which indicates that live L. salivarius were detected in all pregnant women, but only in 4 of the 13 non-pregnant women after probiotic treatment.

[0235] ​The genus-level metabolomics distribution of CVL samples from women in the RM group did not differ from the pregnancy outcomes (results not shown).

[0236] Figure 14

[0237] The mean pH of CVL samples from pregnant women [5.69 (0.48) units] was slightly lower than that of non-pregnant women [5.99 (0.33) units] (p = 0.024; one-way ANOVA) (Table 7). Differences in vaginal cytokine concentrations at the start of the study also existed based on the final pregnancy outcome, but these differences were similar to those already described independently in the RM and INF groups (Table 8). Figure 14 and 13 The only parameters that initially showed no difference between the two groups were Nugent score, detection frequency, and lactobacillus count (Table 8). Overall, lactobacillus was detected in all pregnant women, but only in half of the non-pregnant women (p<0.001; chi-square test). Furthermore, in the pregnant women group, the lactobacillus count increased by 1.6 log units after probiotic treatment, but there was no change in the non-pregnant women (p<0.001; chi-square test). Comparison of vaginal parameters in control group women, all pregnant and non-pregnant women Significant differences were also observed in the detection of *Lactobacillus salivarius* in CVL samples. High concentrations of *Lactobacillus salivarius* were detected in all women with successful pregnancies compared to those with unsuccessful pregnancies [mean (SD) = 6.70 (0.56) copies / swab]. Figure 15 ).

[0238]

[0239]

[0240]

[0241] Table 8. Characteristic differences and comparisons of vaginal parameter changes in women who completed full-term pregnancies (n=25) and those who did not (n=19) after intervention with Lactobacillus salivarius CECT 5713, including all women in the RM and INF groups (n=44).

[0242] #One-way ANOVA was used to assess the differences in means between groups. *Chi-square (Pearson) test for 2×2 contingency tables.

[0243] Fisher's exact probability test for 2×2 contingency tables.

[0244] **Differences in the mean of positive samples.

[0245] Antimicrobial activity of Lactobacillus salivarius CECT 5713 against vaginal and cervical pathogens

[0246] The analysis of the vaginal parameters (pH, Nugent score, TGFβ1, TGFβ2, VEGF, Lactobacilli count) revealed statistically significant differences among the three groups (control, RM and INF groups, in women who experienced a full-term pregnancy, and in women who did not get pregnant or who had a miscarriage, in the RM and INF groups) in all the above parameters (Table 9; Co-aggregation of Lactobacillus salivarius CECT 5713 with vaginal pathogens and adhesion to vaginal cells ).

[0247]

[0248]

[0249]

[0250] Table 9. Comparison of vaginal parameters (pH, Nugent score, cytokines and Lactobacilli count) between participants in the control group (n=14) and in the RM and INF groups (total) who got pregnant (n=25) or did not get pregnant (n=19) after probiotic treatment.

[0251] SD: standard deviation; CI: confidence interval, TGFβ1, transforming growth factor beta 1; TGFβ2, transforming growth factor beta 2; VEGF, vascular endothelial growth factor.

[0252] # One-way ANOVA test was used to assess the differences in the means between the groups. Different letters in bold in the same row indicate statistically significant differences between the groups.

[0253] * Freeman Halton extension of the Fisher's exact probability test for 2 x 3 contingency tables.

[0254] ** Mean value of women from whom Lactobacilli could be isolated from the sample.

[0255] Alpha-amylase activity of Lactobacillus salivarius CECT 5713 ​

[0256] Lactobacillus salivarius CECT 5713 showed antimicrobial inhibitory activity (zone of inhibition > 2 mm) against all the indicated organisms used in this study under aerobic and anaerobic conditions. To explain the compound(s) responsible for the antimicrobial activity, this non-bacterial strain was selected for the production of hydrogen peroxide, lactic acid and / or acetate. Quantitative analysis showed that under aerobic conditions, the average amount (± SD) of hydrogen peroxide produced by the strain was 0.729 μg / ml (± 0.122). When the assay was performed under anaerobic conditions, no production of this compound was observed. The average concentration (± SD) of L-lactic acid in the supernatant of the strain obtained from MRS cultures (initial pH = 6.2) was 11.07 ± 0.42 mg / ml and 11.69 ± 0.58 mg / ml under aerobic and anaerobic conditions, respectively, while the average pH in the supernatant was 3.91 and 3.86 under aerobic and anaerobic conditions, respectively. No D-lactic acid was detected in the culture supernatant. The average concentration (± SD) of acetate in the same supernatant was 0.70 mg / ml (± 0.11) and 0.46 mg / ml (± 0.14) under aerobic and anaerobic conditions, respectively.

[0257]

[0258]

[0259] Table 10. Antimicrobial activity and co-aggregation of Lactobacillus salivarius CECT 5713 against various cervical-vaginal pathogens.

[0260] a Co-aggregation was defined as visible bacterial clusters and was classified according to the criteria of Younes et al. (2012) as (++): large and dense clusters; (+): small and sparse clusters; (-): no visible clusters. Reference: Younes, J. A., van der Mei, H. C, van den Heuvel, E., Busscher, H. J., and Reid, G. (2012). Adhesion forces and co-aggregation between vaginal staphylococci and lactobacilli. PLoS One, 7, e36917. doi: 10.1371 / journal.pone.0036917.

[0261] ​

[0262] The strain was able to form large and clear co-aggregates with all the selected vaginal and cervical pathogens. Co-aggregation with G. vaginalis, S. agalactiae and C. albicans strains was particularly evident. In this study, the tested strain adhered strongly to vaginal epithelial cells, with an average of 329 ± 46 adhered lactobacilli in 20 random microscopic fields. The average (± SD) for L. salivarius CECT 9145, a control strain that adheres highly to vaginal cells, was 336 ± 52 adhered lactobacilli.

[0263] ​ ​

[0264] When treated with iodine solution, the extracellular amylase produced by L. salivarius CECT 5713 was observed by a clearing zone around the colonies (about 2.0 mm). Subsequently, when the a-amylase activity was determined, this strain showed a high level of a-amylase activity after 16 hours (0.83 U / ml) (concentration of L. salivarius CECT 5713: about 8.6 log cfu / ml), and similar levels of this strain were detected in the supernatant within 48 hours (when the determination ended). 10 SEQUENCE LISTING <110> BIOEXPLORED, S.L. <120> Probiotic for the treatment of infertility and recurrent miscarriage <130> P19424PC00 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Universal primer SD-Bact-0341-bS-17 <220> <221> misc_feature <222> (9)..(9) <223> n is a, c, g or t <400> 1 cctacgggng gcwgcag 17 <210> 2 <211> 21 <212> ADN <213> Artificial Sequence ​<220> <223> Universal primer SD-Bact-129 0785-aA-21 <400> 2 gactachvgg gtatctaatc c 21

Claims

1. The use of a strain of *Lactobacillus salivarius* with accession number 5713, deposited at the Spanish Center for Type Culture Collection (CECT), for the preparation of a medicament for the treatment and / or prevention of infertility or recurrent miscarriage in female subjects; wherein infertility refers to infertility of unknown cause; and recurrent miscarriage refers to recurrent miscarriage of unknown cause.

2. The use according to claim 1, wherein the strain is combined with assisted reproductive treatment and administered to women suffering from infertility or recurrent miscarriage.

3. The use according to claim 1, wherein the strain is administered orally.

4. The use according to claim 1, wherein the strain is applied for at least 3 months, or until pregnancy is diagnosed.

5. The use according to claim 4, wherein the application time of said strain is at least 6 months.

6. The use according to claim 4, wherein if pregnancy is diagnosed, the administration of the strain continues for at least until the 15th week of pregnancy.

7. The use according to any one of claims 1 to 6, wherein the strain is at 8.5 log... 10 CFU to 9.5 log 10 Daily dose of CFU.

8. The use according to claim 7, wherein the strain is in a concentration of 9 log... 10 Daily dose of CFU.

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