Can Lactobacillus spp. Be a Factor Reducing the Risk of Miscarriage?

The vagina contains various cells and receptors associated with the immune system that recognize and respond to the presence of microorganisms ( Wira et al. 2005 ). Both commensal and pathogenic bacteria are recognized by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), dectin-1 receptor, and nucleotide-binding oligomerization domain (NOD). Those receptors are present on both the squamous epithelial cells lining the vagina and the columnar cells lining the upper section of the female genitalia ( Villa et al. 2020 ). The decidua cells such as T lymphocytes, macrophages and natural killer cells (NK) under stimulation produce specific cytokines. Cell-to-cell communication at the mother-embryo interface leads to changes in the expression of the type and amount of cytokines. Immune tolerance or immune stimulation may be related to modifications in the cytokine pattern of T lymphocytes. How disruption of the vaginal microbial ecosystem and the endometrium may adversely affect implantation and miscarriage is not fully understood. Al-Nasiry et al. (2020) proposed possible mechanisms that may contribute to the impact of bacteria on the implantation process. First, the dominance of non-commensal bacteria may weaken the integrity of the endometrial mucosal barrier by affecting the tight junctions of the epithelium. It, in turn, may further weaken host defense mechanisms and allow pathogens to penetrate the endometrial stroma and induce an immune response by antigen-presenting cells (APCs) and other immune cells expressing PRRs. Abnormal stimulation of T lymphocytes, either directly by invading pathogens breaching the mucosal barrier or indirectly by absorbed bacterial products, results in an imbalance in cytokine production in favor of pro-inflammatory T helper 1 cells (Th1), dominated by tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interleukin-2 (IL-2) ( Al-Nasiry et al. 2020 ). Lactobacillus spp. is one of the most dominant genera of the healthy vaginal microbiota. Nevertheless, the interactions between this commensal bacterium and the immune system are largely unknown ( Keelan 2011 ). Interactions between the microbiota of the reproductive tract and components of the immune system, located in the vagina and uterus, may influence the production of a specific environment, favorable or unfavorable for the development of pregnancy. It is known that lactic acid bacteria can interact with mucosal immune cells or epithelial cells lining the mucosa to modulate specific immune system functions ( Wells 2011 ). Lactic acid, produced by Lactobacillus spp., shows immunomodulatory properties by inducing an anti-inflammatory response in vaginal and cervical epithelial cells. The balance of cytokines secreted by Th1 and Th2 cells is a critical component of a normal immune response ( Szekeres-Bartho and Wegmann 1996 ; Wells 2011 ; Valenti et al. 2018 ). Cytokines. Microbial stimulation of PRR initiates signaling cascades, leading to the activation of specialized cells, including NK cells, macrophages, CD4 + and CD8 + T cells, and cytotoxic T lymphocytes, and the secretion of specific cytokines ( Genc et al. 2004a ; 2004b ). It has been shown that specific bacterial species in the vagina can affect the pattern of secreted cytokines. The lactic acid and hydrogen peroxide produced by Lactobacillus spp. bacteria can modulate cytokine production. Women whose vaginal microbiota is classified as CST-IV have been shown to have increased levels of cytokines such as TNF-α, IFN-γ, interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-4 (IL-4), and interleukin-8 (IL-8). Similarly, diagnosed BV has been shown to increase levels of immune mediators such as IL-1β, IL-8, TNF-α, IFN-γ, IL-2, interleukin-6 (IL-6) ( Beghini et al. 2015 ), and AV has been shown to increase levels of IL-1β and IL-6 in vaginal secretions, which may promote obstetric complications ( Amabebe and Anumba 2018 ; Donders et al. 2020 ). Anahtar et al. (2015) showed that Prevotella amnii, Mobiluncus mulieris, Sneathia amnii , and Sneathia sanguinegens (CST-IV) induce upregulation of IL-1α, IL-1β, and IL-8 secretion. Additionally, women whose vaginal microbiota is classified as CST-III showed significantly higher IFN-γ and IL-8 levels compared to CST-I. During the transition from CST-I to CST-III and CST-IV, significant increases in IL-1α, IL-1β, and TNF-α were observed ( Anahtar et al. 2015 ). Both in humans and a mouse model, increases in pro-inflammatory cytokines were found to be associated with an increased risk of pregnancy loss ( Clark et al. 1998 ; Raghupathy et al. 1999 ). Production of specific cytokines can affect fertility and pregnancy maintenance ( Marzi et al. 1996 ; Garzia et al. 2013 ). Increased production of IL-2 and decreased interleukin-10 (IL-10) has been observed in reproductive disorders ( Marzi et al. 1996 ; Garzia et al. 2013 ). In a study conducted by Xu et al. (2020) low number of Lactobacillus spp. in the vagina correlated with increased IL-2 in women who had a miscarriage early in pregnancy. The presence of H 2 O 2 produced by lactic acid bacteria appeared to be associated with lower levels of certain vaginal pro-inflammatory cytokines. Additionally, increased amounts of L. crispatus were related to the inhibition of IL-1β production ( Xu et al. 2020 ). In a mouse model of Lactobacillus rhamnosus HN001, Lactobacillus acidophilus LA-14 inhibited G. vaginalis -induced expression of IL-1β, TNF-α, and interleukin-17 (IL-17). In contrast, IL-10 expression increased due to L. rhamnosus HN001 and L. acidophilus LA-14 treatment ( Jang et al. 2017 ). The IL-2, TNF-α and IFN-γ cytokines have been shown to significantly increase in the serum of infertile patients ( An et al. 2015 ). It has also been proven that vulvar and vaginal candidiasis can contribute to reproductive disorders by increasing the production of certain cytokines. Another study shows that the vaginal mucosa has a potential function in local immune responses against pathogens, not only bacterial but also fungal, which may result in obstetric complications ( Niu et al. 2017 ; Abdul-Aziz et al. 2019 ). Natural killer cells. Natural killer (NK) cells are present in peripheral blood (pNK) and uterine tissue (uNK) ( Moffett et al. 2004 ). It has been shown that uNK and pNK cells may be associated with reproductive processes ( Thum et al. 2007 ; Kuon et al. 2017 ). The uNK cells play a key role in the initiation and maintenance of pregnancy. The uNK cells are not cytotoxic, secrete pro-angiogenic factors, and regulate trophoblast invasion. They are involved in the remodeling of spiral arteries thus have a beneficial effect on pregnancy. After successful implantation, the uNK cells reach a peak and constitute about 70% of all uterine lymphocytes in the first trimester of pregnancy but decrease in the second half of pregnancy ( Bulmer et al. 1991 ; Dons’koi et al. 2014 ). A different role in reproductive processes has been attributed to pNK cells. There are reports that increased levels of pNK cells may have a negative effect on reproduction ( Thum et al. 2007 ). It can be hypothesized that the presence of a potentially pathogenic microorganism can stimulate an inflammatory response leading to systemic changes in immune parameters revealed by pNK elevation. It is well known that lipopolysaccharides from Gram-negative bacteria are potent immunostimulators. Indeed, LPS is a potent activator of NK cell activity ( Lindemann 1988 ; Kuon et al. 2017 ). Some studies indicate that women with RM have altered peripheral blood NK parameters (increased numbers and/or activation levels) compared to women without diagnosed RM ( King et al. 2010 ). The role of Lactobacillus spp. in pNK regulation may be related to its function in maintaining proper vaginal pH. Fluctuations in vaginal pH due to changes in the vaginal microbiota have increased susceptibility to infections, which may indirectly affect fertility. Patients with unexplained infertility reported an association of abnormal vaginal flora with increased levels of TNF-α and IFN-γ in cervical mucus, which was related to increased numbers of pNK cells ( Nakano et al. 2015 ). Recent research showed a significantly higher percentage of pNKs correlated with the presence of G. vaginalis in the vagina of women with RM (Seshadri and Sunkara 2014 ), but no association between the presence of G. vaginalis in the vagina and the amount of uNKs. In addition, the lack of Lactobacillus spp. has been shown to correlate with a decreased number of pNK cells ( Kuon et al. 2017 ). However, there is still no pathophysiological explanation as to why pNK is elevated in a group of women with RM ( Park et al. 2010 ; Kuon et al. 2017 ; Fu et al. 2021 ). NK cells are innate lymphocytes with a CD3 ‒ CD56 + phenotype. Studies indicate that CD56 +bright NK cells, which have a high affinity for IL-2 and produce various cytokines, are predominantly present in the uterus during pregnancy ( Vince and Johnson 2000 ; Koopman et al. 2003 ), while the presence of CD56 +dim NK cells, with moderate affinity for IL-2, having cytotoxic activity in pregnancy is related to the risk of miscarriage ( King et al. 2010 ). The relationship between peripheral and uterine NK cells is still unclear. It is commonly argued that blood and uterine NK cells have different phenotypes and that uNK cells are benign, produce cytokines, and are likely essential for normal pregnancy ( Moffett-King 2002 ). However, there is some evidence for the transfer of pNKs to the uterus and their differentiation to uNK-like phenotypes in pregnancy ( King et al. 2010 ; Cerdeira et al. 2013 ). The pNKs are increasingly implemented as a useful diagnostic tool to initiate immunomodulatory therapies in patients with RM. Extracellular trap. In 2004, Brinkmann et al. first described a new protective mechanism of neutrophils, known as a formation of neutrophil extracellular traps (NETs). The NETs, which are composed of DNA strands, histones, neutrophil elastase, myeloperoxidase, other peptides, enzymes such as lactoferrin, lysozyme C, neutrophil defensins, cathepsin G, gelatinase, cathelicidins, leukocyte proteinase 3, and calprotectin are shed under the influence of pathogens ( Nija et al. 2020 ). The NET formation is one of the mechanisms to fight pathogens ( Brinkmann and Zychlinsky 2012 ). Subsequent studies have shown that extracellular traps can be produced not only by neutrophils but also by macrophages ( Aulik et al. 2012 ; Hellenbrand et al. 2013 ), monocytes ( Muñoz-Caro et al. 2015 ), eosinophils ( Yousefi et al. 2008 ), as well as basophils ( Morshed et al. 2014 ). The formation of neutrophil extracellular traps is not always beneficial to health. NETosis is an effective antimicrobial mechanism that protects the host from several infectious diseases. At the same time, it is a double-edged sword of the innate immune system in the sense that if neutrophil extracellular traps are produced in excess or if they are not removed promptly, it can induce many diseases, including autoimmune disorders, coagulation disorders, and even cancer metastasis ( Nija et al. 2020 ). It has been suggested that neutrophils have a protective role at the maternal-fetal tissue interface. In the case of infection or other stimuli not yet studied, neutrophils become over-activated and cause damage to the placenta and fetal membranes ( Tong and Abrahams 2020 ). Preliminary studies show that overproduction of NETs in pregnancy is a detrimental phenomenon to pregnancy and can cause, among others, pre-eclampsia at the end of pregnancy. It is hypothesized that NETs occupy space within the trophoblast villi, reduce blood flow in the placental vessels, and ultimately cause fetal hypoxia. NETosis, aided by activated vascular endothelial cells, can destroy maternal endothelial cells ( Brinkmann and Zychlinsky 2012 ; Niedźwiedzka-Rystwej et al. 2019 ). The study conducted by Omeljaniuk et al. (2020) evaluated neutrophil extracellular traps in women who had a miscarriage during the first trimester of pregnancy. The study material consisted of the woman’s blood serum and trophoblast fragments after miscarriage. The presence of essential structural elements of NET was observed in the trophoblast fragments. According to the author, the presence of NET structural elements in the placenta correlated with their presence in the mother’s peripheral blood suggests a relationship between NETosis and miscarriage ( Omeljaniuk et al. 2020 ). The study conducted by Doster et al. (2018) examined whether ex vivo infection of fetal membrane fragments with GBS could affect the formation of macrophage extracellular traps (METs). Extracellular trap-associated structures were found in fetal membrane fragments, confirming MET formation after GBS stimulation. Thus, infection with pathogenic bacteria can cause extracellular traps in the placenta and thus affects the fetus ( Doster et al. 2018 ). Can Lactobacilli regulate extracellular trap formation? A report by Vong et al. (2014) indicates that in a mouse model using bone marrow-derived cells, the probiotic strain L. rhamnosus GG inhibits S. aureus -induced NET for neutrophil motion. Moreover, LGG suppressed reactive forms of oxygen production and phagocytic capacity of neutrophils, thus possibly providing some level of hyporeactivity ( Vong et al. 2014 ; Mutua and Gershwin 2021 ). The ability of LGG to inhibit S. aureus -induced NETs also translates into protection against cellular cytotoxicity. The S. aureus secretes pore-forming toxins that cause lysis of neutrophils, including leukocidin, which has been shown previously to induce NET formation ( Pilsczek et al. 2010 ). It remains to be determined whether LGG secretes bacteriocins that have antimicrobial activity against S. aureus or whether it directly interferes with the production of toxins secreted by S. aureus ( Vong et al. 2014 ). It is not clear whether Lactobacillus spp. can exert beneficial effects on pregnancy by inhibiting neutrophil extracellular traps formation. Further studies are needed to address whether a similar mechanism to Vong et al. (2014) may occur in placental membranes. These studies could provide evidence of new immunomodulatory properties of Lactobacillus spp. in pregnancy by regulating the formation of extracellular traps.

Miscarriage is one of the most common obstetric complications. The abnormal vaginal and uterine microbial composition may be one of the factors that increase the risk of miscarriage. Lactobacillus spp. is the most common bacteria within the reproductive tract. Microbiological tests before conception and in early pregnancy to determine the vaginal microbial composition may be important to understand the mechanisms that promote proper embryo implantation, placenta formation, and reduce the incidence of miscarriage. Although the presence of Lactobacilli in the vagina has long been confirmed, its presence in the uterus continues to raise some doubts. Future research should focus on determining whether BV diagnosed among women who have had a miscarriage is its result or has developed independently. It is important to determine whether the presence of Lactobacilli significantly prevents pregnancy loss. Furthermore, it is necessary to assess the prevalence of different clonal variants of L. iners , which in some cases promote vaginal health and in others are associated with dysbiosis and gynecologic complications in pregnancy. In addition, a thorough understanding of both the molecular and immunological mechanisms of host- Lactobacillus spp. interaction is required. Only by considering these relationships will it be possible to answer the question of the importance and extent of the protective role of Lactobacillus spp. in miscarriage.

Pregnancy loss is a common obstetric problem, affecting up to 25% of pregnancies worldwide ( Larsen et al. 2013 ; Al-Memar et al. 2020 ). A miscarriage is the expulsion of a fetal egg from the uterus up to 22 weeks of gestation. Miscarriages can be divided into early miscarriages, up to 12 weeks of gestation, and late miscarriages, occurring between 12 and 22 weeks of gestation ( Larsen et al. 2013 ). The European Society of Human Reproduction and Embryology (ESHRE) has introduced the additional term recurrent miscarriage (RM) when there are three or more consecutive pregnancy losses ( Farquharson et al. 2005 ; Jauniaux et al. 2006 ; Christiansen et al. 2008 ). The occurrence of early miscarriage is dependent on the woman’s age. Among women aged 20–24 years, it is 10% of pregnancies, while in women aged 40 to 44 years, it is 51% of pregnancies. It is related to the higher incidence of genetic aberrations in embryos of older women ( Nybo Andersen et al. 2000 ). Late miscarriages occur less frequently and account for about 4% of all miscarriages ( Ugwumadu et al. 2003 ). Major causes of miscarriage include genetic ( Franssen et al. 2006 ; Branch et al. 2010 ) and epigenetic disorders of the embryo ( Daher et al. 2012 ; Yin et al. 2012 ), immunological ( Holers et al. 2002 ; Calleja-Agius et al. 2012 ), and endocrine factors ( Cocksedge et al. 2009 ), uterine malformations ( Chan et al. 2011 ), improper embryo selection ( Salker et al. 2010 ), and lifestyle ( Larsen et al. 2013 ) ( Fig. 1 ). Perhaps a hitherto underappreciated cause of miscarriage may be an abnormal microbiota composition of the female reproductive system. Currently, the normal state of vaginal and uterine microbiota that would promote a physiological pregnancy is being sought. So far, it has been shown that a normal pregnancy is characterized by a stable vaginal bacterial composition with a dominance of Lactobacillus spp. and low diversity of other bacteria ( Ravel et al. 2011 ; MacIntyre et al. 2015 ) ( Fig. 2 ). Numerous studies show a possible relationship between preterm delivery, a decrease in Lactobacillus spp. and an increase in bacterial biodiversity in the vagina ( Brown et al. 2018 ; Freitas et al. 2018 ; Al-Memar et al. 2020 ), bacterial vaginosis (BV), or aerobic vaginitis (AV). However, the relationship between miscarriage and the vaginal and uterine microbial composition is relatively poorly understood ( Zhang et al. 2019 ; Al-Memar et al. 2020 ; Xu et al. 2020 ). Factors affecting the risk of miscarriage. Normal vaginal microbiota and vaginal dysbiosis. The aim of this study is an attempt to answer the question of whether, in the light of available literature, the Lactobacillus spp. can be a factor reducing the risk of miscarriage.