Probiotic lactobacilli in formulas and hygiene products for the health of the urogenital tract

Abstract Lactobacilli are the predominant microorganisms of the healthy human vagina. A novel alternative for the prevention and treatment of female urogenital tract infections (UGTI) is the inclusion of these microorganisms as active pharmaceutical ingredients in probiotic formulas, and more recently in female hygienic products. Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” A list of requirements must be considered during the development of probiotic product/formula for the female urogenital tract (UGT). This review aims to resume the requirements, probiotic characteristics, and clinical trial applied to determine the effect of probiotic and potentially probiotic strains on different woman’s physiological and pathological conditions, and in preterm birth prevention. A revision of female hygienic products available in the world market is included, together with novel studies applying nanotechnology for Lactobacillus incorporation in hygienic products. Further studies and well‐designed clinical trials are urgently required to complement the current knowledge and applications of probiotics in the female UGT. The use of probiotic formulas and products will improve and restore the ecological equilibrium of the UGT microbiome to prevent and treat UGTI in women under different conditions.


| HE ALTHY VAG INAL MICROB IOTA-L AC TOBACILLUS PREDOMINAN CE: FUN C TIONS
The human body and their mucosa are lately considered as highly active ecosystems, where microorganisms play a very diverse list of functions and contribute to host nutrition, overall development, defenses, and immune system, response to pathogens, and mucosal cell differentiation and proliferation. The related research and the knowledge of these active communities and their gene contents have been referred collectively as the human microbiome (HM), supported mainly by an NIH-funded project consortium. 1

The Human
Microbiome Project and the European MetaHIT consortium initiated almost two decades ago, aimed at detailed characterization of the structure and the composition of the microbiota from various body areas. 1 It is interesting to remark that microbial numbers within an individual are estimated higher than the human cell number by an order of magnitude. In such a way that the HM is a complex system of many microbial communities, deeply described in terms of composition, diversity, and dynamics, known thanks to the use and update of massively parallel sequencing and other high throughput approaches available, indicating the long list of eukaryotes, archaea, bacteria, and viruses detected. These molecular techniques include Sanger sequencing of 16S rRNA of bacterial colonies, terminal restriction fragment length polymorphism of 16S rRNA, qPCR, and next-generation sequencing that have modified the concept of the microbiome versatilities and more specifically Lactobacillus identification. [2][3][4][5][6][7][8][9][10][11][12][13] Then, the majority of the indigenous microbiota exists in a mutually beneficial relationship with the host, while few are opportunistic pathogens. 14 An important body site providing a habitat for the development of structured microbial communities is the vaginal tract, which is broadly colonized by microorganisms known as the vaginal microbiota (VM). Unique conditions of the vagina are characterized by a few microbial species, being the vaginal microbiome a specific compartment of the HM. 15 The predominance of lactobacilli was described deeply in the vaginal tract in the NIH Project, and this concept agrees with the first proclaimed by Doderlein in the early 1900. 16 The most frequently isolated species are Lactobacillus crispatus, L. gasseri, L. jensenii, and L. iners.
Their relative dominance was studied by Ravel et al. 8  These results indicate that a potential key ecological function, the production of lactic acid, seems to be conserved in all communities, and support at the same time the application of lactic acid producer and immunomodulatory-probiotic bacteria, and are concordant with α diversity studies published later. [17][18][19] Ma and Li 20 have published recently a review on the association between vaginal community state types and species specificity index reclassifying in five groups.
The second phase of the NIH-Human Microbiome Project was the Integrative Human Microbiome Project 21 designed to explore hostmicrobiome interplay, including immunity, metabolism, and dynamic molecular activity to gain a more holistic view of host-microbe interactions over time, in a way to address the relationships between host and microbiome mechanistically. The enormous importance of vaginal microbiome supported its inclusion as one of the systems proposed to be studied in this second phase: pregnancy and preterm birth, to go further in the dynamics of human health and disease-related with known microbiome interactions. In such a way that the concept of reproductive microbiome has been conceived, joining the microbiome of the vaginal tract, placenta and milk/mammary gland, and the direct relationship with fetal development, birth, and newborn features. 22 In addition, the VM of the mother plays an essential role in the initial colonization of newborn babies and therefore the development of a healthy gastrointestinal and skin microbiota. The maternal microbiota exerts an indirect effect on the fetus via maternal factors, such as maternal immune responses or microbial metabolites that cross the placenta, 23,24 or other indirect factors mediating epigenetic programming in the fetus (diet, stress, or neuroendocrine exposure).
Kaminska and Gajecka 25 discuss the influence of human VM, not only bacteria but also viruses and fungi that constitute important components of the reproductive tract microbiome. The impact of the maternal microbiome on fetal development, and the establishment of neonatal microbiomes, including the placenta microbiome, and the hematogenous source of intrauterine infection on the health status of women were analyzed. On the other side, some evidence indicates that infertile patients harbor a different reproductive tract microbiome compared with healthy and fertile women. 26 Fertilization occurs in the uterus, an immune-protected organ, considered a sterile site maintained by the cervical plug for centuries. But the microbial communities in the endometrial cavity and its implications in reproductive health and disease, particularly chronic endometritis was published. 27 Even though bacterial DNA in blood in placenta was demonstrated, mechanisms and functions of transplacental trafficking of free nucleic acids are still in discussion. Then, microbes interact with the host cells along the female reproductive tract, generating the physical, chemical, and biological environment that embryo will encounter during the peri-implementation period and throughout pregnancy. Later, birth represents the first major exposure to a complex microbiota, because the birth canal is always adjacent to the rectum, providing an efficient mechanism for intergenerational transmission of both vaginal and gut microbes. 28 The baby swallows these microorganisms, supported by DNA and live bacteria in the meconium. 29 The maternal gut microbes immediately start to colonize the newborn's own gut, engaging in a kind of conversation with developing immune cells. In this way, the very early microbiome prepares the immune system for healthy functioning later in life. When a baby is born by cesarean section, the gut is seeded with different microbes not those from the mother's gut and vagina, but from her skin and breast milk, the nurse's hands, and other sources. These early differences might have implications that last a lifetime and provides differential colonization and diversity of microorganisms in the intestinal microbiome in C-sections or vaginal born babies later in their lives. 30,31 On the other hand, the urinary tract, previously considered a sterile body niche, has emerged as the host of an array of bacteria in healthy individuals, revolutionizing the urology research field. 32 Specific bacterial communities have found in the healthy urinary tract, which can change in a wide variety of urologic disorders. Then, it is also of main importance to resolve the modulation of the microbiome to improve urinary tract health.

| FAC TOR S AFFEC TING THE VM EQUILIB RIUM
The equilibrated/healthy ecological systems of the reproductive microbiota can be affected by a long list of intrinsic or extrinsic factors. Intrinsic factors or host factors include race, physiologic (hormonal changes, menstruation, pregnancy), immune system imbalances, maturation, and the relationship with genetic susceptibility, cancer, and the phages isolated in the tract. During the menstrual period there is an increment of pH in the area, and high availability of nutrients derived from menstrual bleeding for microbial growth. This period usually causes disturbance and discomfort, with lower number of lactobacilli, consequent undesirable microorganisms, increased infection rates, and recurrences. 8,36,37 In menopause, the estrogens are no longer present and glycogen level decreases, leading to a decrease in lactobacilli. 38 The human immune system restricts microbiota to their natural niches. 28 There is growing evidence that the innate immune   14 The main viral sexually transmitted diseases are human papilloma virus (HPV), human immunodeficiency virus, and herpes simplex virus type 2 (HSV-2). Each pathogen has its unique infection kinetics, pathology, and host evasion mechanisms. In a similar way, the genital mucosal immunity reacts differently toward each of them.
The urinary tract has emerged as the host of an array of bacteria in healthy individuals. There is a microbiome associated with the healthy urinary tract that can change in certain urological disorders. 32, 56 The vagina is a key anatomical site in the pathogenesis of UTI in women, serving as a potential reservoir for infecting bacteria, and also the organ where the ascendant colonization from rectum begins. 57 UTIs in females usually start as vaginal infections and ascend to the urethra and bladder. 58 Women are affected with an estimated UTI lifetime risk of 60%, and 50%-fold predominance compared with adult men 59 increasing with age. 60 Single episodes are very common, being Escherichia coli the most common cause of UTI 57,61 or different Gram-positive bacteria. 62 Recurrent UTI occurring in up to one-third of women after the first episode, 63 showed to be related to a decreased vaginal lactobacilli. 64 Rates of UTI begin to rise at the climateric, and recurrences are one of the features of menopause. 65 Vaginal tract is then a site at which probiotic interventions may decrease the risk of UTI. 57,58,66 A wide diversity of antimicrobial compounds is applied to treat UGTI by vaginal or oral administration, including antibacterial, antifungal, antiparasitic, and antiviral agents. The antibiotic use decreases the vaginal Lactobacillus number, promotes resistance, and sometimes infection recurrences. In such a way that the resistance to antimicrobials can be transmitted either transversally or horizontally to other members of the microbiome, generating multi-resistant microorganisms. The "resistome" concept has emerged, representing the collection and variety of genes that confer antimicrobial resistance (biocides, heavy metals, plasmids) in different microbiomes, spread over multiple areas, 67 analyzed lately by targeted metagenomics techniques. 68 For this reason, and supported by different concerns related to public health and the prevention of infections, the urgent requirement of adequate alternatives emerged in the last two decades.

| PROB I OTI C A S PROP OSAL THER APIE S . S TR AIN , AND HOS T S PECIFI CIT Y
One of the therapies applied for UGTI are probiotics. The definition was submitted to many discussions by government and scientific organizations during the last 30 years, and at the end is live microorganisms that, when administered in adequate amounts, confer a physiological health benefit on the host. 69,70 However, more recently, new definitions have complemented the probiotic area. The term "paraprobiotics" means dead or inactive cells of probiotics that have shown a significant effect on human health [71][72][73][74] ; while the term "postbiotics" is used to describe healthful metabolites of probiotics also showing effect on the host. [75][76][77][78] Lately, Zendeboodi et al. 79 proposed three main classes of probiotic products, including "true probiotic" as viable and active probiotic cell, "pseudo-probiotic" as viable and inactive cells in vegetative or spore forms, and "ghost probiotic" as dead/nonviable cell, either intact or ruptured.
Probiotics have been widely studied and applied in the field of food and food supplements, with no particular negative effect. [80][81][82] In the last few years, the pharmaceutical industry showed a growing interest in new formulations containing beneficial microorganisms, in many cases specific to the host. 83,84 However, the increased interest in the clinical application of probiotics requires specific attention by their administration to non-healthy population. More recently, the Food and Drug Administration has defined a new "live biotherapeutic products" (LBP) category. Then, the documents and demonstration of quality, safety, and efficacy of new products, including LBP, are framed by the characteristics and risks of the aimed population, as well as those of the strains and product components, in such a way that the global benefit-risk ratio can be assessed regarding their intended use. 85 Not all the Lactobacillus strains can be considered as probiotics because the beneficial effect must be evidenced in the specific host as stated. The strains included in different products/formulas must be submitted to a long list of protocols and trials to be named as probiotics. Also, a body of different criteria applied, reviewed by different authors, to be considered as probiotic strains. 69,86 The range of functional genomics to identify genes and gene products that govern the distinctive phenotypes and health associations of probiotic strains was reviewed, recognizing the strain specificity of probiotic effects. 87 This strain specificity and the disease specificity of probiotic efficacy, and the need to take these two factors: both specific strains and type of disease, when recommending the appropriate probiotic effect for each patient is also recommended. 88,89 One of the first considerations is the host specificity, which means that the strains should be isolated from the same host where will be applied. Even though some strains are used historically as probiotic or included in foods, the host specificity indicates a higher possibility to be adapted to the host conditions. Referred to the vaginal tract or mucosal specificity, Yildirim et al. 90 have shown that all primates exhibited host-specific VM and that humans were distinct from other primates in both microbiome composition and diversity. Mendes-Soares et al. 91 demonstrated that genomes of vaginal species were significantly smaller and had significantly lower GC content than those of non-vaginal species.
More specifically, Van Der Veer et al. 92

| REQU IREMENTS FOR PROB I OTI C S FORMUL A S/PRODUC TS
In the design of probiotic formulas/products, different sets of criteria and protocols must be applied according to the requirements and exigencies of regulatory organisms, described in different revisions, added to the host and tract specificity described should be assumed of sharing the same documented safety history with preexisting ones. 96 Different efficacy and safety assessment protocols were recommended by the experts for a putative probiotic candidate, before confirmation and acceptance for public consumption. In order to guarantee the probiotic safety, each strain must be assayed in some aspects: antibiotic-resistance patterns; assessment of certain metabolic activities; toxin production; determination of hemolytic activity; side-effects in experimental animal models; phase I trials; epidemiological surveillance of adverse incidents in consumers, between other. 69 Refered to the potential dissemination of the resistance mediated by genetic mechanisms, such as horizontal gene transfer where plasmids, transposons and integrons may be involved, 97-101 is essential to evaluate the susceptibility patterns of potentially probiotic bacteria. 102 Microbial resistance to clinically relevant antibiotics must be absent as part of the QPS assessment of bacteria deliberately introduced into the food chain. 103 Also it is important to screen the enzymes acting as potential virulence factors, as hydrolytic enzymes (hemolysin, lecithinase, gelatinase, etc.) able to produce damage to the host. [104][105][106][107] The temporal persistence and colonization or permanence capability of beneficial microorganisms, and their cellular and molecular effects on the integrity of host mucosa and immune system must also be assayed. [108][109][110] The formulas for the UGT are designed for oral or local administration. The oral delivery is supported by the microbial ability to survive through gastrointestinal tract, and to ascend to vagina after their excretion from the rectum, in ascendant colonization. [111][112][113] Whereas vaginal application allows direct and tar- as well as also in the prevention of preterm birth (Table 1). In the present review, most of the available clinical trials published between 2015 and 2020 were included, because previous trials were analyzed before. 86,142 In non-pregnant, fertile women (Table 1, white panel), probiotic strains were assayed to: evaluate their safety, and to improve the VM of healthy women 129,139,143,151 ; restore the abnormal VM 129,140,159,165 ; prevent recurrent UGTI 141,166 ; as adjunct treatment to BV, 37,160,161,167 AV 160 antimicrobial therapy, recurrent-VVC (R-VVC), 152,162 and TV in BV presence 144 ; as BV and R-VVC subsequent treatment 141,162,169 ; and HPV clearance. 145,153 Most of the publications have evaluated commercial, or potential probiotic formulas, including oral and vaginal capsules, vaginal tablets, suppositories or powders, and even yogurt, containing individual or combined Lactobacillus strains of urogenital, intestinal, or food origins, as shown in Table 1.
Lactobacillus strains combined with bovine-lactoferrin were also as- Ou et al. 153 published that the application of probiotic strains did not influence genital high risk-HPV clearance, but decreased the rates of mildly abnormal and unsatisfactory cervical smears.
Probiotic application in pregnant women (Table 1,  Streptococcus (GBS) was reported. 150,157,168 However, oral probiotics taken from early pregnancy showed no effect on vaginal health during mid and end gestation, 155,156 or 3 weeks doses or until birth in 36-week-pregnancy were ineffective in impacting GBS vaginal colonization. 158 The addition of vaginal probiotics to standard antibiotic treatment on perinatal outcome in preterm premature rupture of membranes pregnancy prolonged the bith gestational age, the latency period length, improving newborn weight and health. 163 On the other hand, the oral application of probiotic in maternal health during pregnancy, postpartum, and in infant eczema and allergy prevention was evaluated. 146-149 L. rhamnosus HN001 during pregnancy reduced gestational diabetes mellitus (GDM) prevalence, particularly among older women and those with previous GDM, 147 and the depression and anxiety scores in women in the postpartum period. 148 However, L. rhamnosus HN001 did not reduce the infant eczema. 149 In other way, Anoshina 168 showed that combined Lactobacillus strains to HSV-women decreased the complaints incidence of bloating, discomfort, constipation, mucus in stool, excessive vaginal discharge, itching, swelling and mucosa redness, reducing the placental insufficiency and preeclampsia as well as fetal distress incidence.
In postmenopausal women, the application of probiotic strains (Table 1,

| HYG IENE PRODUC TS FOR THE PRE VENTI ON OF UG TI
The probiotic administration in hygiene products, as tampons and pads, has emerged as a new possibility to carry beneficial strains and exert the claimed effect. The pharmacological aspects of the probiotic products for feminine hygiene including sanitary towels and tampons, classified in several countries as medical devices, are scarcely described. They can act in the VM restoration, and in some cases, could reduce the risk associated with toxic shock syndrome produced by Staphylococcus aureus strains, with higher incidence in women who use tampons. 171 The availability of everyday feminine hygiene products (e.g., tampons, sanitary napkins, panty liners) containing probiotic microorganisms from vaginal origin is limited in the market and available only in some regions of the world. Table 2 shows the feminine hygiene products containing vaginal probiotic lactobacilli assayed for pH balancing and protection during men-    The coating system was successfully applied in several products, as stent, soybeans, cellulose paper, with a wide variety of bioactive immobilized in nanofibers through electrospinning. [180][181][182] Then,