Application of Ligilactobacillus salivarius CECT5713 to Achieve Term Pregnancies in Women with Repetitive Abortion or Infertility of Unknown Origin by Microbiological and Immunological Modulation of the Vaginal Ecosystem

In this study, the cervicovaginal environment of women with reproductive failure (repetitive abortion, infertility of unknown origin) was assessed and compared to that of healthy fertile women. Subsequently, the ability of Ligilactobacillus salivarius CECT5713 to increase pregnancy rates in women with reproductive failure was evaluated. Vaginal pH and Nugent score were higher in women with reproductive failure than in fertile women. The opposite was observed regarding the immune factors TGF-β 1, TFG-β 2, and VEFG. Lactobacilli were detected at a higher frequency and concentration in fertile women than in women with repetitive abortion or infertility. The metataxonomic study revealed that vaginal samples from fertile women were characterized by the high abundance of Lactobacillus sequences, while DNA from this genus was practically absent in one third of samples from women with reproductive failure. Daily oral administration of L. salivarius CECT5713 (~9 log10 CFU/day) to women with reproductive failure for a maximum of 6 months resulted in an overall successful pregnancy rate of 56%. The probiotic intervention modified key microbiological, biochemical, and immunological parameters in women who got pregnant. In conclusion, L. salivarius CECT5713 has proved to be a good candidate to improve reproductive success in women with reproductive failure.

[1]  L. Calvo,et al.  Structural Variations of Vaginal and Endometrial Microbiota: Hints on Female Infertility , 2020, Frontiers in Cellular and Infection Microbiology.

[2]  P. Vandamme,et al.  A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. , 2020, International journal of systematic and evolutionary microbiology.

[3]  V. D’Argenio,et al.  Microbiota and Human Reproduction: The Case of Male Infertility , 2020, High-throughput.

[4]  V. D’Argenio,et al.  Microbiota and Human Reproduction: The Case of Female Infertility , 2020, High-throughput.

[5]  A. Fazleabas,et al.  Endometrial Immune Dysfunction in Recurrent Pregnancy Loss , 2019, International journal of molecular sciences.

[6]  Zongxin Ling,et al.  Lactobacillus iners Is Associated with Vaginal Dysbiosis in Healthy Pregnant Women: A Preliminary Study , 2019, BioMed research international.

[7]  V. D’Argenio,et al.  The evolving role of genetic tests in reproductive medicine , 2019, Journal of Translational Medicine.

[8]  William A. Walters,et al.  Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2 , 2019, Nature Biotechnology.

[9]  M. Stojanov,et al.  Bacterial Colonization of the Female Upper Genital Tract , 2019, International journal of molecular sciences.

[10]  James A. Foster,et al.  What's Normal? Microbiomes in Human Milk and Infant Feces Are Related to Each Other but Vary Geographically: The INSPIRE Study , 2019, Front. Nutr..

[11]  J. M. Rodríguez,et al.  Rectal and Vaginal Eradication of Streptococcus agalactiae (GBS) in Pregnant Women by Using Lactobacillus salivarius CECT 9145, A Target-specific Probiotic Strain , 2019, Nutrients.

[12]  T. Zemskaya,et al.  The effect of 16S rRNA region choice on bacterial community metabarcoding results , 2019, Scientific Data.

[13]  J. M. Rodríguez,et al.  Prevention of Recurrent Acute Otitis Media in Children Through the Use of Lactobacillus salivarius PS7, a Target-Specific Probiotic Strain , 2019, Nutrients.

[14]  T. Moench,et al.  Vaginal pH measured in vivo: lactobacilli determine pH and lactic acid concentration , 2019, BMC Microbiology.

[15]  I. Moreno,et al.  Deciphering the effect of reproductive tract microbiota on human reproduction , 2018, Reproductive medicine and biology.

[16]  Laura M. Heiser,et al.  Common Cervicovaginal Microbial Supernatants Alter Cervical Epithelial Function: Mechanisms by Which Lactobacillus crispatus Contributes to Cervical Health , 2018, Front. Microbiol..

[17]  J. Ravel,et al.  Cervicovaginal microbiota, women's health, and reproductive outcomes. , 2018, Fertility and sterility.

[18]  J. Ravel,et al.  Anti-HIV-1 Activity of Lactic Acid in Human Cervicovaginal Fluid , 2018, mSphere.

[19]  P. O’Toole,et al.  Comparative Genomics of the Genus Lactobacillus Reveals Robust Phylogroups That Provide the Basis for Reclassification , 2018, Applied and Environmental Microbiology.

[20]  D. Anumba,et al.  The Vaginal Microenvironment: The Physiologic Role of Lactobacilli , 2018, Front. Med..

[21]  P. Gajer,et al.  A retrospective pilot study to determine whether the reproductive tract microbiota differs between women with a history of infertility and fertile women , 2018, The Australian & New Zealand journal of obstetrics & gynaecology.

[22]  M. Arasu,et al.  In vitro investigation on probiotic, anti-Candida, and antibiofilm properties of Lactobacillus pentosus strain LAP1. , 2018, Archives of oral biology.

[23]  T. Padmavathi,et al.  Screening of potential probiotic lactic acid bacteria and production of amylase and its partial purification , 2018, Journal, genetic engineering & biotechnology.

[24]  Benjamin D. Kaehler,et al.  Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin , 2018, Microbiome.

[25]  S. Kovachev.  Defence factors of vaginal lactobacilli , 2018, Critical reviews in microbiology.

[26]  M. Vaneechoutte,et al.  Lactobacillus iners, the unusual suspect. , 2017, Research in microbiology.

[27]  W. Cheng,et al.  Expression of C-type lectin receptors and Toll-like receptors in decidua of patients with unexplained recurrent spontaneous abortion. , 2017, Reproduction, fertility, and development.

[28]  G. Ricci,et al.  Subclinical alteration of the cervical–vaginal microbiome in women with idiopathic infertility , 2017, Journal of cellular physiology.

[29]  R. Cone,et al.  The role of lactic acid production by probiotic Lactobacillus species in vaginal health. , 2017, Research in microbiology.

[30]  J. M. Rodríguez,et al.  Microbial Diversity in Milk of Women With Mastitis: Potential Role of Coagulase-Negative Staphylococci, Viridans Group Streptococci, and Corynebacteria , 2017, Journal of human lactation : official journal of International Lactation Consultant Association.

[31]  M. Vaneechoutte,et al.  Lactobacillus iners: Friend or Foe? , 2017, Trends in microbiology.

[32]  S. Witkin,et al.  Why do lactobacilli dominate the human vaginal microbiota? , 2017, BJOG : an international journal of obstetrics and gynaecology.

[33]  J. Nicholson,et al.  The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk , 2017, Microbiome.

[34]  J. Jimenez-Almazan,et al.  Evidence that the endometrial microbiota has an effect on implantation success or failure. , 2016, American journal of obstetrics and gynecology.

[35]  L. Forney,et al.  Genomic Comparisons of Lactobacillus crispatus and Lactobacillus iners Reveal Potential Ecological Drivers of Community Composition in the Vagina , 2016, Applied and Environmental Microbiology.

[36]  J. Lepargneur.  Lactobacillus crispatus as biomarker of the healthy vaginal tract. , 2016, Annales de biologie clinique.

[37]  Paul J. McMurdie,et al.  DADA2: High resolution sample inference from Illumina amplicon data , 2016, Nature Methods.

[38]  L. Samaranayake,et al.  Probiotic lactobacilli inhibit early stages of Candida albicans biofilm development by reducing their growth, cell adhesion, and filamentation , 2016, Applied Microbiology and Biotechnology.

[39]  P. Humaidan,et al.  Abnormal vaginal microbiota may be associated with poor reproductive outcomes: a prospective study in IVF patients. , 2016, Human reproduction.

[40]  J. H. van de Wijgert,et al.  Unique Insights in the Cervicovaginal Lactobacillus iners and L. crispatus Proteomes and Their Associations with Microbiota Dysbiosis , 2016, PloS one.

[41]  Reet Mändar,et al.  Characterisation of probiotic properties in human vaginal lactobacilli strains , 2016, Microbial ecology in health and disease.

[42]  Jean M. Macklaim,et al.  Changes in vaginal microbiota following antimicrobial and probiotic therapy , 2015, Microbial ecology in health and disease.

[43]  H. Seow,et al.  In vitro modulation of probiotic bacteria on the biofilm of Candida glabrata. , 2015, Anaerobe.

[44]  R. Cone,et al.  Antimicrobial and immune modulatory effects of lactic acid and short chain fatty acids produced by vaginal microbiota associated with eubiosis and bacterial vaginosis , 2015, Front. Physiol..

[45]  D. Klionsky,et al.  How to control self-digestion: transcriptional, post-transcriptional, and post-translational regulation of autophagy. , 2015, Trends in cell biology.

[46]  Y. Cheah,et al.  Probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 exhibit strong antifungal effects against vulvovaginal candidiasis-causing Candida glabrata isolates , 2015, Journal of applied microbiology.

[47]  P. Brigidi,et al.  Microbes Central to Human Reproduction , 2014, American journal of reproductive immunology.

[48]  W. Kneifel,et al.  Characterisation of the vaginal Lactobacillus microbiota associated with preterm delivery , 2014, Scientific Reports.

[49]  L. Forney,et al.  Comparative Functional Genomics of Lactobacillus spp. Reveals Possible Mechanisms for Specialization of Vaginal Lactobacilli to Their Environment , 2014, Journal of bacteriology.

[50]  J. Segars,et al.  Potential Influence of the Microbiome on Infertility and Assisted Reproductive Technology , 2014, Seminars in Reproductive Medicine.

[51]  R. Cone,et al.  Vaginal pH and Microbicidal Lactic Acid When Lactobacilli Dominate the Microbiota , 2013, PloS one.

[52]  P. De Sutter,et al.  Risks associated with bacterial vaginosis in infertility patients: a systematic review and meta-analysis. , 2013, Human reproduction.

[53]  R. Cone,et al.  Vaginal concentrations of lactic acid potently inactivate HIV , 2013, The Journal of antimicrobial chemotherapy.

[54]  J. Brosens,et al.  Molecular aspects of implantation failure. , 2012, Biochimica et biophysica acta.

[55]  Pelin Yilmaz,et al.  The SILVA ribosomal RNA gene database project: improved data processing and web-based tools , 2012, Nucleic Acids Res..

[56]  A. Klindworth,et al.  Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies , 2012, Nucleic acids research.

[57]  R. Gilchrist,et al.  TGF-β Mediates Proinflammatory Seminal Fluid Signaling in Human Cervical Epithelial Cells , 2012, The Journal of Immunology.

[58]  Verena D. Schmittmann,et al.  Qgraph: Network visualizations of relationships in psychometric data , 2012 .

[59]  Edwin van den Heuvel,et al.  Adhesion Forces and Coaggregation between Vaginal Staphylococci and Lactobacilli , 2012, PloS one.

[60]  Katherine H. Huang,et al.  Structure, Function and Diversity of the Healthy Human Microbiome , 2012, Nature.

[61]  S. Delgado,et al.  Characterization of Lactobacillus salivarius CECT 5713, a strain isolated from human milk: from genotype to phenotype , 2012, Applied Microbiology and Biotechnology.

[62]  M. J. García,et al.  Synergistic Effect Between Two Bacteriocin-like Inhibitory Substances Produced by Lactobacilli Strains with Inhibitory Activity for Streptococcus agalactiae , 2012, Current Microbiology.

[63]  Ronald W. Davis,et al.  The dynamics of the vaginal microbiome during infertility therapy with in vitro fertilization-embryo transfer , 2012, Journal of Assisted Reproduction and Genetics.

[64]  J. Bromfield,et al.  Seminal Fluid Regulates Accumulation of FOXP3+ Regulatory T Cells in the Preimplantation Mouse Uterus Through Expanding the FOXP3+ Cell Pool and CCL19-Mediated Recruitment1 , 2011, Biology of reproduction.

[65]  R. Cone,et al.  In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide , 2011, BMC infectious diseases.

[66]  C. Huttenhower,et al.  Metagenomic biomarker discovery and explanation , 2011, Genome Biology.

[67]  L. Qiu,et al.  Decidual CD4+CD25+CD127dim/- regulatory T cells in patients with unexplained recurrent spontaneous miscarriage. , 2011, European journal of obstetrics, gynecology, and reproductive biology.

[68]  F. Pérez-Cano,et al.  In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk. , 2010, Immunobiology.

[69]  Jean M. Macklaim,et al.  At the crossroads of vaginal health and disease, the genome sequence of Lactobacillus iners AB-1 , 2010, Proceedings of the National Academy of Sciences.

[70]  J. Xaus,et al.  Safety and tolerance of the human milk probiotic strain Lactobacillus salivarius CECT5713 in 6-month-old children. , 2010, Nutrition.

[71]  J. M. Rodríguez,et al.  Treatment of infectious mastitis during lactation: antibiotics versus oral administration of Lactobacilli isolated from breast milk. , 2010, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[72]  P. Gajer,et al.  Vaginal microbiome of reproductive-age women , 2010, Proceedings of the National Academy of Sciences.

[73]  R. Cone,et al.  Cervicovaginal fluid and semen block the microbicidal activity of hydrogen peroxide produced by vaginal lactobacilli , 2010, BMC infectious diseases.

[74]  S. Robertson.  Immune regulation of conception and embryo implantation-all about quality control? , 2010, Journal of reproductive immunology.

[75]  Ramazan Demir,et al.  Vasculogenesis and angiogenesis in the endometrium during menstrual cycle and implantation. , 2010, Acta histochemica.

[76]  J. Marrazzo,et al.  Temporal Variability of Human Vaginal Bacteria and Relationship with Bacterial Vaginosis , 2010, PloS one.

[77]  P. Bork,et al.  A human gut microbial gene catalogue established by metagenomic sequencing , 2010, Nature.

[78]  J. Hayball,et al.  Activating T regulatory cells for tolerance in early pregnancy - the contribution of seminal fluid. , 2009, Journal of reproductive immunology.

[79]  Alison S Care,et al.  Seminal Fluid Drives Expansion of the CD4+CD25+ T Regulatory Cell Pool and Induces Tolerance to Paternal Alloantigens in Mice1 , 2009, Biology of reproduction.

[80]  M. Vaneechoutte,et al.  Characterization of indigenous vaginal lactobacilli from healthy women as probiotic candidates. , 2008, International microbiology : the official journal of the Spanish Society for Microbiology.

[81]  J. Xaus,et al.  Oral Administration of Lactobacillus Strains Isolated from Breast Milk as an Alternative for the Treatment of Infectious Mastitis during Lactation , 2008, Applied and Environmental Microbiology.

[82]  D. Clark,et al.  ORIGINAL ARTICLE: Prevention of Spontaneous Abortion in the CBA × DBA/2 Mouse Model by Intravaginal TGF‐β and Local Recruitment of CD4+ 8+ FOXP3+ Cells , 2008, American journal of reproductive immunology.

[83]  V. Ravindran,et al.  Real-Time Quantitative PCR Measurement of Ileal Lactobacillus salivarius Populations from Broiler Chickens To Determine the Influence of Farming Practices , 2007, Applied and Environmental Microbiology.

[84]  A. Mencacci,et al.  Examination of bacterial contamination at the time of embryo transfer, and its impact on the IVF/pregnancy outcome , 2007, Journal of Assisted Reproduction and Genetics.

[85]  Evan R. Ball,et al.  Angiogenesis in implantation , 2007, Journal of Assisted Reproduction and Genetics.

[86]  J. Xaus,et al.  Two Lactobacillus strains, isolated from breast milk, differently modulate the immune response , 2007, Journal of applied microbiology.

[87]  J P Flandrois,et al.  16S rRNA sequencing in routine bacterial identification: a 30-month experiment. , 2006, Journal of microbiological methods.

[88]  J. Xaus,et al.  Lactobacillus salivarius CECT 5713, a potential probiotic strain isolated from infant feces and breast milk of a mother-child pair. , 2006, International journal of food microbiology.

[89]  J. Xaus,et al.  Antimicrobial potential of four Lactobacillus strains isolated from breast milk , 2006, Journal of applied microbiology.

[90]  Guoyao Wu,et al.  Evidence for altered placental blood flow and vascularity in compromised pregnancies , 2006, The Journal of physiology.

[91]  A. Kondo,et al.  Display of alpha-amylase on the surface of Lactobacillus casei cells by use of the PgsA anchor protein, and production of lactic acid from starch. , 2006, Applied and environmental microbiology.

[92]  J. Marrazzo,et al.  Molecular identification of bacteria associated with bacterial vaginosis. , 2005, The New England journal of medicine.

[93]  M. Yudin.  Bacterial vaginosis in pregnancy: diagnosis, screening, and management. , 2005, Clinics in perinatology.

[94]  L. Nardo.  Vascular endothelial growth factor expression in the endometrium during the menstrual cycle, implantation window and early pregnancy , 2005, Current opinion in obstetrics & gynecology.

[95]  Hai-yan Lin,et al.  Involvement of ERK1/2 pathway in TGF‐β1‐induced VEGF secretion in normal human cytotrophoblast cells , 2004, Molecular reproduction and development.

[96]  A. Stavreus-Evers,et al.  Circulating angiogenic factors during periovulation and the luteal phase of normal menstrual cycles. , 2004, Fertility and sterility.

[97]  L. S. Leshin,et al.  Wound Healing and Angiogenic Properties of Supernatants from Lactobacillus Cultures , 2003, Experimental biology and medicine.

[98]  L. Wildt,et al.  Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function. , 2003, Fertility and sterility.

[99]  Alexandra Kaider,et al.  Bacterial vaginosis as a risk factor for preterm delivery: a meta-analysis. , 2003, American journal of obstetrics and gynecology.

[100]  S. Robertson,et al.  Transforming growth factor β—a mediator of immune deviation in seminal plasma , 2002 .

[101]  S. Robertson,et al.  Defining the actions of transforming growth factor beta in reproduction , 2002, BioEssays : news and reviews in molecular, cellular and developmental biology.

[102]  A. Rutherford,et al.  Rates of bacterial vaginosis in women undergoing in vitro fertilisation for different types of infertility , 2002, BJOG : an international journal of obstetrics and gynaecology.

[103]  E. Shalev,et al.  Bacterial colonization of the uterine cervix and success rate in assisted reproduction: results of a prospective survey. , 2002, Human reproduction.

[104]  K. Whaley,et al.  Origins of vaginal acidity: high D/L lactate ratio is consistent with bacteria being the primary source. , 2001, Human reproduction.

[105]  S. Robertson,et al.  The role of semen in induction of maternal immune tolerance to pregnancy. , 2001, Seminars in immunology.

[106]  A. Onderdonk,et al.  Nugent Score Related to Vaginal Culture in Pregnant Women , 2001, Obstetrics and gynecology.

[107]  J. Schnürer,et al.  Lactobacillus coryniformis subsp.coryniformis Strain Si3 Produces a Broad-Spectrum Proteinaceous Antifungal Compound , 2001, Applied and Environmental Microbiology.

[108]  K. Agnew,et al.  Bacteria in the transfer catheter tip influence the live-birth rate after in vitro fertilization. , 2000, Fertility and sterility.

[109]  M. Diamond,et al.  Expression and regulation of vascular endothelial growth factor in a first trimester trophoblast cell line. , 2000, Placenta.

[110]  O. Carpén,et al.  VEGF, its receptors and the tie receptors in recurrent miscarriage. , 2000, Molecular human reproduction.

[111]  J. Grudzinskas,et al.  Prophylactic antibiotics and endocervical microbial inoculation of the endometrium at embryo transfer , 1999, The Lancet.

[112]  M. Benahmed,et al.  Seminal transforming growth factor-beta in normal and infertile men. , 1999, Human reproduction.

[113]  E. Kawinski,et al.  Plasmin, substilisin-like endoproteases, tissue plasminogen activator, and urokinase plasminogen activator are involved in activation of latent TGF-beta 1 in human seminal plasma. , 1998, Biochemical and biophysical research communications.

[114]  R. Fanchin,et al.  Microbial flora of the cervix assessed at the time of embryo transfer adversely affects in vitro fertilization outcome. , 1998, Fertility and sterility.

[115]  J. Suárez,et al.  Adherence of Human Vaginal Lactobacilli to Vaginal Epithelial Cells and Interaction with Uropathogens , 1998, Infection and Immunity.

[116]  S. Fox,et al.  Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenes , 1997, Cancer research.

[117]  J. Piper,et al.  Bacterial vaginosis and intraamniotic infection. , 1997, American journal of obstetrics and gynecology.

[118]  J. Grudzinskas,et al.  Fertilization and early embryology: Incidence of microbial growth from the tip of the embryo transfer catheter after embryo transfer in relation to clinical pregnancy rate following in-vitro fertilization and embryo transfer , 1996 .

[119]  R. Jaffe,et al.  Angiogenesis in the human female reproductive tract. , 1995, Obstetrical & gynecological survey.

[120]  J. Rossant,et al.  Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium , 1995, Nature.

[121]  M. Krohn,et al.  The Genital Flora of Women with Intraamniotic Infection , 1995 .

[122]  T. Chu,et al.  Characterization of Latent Transforming Growth Factor‐β From Human Seminal Plasma , 1995, American journal of reproductive immunology.

[123]  M A Krohn,et al.  Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation , 1991, Journal of clinical microbiology.

[124]  L. Peeters,et al.  Early pregnancy wastage: relationship between chorionic vascularization and embryonic development. , 1988, Fertility and sterility.

[125]  Caiyan Wang,et al.  Distinct pattern of Th 17 / Treg cells in pregnant women with a history of unexplained recurrent spontaneous abortion , 2018 .

[126]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[127]  R. Knight,et al.  Microbiota restoration: natural and supplemented recovery of human microbial communities , 2011, Nature Reviews Microbiology.

[128]  E. Diamandis,et al.  Potential role of multiple members of the kallikrein-related peptidase family of serine proteases in activating latent TGFβ1 in semen , 2010, Biological chemistry.

[129]  S. Wahl,et al.  TGF-beta: a mobile purveyor of immune privilege. , 2006, Immunological reviews.

[130]  K. Agnew,et al.  Relationship of Vaginal Bacteria and Inflammation With Conception and Early Pregnancy Loss Following In-Vitro Fertilization , 2003, Infectious diseases in obstetrics and gynecology.

[131]  T. J. Poole,et al.  The role of FGF and VEGF in angioblast induction and migration during vascular development , 2001, Developmental dynamics : an official publication of the American Association of Anatomists.

[132]  D. Sherer,et al.  Angiogenesis during implantation, and placental and early embryonic development. , 2001, Placenta.

[133]  L. Giudice,et al.  Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine. , 1994, Fertility and sterility.