WO2021176387A1 - Nouvelle utilisation de probiotiques - Google Patents

Nouvelle utilisation de probiotiques Download PDF

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Publication number
WO2021176387A1
WO2021176387A1 PCT/IB2021/051800 IB2021051800W WO2021176387A1 WO 2021176387 A1 WO2021176387 A1 WO 2021176387A1 IB 2021051800 W IB2021051800 W IB 2021051800W WO 2021176387 A1 WO2021176387 A1 WO 2021176387A1
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composition
bacteria
use according
lactobacillus
microbiota
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PCT/IB2021/051800
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English (en)
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Antonio La Marca
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Antonio La Marca
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a composition comprising bacteria for use in the treatment or prevention of a pathology caused by or associated with an imbalance in the microbiota of the female reproductive system.
  • the endometrium i.e. the tissue lining the innermost layer of the uterine cavity, undergoes remodelling under the stimulus of hormonal fluctuations typical of the menstrual cycle and is subject to profound modifications regarding both the number of cells making it up and the structure and function thereof.
  • endometrial stromal cells which undergo a phenomenon known as decidualization, i.e. a process of cell differentiation in which the ESC are transformed from proliferating fibroblasts into specialized cells with secretory activity.
  • Decidualized ESC in fact produce factors capable of increasing endometrial receptivity both directly, by inducing genes coding for growth factors and adhesion proteins, and indirectly, by regulating the activity of different types of cells of the immune and vascular systems.
  • reproductive medicine Since implantation takes place in the endometrial cavity and not in the vagina, reproductive medicine focuses its interest on the endometrial microbiota and its impact in obtaining and maintaining a pregnancy.
  • Uterine infections are a known risk factor for infertility, since a pathogenic environment that provokes inflammation and activation of the immune system at the endometrial level can end up compromising any implantation of an embryo and hence the start of a pregnancy.
  • a first aspect of the present invention relates to a composition comprising bacteria, preferably probiotic or paraprobiotic bacteria, for use in the treatment or prevention of a pathological condition of the female reproductive system caused by or associated with an imbalance in the microbiota of the female reproductive system and/or an insufficient uterine decidualization.
  • a second aspect of the present invention relates to the use of a composition comprising bacteria, preferably probiotic or paraprobiotic bacteria, to increase the levels or proliferation of commensal bacteria, and/or to reduce the levels or reduce the proliferation of pathobiontic microorganisms.
  • said use comprises at least one step of administering an effective amount of a composition comprising bacteria, preferably probiotic or paraprobiotic bacteria, to an individual who has a need therefor.
  • a third aspect of the present invention relates to a kit comprising a composition based on bacteria, preferably probiotic or paraprobiotic bacteria, and a device for an intrauterine or intratubal administration of the composition.
  • a fourth aspect of the present invention relates to a method for the treatment or prevention of a pathological condition of the female reproductive system caused by or associated with an imbalance in the microbiota of the female reproductive system and/or an insufficient decidualization.
  • Said method comprises at least one step of administering an effective amount of a composition comprising bacteria, preferably probiotic or paraprobiotic bacteria, as described above in detail, to an individual who has a need therefor.
  • Figure 1 shows the analysis, in a denaturing gel (SDS-PAGE), of protein extracts originating from viable or tyndallized cultures of L. acidophilus and L. plantarum
  • Figure 2 shows the expression of the decidualization marker prolactin (, PRL ) in cultures of ESC treated with viable or tyndallized L. acidophilus
  • Figure 3 shows the expression of the decidualization marker insulin-like growth factor binding protein 1 (IGFBP-1) in cultures of ESC treated with viable or tyndallized L. acidophilus;
  • IGFBP-1 insulin-like growth factor binding protein 1
  • FIG. 4 shows the expression of the marker of leukaemia inhibiting factor (LIF) in cultures of ESC treated with viable or tyndallized L. acidophilus
  • Figure 5 shows the quantification, using the ELISA method, of PRL secreted in the culture medium of ESC cells treated with viable or tyndallized L. acidophilus;
  • LIF leukaemia inhibiting factor
  • Figure 6 shows the quantification, using the ELISA method, of IGFBP-1 secreted in the culture medium of ESC cells treated with viable or tyndallized L. acidophilus;
  • Figure 7 shows the quantification, using the ELISA method, of LIF secreted in the culture medium of ESC cells treated with viable or heat- killed L. acidophilus;
  • Figure 8 refers to the immunoblot analysis of the proteins secreted in the ESC culture medium: 1) ESC control; 2) ESC+ viable L. acidophilus; 3) ESC +progesterone + cAMP; 4) ESC + proteins extracted from tyndallized L. acidophilus;
  • Figure 9 shows the results of immunohistochemical staining of the count of 1000 cells per field per slide and expressed as a percentage of the average count of cells positive for the anti-PRL antibody: 1) ESC control; 2) ESC+ viable L. acidophilus; 3) ESC + progesterone + cAMP; 4) ESC + proteins extracted from heat-killed L. acidophilus;
  • Figure 10 shows the average composition, expressed as a percentage, of the microorganisms making up the endometrial bacterial flora, in the sample of 14 women examined prior to the treatments;
  • Figure 11 shows the average composition, expressed as a percentage, of the microorganisms making up the endometrial bacterial flora, in the sample of women treated with viable lactobacilli administered via the vaginal route;
  • Figure 12 shows the average composition expressed as a percentage of the microorganisms making up the endometrial bacterial flora, in the sample of women treated with tyndallized lactobacilli administered via the intrauterine route;
  • Figure 13 shows the composition expressed as a percentage of the microorganisms making up the endometrial bacterial flora, in the sample of 14 women examined prior to the treatments with intrauterine tyndallized lactobacilli;
  • Figure 14 shows the average composition expressed as a percentage of the microorganisms making up the endometrial bacterial flora, in the sample of 14 women examined after treatments with intrauterine tyndallized lactobacilli;
  • Figure 15 shows: (A) an intrauterine catheter and (B) a syringe associable with said intrauterine catheter;
  • Figure 16 shows: (A) an intratubal catheter and (B) a syringe associable with said intratubal catheter.
  • the term “decidualization” means the process of cell differentiation in which the endometrial stromal cells (ESC) are transformed from proliferating fibroblasts into specialized cells with secretory activity.
  • the term “microbiota” means the set of symbiotic microorganisms which coexist with the human body without harming it.
  • microbiome means the totality of the genetic resources possessed by the microbiota, i.e. the genes that the latter is capable of expressing.
  • pathobiont refers to bacteria which are present in the female reproductive system and are potentially pathogenic if present in large amounts.
  • probiotic means, according to what has been established by the FAO and WHO: “Live microorganisms which when administered in adequate amounts confer a health benefit on the host.
  • Probiotics are microorganisms that show to be capable, once ingested in adequate amounts, of exercising functions that are beneficial to the body, which is substantially a reiteration of the definition of the two above- mentioned organizations.
  • paraprobiotic means non-viable microbial cells (intact or broken) or raw cell extracts (i.e. with a complex chemical composition), which, when administered (orally or topically) in adequate amounts, provide an advantage for the health of human or animal consumers.
  • postbiotics means the metabolic bioproducts generated by probiotic microorganisms.
  • the term “tyndallized” refers to a microorganism inactivated by means of heat, in particular by means of a sterilization process in discontinuous steps.
  • a first aspect of the present invention relates to a composition comprising bacteria, preferably probiotic or paraprobiotic bacteria, for use in the treatment or prevention of a pathological condition of the female reproductive system caused by or associated with an imbalance in the microbiota of the female reproductive system and/or an insufficient uterine decidualization.
  • Said pathological condition is preferably caused by or associated with an imbalance in the endometrial and/or vaginal microbiota and/or tubal mucosa, preferably in the endometrial microbiota and/or microbiota of the tubal mucosa.
  • said pathological condition is caused by or associated with a decrease, in the endometrial microbiota and/or tubal mucosa, in the levels of commensal bacteria and/or the proliferation of pathobiontic microorganisms.
  • said pathological condition is caused by or associated with a decrease, in the endometrial microbiota and/or microbiota of the tubal mucosa, in the levels of bacteria of the genus Lactobacillus and/or of the genus Bifidobacterium, preferably of bacteria belonging to the genus Lactobacillus, more preferably of the species Lactobacillus crispatus and/or Lactobacillus gasseri.
  • said pathological condition is caused by or associated with an increase in the proliferation, in the endometrial microbiota and/or microbiota of the tubal mucosa, of at least one microorganism selected from: Streptococcus, preferably the species S. pyogenes, S agalactiae, S. faecalis, S. pneumoniae, S.
  • Streptococcus preferably the species S. pyogenes, S agalactiae, S. faecalis, S. pneumoniae, S.
  • Staphilococcus preferably the species Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus cohnii, Staphylococcus delphini, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus pseudintermedius, Staphylococcus saprophyticus, Staphylococcus warneri, Staphylococcus xylosus, Enterobatteriacee, preferably E. coli, Klebsiella and Enterobacter, Gardenerella vaginalis, Candida, Trichomonas, Clamidia Trachomatis and combinations thereof.
  • Said pathological condition is preferably caused by or associated with an insufficient decidualization, i.e. an insufficient differentiation of endometrial fibroblasts into endometrial stromal cells (ESC).
  • Decidualized ESC produce factors capable of increasing endometrial receptivity and regulate the activity of different types of cells of the immune and vascular systems.
  • said pathological condition is selected from: infertility, sterility, acute endometritis, chronic endometritis, threat of miscarriage, miscarriage, recurrent miscarriage, implantation failure, ectopic pregnancy, placenta previa, placental abruption, intrauterine growth restriction, pre-term delivery, preeclampsia, premature membrane rupture and combinations thereof.
  • composition of the present invention preferably comprises bacteria, preferably probiotic or paraprobiotic, of the genus Lactobacillus and/or of the genus Bifidobacterium.
  • the composition comprises bacteria of the genus Lactobacillus, more preferably belonging to at least one of the following species: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus paracasei,
  • Lactobacillus rhamnosus Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus alimentarius, Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gallinarum,
  • the bacteria of the genus Lactobacillus belong to at least one species selected from L. acidophilus, L. plantarum and L. paraplantarum.
  • the bacteria of the genus Lactobacillus belong to the species L. acidophilus and/or L. plantarum. More preferably, the bacteria are of the species L. acidophilus, preferably strain ATCC 4357 and/or strain ATCC BAA-2832 and/or strain ATCC 11975 and/or strain ATCC 53544.
  • the bacteria are of the species L. plantarum, preferably strain ATCC BAA-793 and/or strain ATCC 700210 and/or strain ATCC 55324.
  • the composition comprises bacteria of the genus Bifidobacterium, more preferably belonging to at least one of the following species: B. animalis, B. bifidum, B. breve, B. infantis, B. longum, B. adolescentis, B. catenulatum, B. angulatum, B. asteroides, B. bourn, B. choerinum, B. coryneforme, B. cuniculi, B. denticolens, B. dentium, B. gallicum, B. gallinarum, B. indicum, B. inopinatum, B. lactis, B. magnum, B. merycicum, B.
  • B. animalis B. bifidum, B. breve, B. infantis, B. longum, B. adolescentis, B. catenulatum, B. angulatum, B. asteroides, B. bourn, B
  • B. pseudocatenulatum, B. pseudolongum, B. pullorum, B. ruminantium, B. saeculare, B. subtile, B. thermacidophilum, B. thermophilum and B. tsurumiense more preferably selected from: Bacillus clausii, Bacillus subtilis, Bacillus coagulans, Bacillus megaterium Bacillus halodurans, Bacillus thuringiensis, Bacillus insolitus and Bacillus marinus.
  • the bacteria as described above preferably of the genus Lactobacillus, are used live, or they are used as probiotics.
  • the bacteria preferably of the genus Lactobacillus, are dead or inactivated, for example by tyndallization, or in the form of a lysate and/or extract, i.e. they are used as paraprobiotics.
  • the bacteria used are tyndallized.
  • the bacteria are used as an extract, preferably as a protein extract, more preferably as a protein extract of tyndallized bacteria.
  • composition further comprises the metabolic bioproducts generated by microorganisms defined as postbiotic.
  • Said metabolic bioproducts are preferably selected from: supernatant, metabolites, cell wall and components thereof, exopolysaccharides, proteins, and any component of the supernatant.
  • the bacteria preferably of the genus Lactobacillus, are present in the composition in an minimum amount sufficient to enable a temporary colonization of the vagina and/or endometrium and/or other regions of the female reproductive system. Said amount preferably ranges between 10 8 and 10 12 units of microorganisms, more preferably between 10 9 and 10 11 units of microorganisms.
  • the bacteria of the present invention are administered in a variable amount comprised between 1 * 10 s and 1 * 10 12 , preferably between 2 * 10 s and 5 * 10 9 , more preferably between 5 * 10 8 and 3 * 10 9 per administration.
  • the administration of bacteria according to the present invention takes place at least 1-2 times a week, preferably at least 3 times a week.
  • composition further comprises excipients and/or further pharmaceutically acceptable substances and/or carriers.
  • the administration can take place through any route.
  • the composition is preferably administered via the topical route, more preferably via the vaginal or intrauterine or intratubal route.
  • the composition is administered via the intrauterine route.
  • the composition is preferably administered via the intrauterine route through injection by means of an intrauterine catheter or a transcervical catheter.
  • the composition is administered into the tubes of the uterus (salpinges).
  • the composition is preferably administered via the intratubal route through injection by means of an intratubal catheter.
  • the composition is formulated for applications of a topical type, preferably in the form of a solution, suspension, powder for gynaecological suspensions, vaginal suppositories, vaginal capsules, vaginal douches, vaginal creams or enemas.
  • the composition is prepared extemporaneously by mixing the composition with water or with a saline solution.
  • Maintaining levels of lactobacilli in the endometrial and/or vaginal microbiota is of particular interest, as it is correlated with the possible occurrence of infections in the female reproductive system.
  • the presence of bacteria, in particular of the genus Lattobacillus, in the endometrial microbiota is capable of controlling the proliferation of pathobiontic microorganisms.
  • the balance of the microbiota, in particular of the endometrial microbiota seems to be involved in the decidualization mechanisms of the endometrium.
  • the Applicant has demonstrated that the administration of bacteria, in particular of the genus Lactobacillus and/or Bifidobacterium, is able to restore a correct balance in the endometrial environment, which contributes to a correct decidualization.
  • the administration of bacteria which are viable or inactivated, preferably tyndallized, or of protein extracts of said bacteria are capable of favouring decidualization.
  • cultures of viable bacteria induce undifferentiated ESC to assume a decidualized phenotype with times and levels of activation of marker genes comparable to those obtained in primary cultures of ESC subjected to the “classic” protocol of decidualization induced by progesterone and cAMP.
  • the levels of gene expression and proteins tied to decidualization increase significantly compared both to the culture of ESC treated according to the classic decidualization protocol and ESC treated in co-culture with viable Lactobacilli, resulting in an earlier attainment of the decidualized state of the ESC.
  • an administration of bacteria, preferably of the genus Lactobacillus, directly into the uterus via intrauterine administration is particularly effective in restoring a correct balance of the endometrial microbiota.
  • a second aspect of the present invention relates to the use of a composition comprising bacteria, preferably probiotic or paraprobiotic bacteria, as described above in detail, to increase the levels or the proliferation of commensal bacteria, preferably of the species Lactobacillus crispatus and/or Lactobacillus gasseri, and/or to reduce the levels or reduce the proliferation of at least one microorganism selected from: Streptococcus, preferably the species S. pyogenes, S agalactiae, S. faecalis, S. pneumoniae, S.
  • Staphylococcus preferably the species Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus cohnii, Staphylococcus delphini, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus pseudintermedius, Staphylococcus saprophyticus, Staphylococcus warneri, Staphylococcus xylosus, and Enterobatteriacee selected from E.
  • Staphylococcus preferably the species Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococc
  • composition of the invention can be used to restore the balance of the endometrial microbiota and/or tubal mucosa also in the absence of a clinically evident pathological condition.
  • said use comprises at least one step of administering an effective amount of a composition comprising bacteria, preferably probiotic or paraprobiotic bacteria, as described above in detail, to an individual who has a need therefor.
  • a third aspect of the present invention relates to a kit comprising a composition based on bacteria, preferably probiotic or paraprobiotic bacteria, as described above in detail and a device for intrauterine or intratubal administration of the composition.
  • said device comprises at least one syringe, preferably preloaded with the composition described above in detail, and an intrauterine or intratubal catheter.
  • an intrauterine catheter is shown in Figure 15A together with a syringe associable therewith ( Figure 15B)
  • an example of an intratubal catheter is shown in Figure 16A together with a syringe associable therewith ( Figure 16B).
  • a fourth aspect of the present invention relates to a method for the treatment or prevention of a pathological condition of the female reproductive system caused by or associated with an imbalance in the microbiota of the female reproductive system and/or an insufficient decidualization.
  • Said pathological condition is preferably caused by or associated with an imbalance in the endometrial and/or vaginal microbiota and/or in the microbiota of the tubal mucosa, preferably in the endometrial microbiota and/or in the microbiota of the tubal mucosa.
  • said pathological condition is caused by or associated with a decrease, in the endometrial microbiota and/or in the microbiota of the tubal mucosa, in the levels of commensal bacteria and the proliferation of pathobiontic microorganisms, as described above.
  • said method comprises at least one step of administering an effective amount of a composition comprising bacteria, preferably probiotic or paraprobiotic bacteria, as described above in detail, to an individual who has a need therefor.
  • the endometrial biopsies were collected during the proliferative phase of the menstrual cycle (from day 5 to day 14 of the menstrual cycle) from healthy women with a regular menstrual cycle who underwent diagnostic hysteroscopy prior to in vitro fertilization (IVF), recommended for reasons connected to the infertility of the male partner.
  • IVF in vitro fertilization
  • the endometrial biopsies were collected in 1X Dulbecco’s phosphate buffered saline (DPBS, Sigma-Aldrich, St. Louis, MO, USA) and immediately processed at room temperature under sterile conditions.
  • the endometrial biopsies, originally about 5-7 mm 3 were further washed twice with 1X DPBS and finally deposited in a sterile Petri dish to enable purification of the endothelial stromal cells according to the protocol described by Ciarmela et al., 2011.
  • the endometrial biopsy was treated at room temperature for 2 minutes with 1 mg/ml of Collagenase Type II (Sigma, St. Louis, MO) dissolved in a buffer for collagenase (DMEM medium; 100 U/ml penicillin; 100 pg/ml streptomycin; 50 pg/ml gentamycin; 2 mM L-glutamine; all of these products were purchased from Life Technologies, UK) to allow enzymatic digestion of the tissue. After removal of the thin outer layer of connective tissue, the endometrial biopsy immersed in the collagenase solution was chopped as finely as possible using a sterile scalpel.
  • Collagenase Type II Sigma, St. Louis, MO
  • the resulting suspension was transferred into a 50 ml tube containing 8 ml of collagenase solution and tissue digestion was made to take place in a temperature-controlled bath at 37°C under gentle stirring, avoiding the formation of foam, until complete digestion of the tissue (the process generally takes 1-4 hours, depending on the initial size of the biopsy).
  • the digested endometrium was then washed by centrifugation at 600 xg for 12 minutes and the supernatant was subsequently removed.
  • the resulting cell pellet was resuspended in 3 ml of foetal bovine serum (FBS), South American (EU Approved, Carlo Erba, Italy), and incubated for 20 minutes at 37°C (5% CO2) to allow the cells a physiological recovery.
  • FBS foetal bovine serum
  • South American EU Approved, Carlo Erba, Italy
  • the ESC thus recovered were washed as previously described and finally suspended in the culture medium (DMEM medium supplemented with 10% FBS and 2 mM L-glutamine), counted using a haemocytometer and plated at a concentration of 10 5 cells/well in a 12-well multiwell plate.
  • the primary cultures thus prepared were maintained in culture (37°C, 5% CO2) until reaching confluence (about 70% of the available surface occupied) and then used for the experiments or treated according to the classic decidualization protocol (Dimitriadis et al., 2005).
  • the primary cultures of ESC were maintained in a decidualization medium (culture medium previously described supplemented with 600 ng/ml progesterone and 10 ng/ml of cAMP; Sigma, St. Louis, MO) until reaching the decidualized phenotype.
  • a decidualization medium culture medium previously described supplemented with 600 ng/ml progesterone and 10 ng/ml of cAMP; Sigma, St. Louis, MO
  • the primary cultures of ESC were subjected daily to: a microscopic evaluation of the cell morphology, replacement of the exhausted culture medium with fresh medium, and sampling of the cultures to allow for future molecular analysis.
  • MRS deMan-Rogosa-Sharp
  • the centrifuged Lactobacilli were washed with 1X PBS and inactivated in an autoclave for 1 h at 70°C.
  • the effective lack of viability of the Lactobacilli was confirmed by inoculating an aliquot of 10 6 heat-killed bacteria in the MRS culture broth and checking for their growth (O.D.) by means of a spectrophotometer.
  • the pellets of viable and heat-killed Lactobacilli were processed for the direct extraction of proteins using RIPA buffer (Sigma, St. Louis, MO). The proteins thus extracted were quantified by means of the Bradford assay.
  • 1X loading buffer 4% SDS; 20% glycerol; 0.004% bromophenol blue; 0.125 M Tris- Cl, pH 6.8; 10% 2-mercaptoethanol
  • 1X protease inhibitor cocktail Sigma Aldrich, St. Louis, MO, USA
  • PMSF phenylmethylsulfonyl fluoride
  • the protein extracts were separated by means of an electrophoretic run through a 12% acrylamide/bis-polyacrylamide gel under denaturing conditions.
  • the known molecular weight marker ColorBurst Electrophoresis Marker (Sigma Aldrich, St. Louis, MO, USA) was used as a reference for band identification.
  • the samples were loaded in duplicate in order to generate two identical gels. The run was carried out for 90 minutes at 120 V.
  • the first gel was transferred with an electroblotter (semi dry mode) to a nitrocellulose membrane to enable the subsequent immunoblot.
  • the gel used for the transfer was dyed with Coomassie brilliant blue (Sigma, St. Louis, MO) in order to visualize the position of the bands corresponding to the proteins.
  • the second gel was used for the extraction and purification of the proteins of interest from the gel. Extraction/purification of proteins from polyacrylamide gel
  • the proteins of interest separated by electrophoresis on the basis of molecular weight, were identified in the second gel (undyed) using the gel dyed with Coomassie as a reference.
  • the bands of interest after alignment of the gel, were excised from the undyed gel using a sterile scalpel. In order to confirm the accuracy of the excision, the remaining part of the gel was dyed and compared with the gel dyed with Coomassie.
  • the bands corresponding to the proteins of interest were passively eluted from the gel after it had been fragmented using a sterile pestle, in 1 ml of elution buffer (50 mM Tris-HCI, 150 mM NaCI, 0.1 mM EDTA; pH 7.5).
  • the elution was conducted overnight in a rotary shaker at 30 °C.
  • the elution mixture was then centrifuged at 10,000 xg for 10 minutes and the supernatant containing the eluted proteins was transferred to a new microcentrifuge tube. Aliquots of supernatant were run in a new SDS- PAGE to confirm the presence of the proteins of interest.
  • the proteins eluted and purified with this method lend themselves to being used in different applications such as experiments (directly dissolved in the medium) or N-terminal sequencing (to define the type of protein).
  • the primary cultures of ESC were incubated with viable or heat-killed L. acidophilus or L. plantarum or with the protein extracts thereof for 10 days at 37 °C and 5% CO2, according to the scheme described below:
  • ESC (10 ® cells/well) + protein lysate obtained from viable L. acidophilus or L. plantarum (1 pg/ml) 5.
  • ESC (10 6 cells/well) + protein lysate obtained from heat-killed L. acidophilus or L. piantarum (1 pg/ml)
  • the ESC and dESC treated as described were processed daily according to the protocols described below.
  • RNA samples were directly lysed in the plate well with 1 ml of the commercial product Tri-Reagent® (Sigma Aldrich, St Louis, MO, USA) and immediately processed for total RNA extraction, following the instructions given in the datasheet.
  • the total RNA extracted was suspended in 20 pi of RNase free water and digested with DNase I (Promega, Madison, Wl, USA) for 30 minutes at 37 °C to remove any contaminating genomic DNA that might be present.
  • the RNA was evaluated by spectrophotometry using the Nanodrop ND-1000 instrument (Thermo Fisher Scientific, Waltham, MA, USA) to determine its quality and concentration.
  • RNA per sample Two pg of total RNA per sample was reverse transcribed to cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), abiding by the protocol provided by the manufacturer.
  • the temperature profile of the reaction used was 5 minutes at 25 °C, 30 minutes at 42 °C and 5 minutes at 85 °C using the 2720 Thermal Cycler instrument (Applied Biosystem, Waltham, MA, USA).
  • RT-guantitative PCR (RT-gPCR)
  • the cDNAs obtained from each sample were used (2 mI) as the template in RT-qPCR reactions.
  • the reactions were conducted in triplicate for every sample and expressed as an average to enable an analysis of gene expression.
  • the reactions were assembled in ice using SsoAdvanced Universal SYBR ® Green Supermix (Bio-Rad, Hercules, CA, USA) as the enzyme, according to the manufacturer’s directions.
  • the primers used (Table I) were designed to pair with the joining sequence between two exons, thereby preventing the possible amplification of any contaminating genomic DNA (containing the introns) that might still be present after digestion with DNase I.
  • All of the primers used have very similar melting temperatures (about 60°), so that the reactions were conducted with the same temperature profile: 30 seconds at 95°C for the initial activation of the enzyme, followed by 40 cycles of 5 seconds (denaturing) at 95°C and annealing/extension at 60°C for 20 second using the StepOne Real-Time PCR System (Applied Biosystems, Waltham, MA, USA).
  • the results were normalized against the constitutively expressed gene b-actin (reference gene).
  • the negative controls of the reactions in triplicate were prepared using corresponding volumes of molecular biology grade water in the place of the cDNAs.
  • the specificity of each reaction was checked by evaluating the resulting dissociation curve and running part of the amplified products (10 mI) in an agarose gel at 1.5% in 1X Tris-Acetic acid-EDTA (TAE).
  • the proteins PRL, IGFBP-1 and LIF produced and translated following the treatments with Lactobacilli or the proteins thereof were quantified by immunoenzymatic assay (ELISA) as described in Rose et al., 1978.
  • ELISA immunoenzymatic assay
  • the corresponding ELISA assay for each molecule was purchased from R&D Biosystems and used according to the procedure recommended by the manufacturer.
  • the optical density (O.D.) related to each sample was measured at a wavelength of 450 nm using a MultiSkan FC plate reader (Thermo Fisher).
  • the protein concentration was calculated using the bioinformatic program MyAssay, specifically designed for the type of protein under examination.
  • the membranes were incubated for 1 hour with the relevant primary antibodies derived in goat anti-human PRL or IGFBP-1 or LIF (Santa Cruz Biotechnology, Dallas, TX, USA), diluted 1 :200. After three washes with 0.05% Tween 20 (Sigma Aldrich, St. Louis, MO, USA) in TBS to remove the unbound primary antibody, the membranes were further incubated with the secondary antibody derived in rabbit anti-goat conjugated with the enzyme horseradish peroxidase (HRP), diluted 1 :10000 (Bethyl, Montgomery, TX, USA).
  • HRP horseradish peroxidase
  • the membranes were immersed in ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA) and the bands were detected under chemiluminescence using the ChemiDoc XRS+ instrument (Bio-Rad, Hercules, CA, USA). The signals were acquired and semi-quantified with image analysis software (VersaDoc Imaging System and QuantityOne software, Bio-Rad Laboratories Inc.).
  • the membranes were “stripped”, i.e. the antibodies used were detached, re-incubated with a primary antibody derived in rabbit anti-human b-tubulin (Abeam, Cambridge, UK), diluted 1 :500, and finally processed as described above. After development, the bands corresponding to b-tubulin were used as an internal control on the amount of protein lysate initially loaded into the gel. Immunohistochemical analysis
  • the cell suspensions corresponding to each individual sample were transferred onto slides using a cytospin (Shandon Cytospin4, Thermo Scientific) at 500 xg for 5 minutes and, finally, fixed at 4 °C for 1 hour in a paraformaldehyde solution (4% paraformaldehyde dissolved in 1X PBS at 7.4 pH).
  • the slides containing the fixed ESC were washed 3 times with 1X PBS and used in immunohistochemical reactions as described below.
  • Antigen unmasking was obtained by treating the slides with protease (Pronase 1 :20, DakoCytomation, CA, USA) for 7 minutes at 37 °C.
  • protease Pronase 1 :20, DakoCytomation, CA, USA
  • the slides were treated afterwards with 3% bovine serum albumin (BSA) dissolved in 1X PBS for 30 minutes at room temperature.
  • BSA bovine serum albumin
  • the slides were incubated with the following polyclonal antibodies derived in rabbit anti human PRL, ILGFB-1 and LIF diluted 1 :25 in 1X PBS containing 3% BSA for 1 hour at room temperature (all of the antibodies were purchased from Santa Cruz, Dallas, TX, USA).
  • the images were acquired with a confocal microscope (Leica TCS SP2 AOBS confocal laser scanning microscope). Excitation and detection of the samples were performed sequentially to avoid overlap between the various signals.
  • the positive ESC were counted by two different operators using the double-blind method; random fields with a total of 100 ESC were selected.
  • the protein lysates originating from viable or heat-killed L. acidophilus and L. plantarum were fractionated in a 12% polyacrylamide gel under denaturing conditions in order to reveal any differences in terms of representative bands.
  • ESC purified as described above were placed in culture with viable or heat-killed L. acidophilus or L. plantarum (10 8 cells/ml) or with the protein lysates thereof.
  • viable or heat-killed L. acidophilus or L. plantarum (10 8 cells/ml) or with the protein lysates thereof.
  • purified ESC were incubated with progesterone and cAMP according to the classic decidualization protocol.
  • the expression of 2 marker genes PRL and IGFBP-1 was analysed by qPCR. As shown in Figures 2 and 3, the addition of viable or heat-killed L.
  • the expression of the marker LIF was monitored over the course of the treatment and analysed with qPCR.
  • the levels of expression of LIF in ESC treated with the decidualization protocol and ESC in a simple co-culture with L. acidophilus (or with L. plantarum) are comparable.
  • the levels of expression of LIF are significantly higher in the samples where the co-culture with L. acidophilus took place in the presence of the bacterial lysate originating from heat- killed L. acidophilus, thus confirming a possible beneficial role of L. acidophilus in improving the receptivity of the endometrium during decidualization.
  • the results of immunohistochemical staining are shown in Figure 9.
  • the results refer to the count of 1000 cells per slide per field and expressed as a percentage of the average count of cells positive to the anti-PRL antibody.
  • the percentages of positive cells decidualized with viable L. acidophilus (or L. plantarum) are comparable to the percentages obtained when applying the classic decidualization protocol.
  • the percentages of positivity and thus decidualization found are higher than those recorded with ESC subjected to the decidualization protocol.
  • the cervix was cleansed with a moistened cotton wad and 20 to 80 pL of endometrial fluid were aspirated using a catheter introduced through the cervix into the uterine cavity, avoiding any contact with the vaginal walls.
  • cervical mucous was aspirated before drawing endometrial liquid and the suction was interrupted at the entry of the internal uterine ostium during removal of the catheter.
  • the aspiration of endometrial fluid is painless and minimally invasive; it causes no risk for the patient.
  • the microbiome was studied by means of sequencing techniques based on 16S ribosomal DNA (rDNA) genes, which represent the region of interest (target sequence), unique for all bacterial species, in microbiome research.
  • rDNA 16S ribosomal DNA
  • target sequence region of interest
  • NGS technologies new-generation sequencing
  • 16S rDNA sequencing was performed using the Ion 16S metagenomics kit (Ion Torrent of Life Technologies, Grand Island, New York, USA). The amplified fragments were sequenced in the Ion PGMTM system and the results were analysed using Ion ReporterTM software according to the manufacturer’s guidelines.

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Abstract

La présente invention concerne une composition comprenant des bactéries destinée à être utilisée dans le traitement ou la prévention d'une pathologie provoquée par ou associée à un déséquilibre dans le microbiote du système reproducteur féminin, de préférence dans l'endomètre ou la muqueuse tubaire. La présente invention concerne en outre un kit pour le traitement ou la prévention de ladite pathologie.
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CN116804176A (zh) * 2023-06-14 2023-09-26 南昌大学 一株阴道卷曲乳酸杆菌及其在制备防治慢性子宫内膜炎和由ce引起的女性不孕症的药物中的应用

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WO2014184643A1 (fr) * 2013-05-14 2014-11-20 Probiotical S.P.A. Composition contenant des bactéries lactiques, utilisable dans le traitement préventif et/ou curatif de la vaginose bactérienne
WO2016100086A1 (fr) * 2014-12-15 2016-06-23 The Johns Hopkins University Transplantation de sécrétions cervico-vaginales pour traiter la vaginose bactérienne
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WO2017196006A1 (fr) * 2016-05-10 2017-11-16 Haudongchun Co., Ltd Composition comprenant une bactérie lactique pour la prévention et le traitement de la vaginose et son utilisation
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023090552A1 (fr) * 2021-11-19 2023-05-25 주식회사 지놈앤컴퍼니 Nouvelle souche de lactobacillus plantarum et utilisation associée
CN116804176A (zh) * 2023-06-14 2023-09-26 南昌大学 一株阴道卷曲乳酸杆菌及其在制备防治慢性子宫内膜炎和由ce引起的女性不孕症的药物中的应用

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