Ureaplasma parvum alters the immune tolerogenic state in placental tissue and could cause miscarriage.

OBJECTIVE: To study the inflammatory profile and genes involved in the response to bacterial infections in women who developed spontaneous abortion in the presence of Ureaplasma parvum. DESIGN: Cross-sectional study. SETTING: A maternal and child referral center. PATIENT(S): Eighty-nine women with spontaneous abortion and 20 women with normal vaginal delivery (control group) were studied. INTERVENTION(S): Samples of biopsied placental tissue were collected for Mollicutes detection. MAIN OUTCOME MEASURE(S): The samples were subjected to histologic analysis, immunohistochemical evaluation for macrophages and lymphocytes, cytokine quantification, and quantitative polymerase chain reaction array to evaluate the expression of 84 genes related to the innate and adaptive immune responses. RESULT(S): The presence of U. parvum in the abortion group was positively associated with the influx of polymorphonuclear cells in the placental tissue and increased concentrations of interleukin-6 and interleukin-12p70. U. parvum caused downregulation of genes involved in the immune response, such as attraction of immune cells, activation of an inflammatory response, T-helper cell 17 response activation, and activation of the complement system at the beginning and end of pregnancy. CONCLUSION: The direct action of U. parvum on placental tissue altered the gestational tolerogenic state, reducing the immune response against pathogens and activating the extrinsic apoptotic pathway, causing spontaneous abortion.

[Advances in immune escape mechanism of Ureaplasma species: Review].

Ureaplasma urealyticum and Ureaplasma parvum are the most common Ureaplasma species causing repeated or persistent infection of the urogenital tract. The host can mobilize innate and adaptive immunity to defend and eliminate pathogens. However, under certain conditions, the host's immune protection cannot completely clear Ureaplasma species. Ureaplasma species have evolved a complex and sophisticated escape mechanism in the long-term defense against host immune protection. This article summarizes the research progress on Ureaplasma species' immune escape mechanism from several aspects such as evading host autophagy mechanism, antagonizing host nutritional immunity and regulating host cell gene expression.

GrpE Immunization Protects Against Ureaplasma urealyticum Infection in BALB/C Mice.

Nucleotide exchange factor (GrpE), a highly conserved antigen, is rapidly expressed and upregulated when Ureaplasma urealyticum infects a host, which could act as a candidative vaccine if it can induce an anti-U. urealyticum immune reaction. Here, we evaluated the vaccine potential of recombinant GrpE protein adjuvanted by Freund's adjuvant (FA), to protect against U. urealyticum genital tract infection in a mouse model. After booster immunization in mice with FA, the GrpE can induced both humoral and cellular immune response after intramuscular injection into BALB/c mice. A strong humoral immune response was detected in the GrpE-immunized mice characterized by production of high titers of antigen-specific serum IgG (IgG1, IgG2a, and IgG3) antibodies. At the same time, the GrpE also induced a Th1-biased cytokine spectrum with high levels of IFN-γ and TNF-α after re-stimulation with immunogen GrpE in vitro, suggesting that GrpE could trigger the Th1 response when used for vaccination in the presence of FA. Although GrpE vaccination in the presence of a Th1-type adjuvant-induced had readily detectable Th1 responses, there wasn't increase inflammation in response to the infection. More importantly, the robust immune responses in mice after immunization with GrpE showed a significantly reduced U. urealyticum burden in cervical tissues. Histopathological analysis confirmed that tissues of GrpE-immunized BALB/c mice were protected against the pathological effects of U. urealyticum infection. In conclusion, this study preliminarily reveals GrpE protein as a promising new candidate vaccine for preventing U. urealyticum reproductive tract infection.

Multilocus sequence typing characterizes diversity of Ureaplasma diversum strains, and intra-species variability induces different immune response profiles.

BACKGROUND: Ureaplasma diversum is a pathogen found in the genital tract of cattle and associated with genital disorders such as infertility, placentitis, abortion, birth of weak calves, low sperm motility, seminal vesiculitis and epididymitis. There are few studies evaluating the genetic diversity of U. diversum strains and their influence on the immune response in cattle. Therefore, to better understand genetic relationships of the pathogenicity of U. diversum, a multilocus sequence typing (MLST) scheme was performed to characterize the ATCC 49782 strain and another 40 isolates recovered from different Brazilian states. RESULTS: Primers were designed for housekeeping genes ftsH, polC, rpL22, rpoB, valS and ureA and for virulence genes, phospholipase D (pld), triacylglycerol lipase (tgl), hemolysin (hlyA), MIB-MIP system (mib,mip), MBA (mba), VsA (VsA) and ribose transporter (tABC). PCRs were performed and the targeted gene products were purified and sequenced. Sequence types (STs), and clonal complexes (CCs) were assigned and the phylogenetic relationship was also evaluated. Thus, a total of 19 STs and 4 CCs were studied. Following the molecular analysis, six isolates of U. diversum were selected, inoculated into bovine monocyte/macrophage culture and evaluated for gene expression of the cytokines TNF-α, IL-1, IL-6, IL-10 and IL-17. Differences were detected in the induction of cytokines, especially between isolates 198 and BA78, promoted inflammatory and anti-inflammatory profiles, respectively, and they also differed in virulence factors. CONCLUSION: It was observed that intra-species variability between isolates of U. diversum can induce variations of virulent determinants and, consequently, modulate the expression of the triggered immune response.

Maternal azithromycin therapy for Ureaplasma parvum intraamniotic infection improves fetal hemodynamics in a nonhuman primate model.

BACKGROUND: Ureaplasma parvum infection is a prevalent cause of intrauterine infection associated with preterm birth, preterm premature rupture of membranes, fetal inflammatory response syndrome, and adverse postnatal sequelae. Elucidation of diagnostic and treatment strategies for infection-associated preterm labor may improve perinatal and long-term outcomes for these cases. OBJECTIVE: This study assessed the effect of intraamniotic Ureaplasma infection on fetal hemodynamic and cardiac function and the effect of maternal antibiotic treatment on these outcomes. STUDY DESIGN: Chronically catheterized pregnant rhesus monkeys were assigned to control (n=6), intraamniotic inoculation with Ureaplasma parvum (107 colony-forming units/mL, n=15), and intraamniotic infection plus azithromycin treatment (12.5 mg/kg twice a day intravenously, n=8) groups. At approximately 135 days' gestation (term=165 days), pulsed and color Doppler ultrasonography was used to obtain measurements of fetal hemodynamics (pulsatility index of umbilical artery, ductus venosus, descending aorta, ductus arteriosus, aortic isthmus, right pulmonary artery, middle cerebral artery and cerebroplacental ratio, and left and right ventricular cardiac outputs) and cardiac function (ratio of peak early vs late transmitral flow velocity [marker of ventricular function], Tei index [myocardial performance index]). These indices were stratified by amniotic fluid proinflammatory mediator levels and cardiac histology. RESULTS: Umbilical and fetal pulmonary artery vascular impedances were significantly increased in animals from the intraamniotic inoculation with Ureaplasma parvum group (P<.05). Azithromycin treatment restored values to control levels. Amniotic fluid prostaglandin F2 alpha levels were significantly higher in animals with abnormal umbilical artery pulsatility index (>1.1) than in those with normal blood flow (P<.05; Spearman ρ=0.6, P<.05). In the intraamniotic inoculation with Ureaplasma parvum group, left ventricular cardiac output was significantly decreased (P<.001), and more animals had abnormal right-to-left ventricular cardiac output ratios (defined as >1.6, P<.05). Amniotic fluid interleukin-6 concentrations were elevated in cases of abnormal right-to-left ventricular cardiac output ratios compared with those in normal cases (P<.05). CONCLUSION: Fetal hemodynamic alterations were associated with intraamniotic Ureaplasma infection and ameliorated after maternal antibiotic treatment. Doppler ultrasonographic measurements merit continuing investigation as a diagnostic method to identify fetal cardiovascular and hemodynamic compromise associated with intrauterine infection or inflammation and in the evaluation of therapeutic interventions or clinical management of preterm labor.

[Association of Ureaplasma urealyticum with the types of antisperm antibody in infertile men].

OBJECTIVE: To investigate the prevalence of Ureaplasma urealyticum (UU) infection in infertile men, its influence on routine semen parameters and the distribution of antisperm antibody (AsAb) and its types in infertile patients with UU infection. METHODS: We detected the positive rate of UU infection, semen parameters, and the distribution of AsAb and its types in 662 infertile men and 25 normal fertile male controls followed by comparison of the obtained data between the two groups of subjects. RESULTS: The positive rate of UU infection was significantly higher in the infertile men than in the normal controls (52.87% ï¼»350/662ï¼½ vs 16.00% ï¼»4/25ï¼½, χ2 = 11.68, P <0.05). The semen volume, sperm count, sperm concentration and percentage of progressively motile sperm were remarkably lower in the UU-positive infertile males than in the control group (P <0.05). No statistically significant difference was observed between the UU-positive and UU-negative groups in the positive rates of total AsAb (43.4% vs 36.5%, χ2 = 3.25, P >0.05) and AsAb IgA, IgM and IgG in the seminal plasma, or in the percentages of serum AsAb IgM (16.9% vs 20.5%, χ2 = 1.22, P >0.05) and IgG (32.7% vs 28.9%, χ2 = 0.99, P >0.05) except in that of serum AsAb IgA (23.6% vs 17.0%, χ2 = 4.03, P <0.05). CONCLUSIONS: The UU infection rate is high in infertile males, which decreases the semen volume, total sperm count, motile sperm concentration and percentage of progressively motile sperm and increases the positive rate of serum AsAb IgA.

Foetal Ureaplasma parvum bacteraemia as a function of gestation-dependent complement insufficiency: Evidence from a sheep model of pregnancy.

PROBLEM: Complement is a central defence against sepsis, and increasing complement insufficiency in neonates of greater prematurity may predispose to increased sepsis. Ureaplasma spp. are the most frequently cultured bacteria from preterm blood samples. METHOD OF STUDY: A sheep model of intrauterine Ureaplasma parvum infection was used to examine in vivo Ureaplasma bacteraemia at early and late gestational ages. Complement function and Ureaplasma killing assays were used to determine the correlation between complement potency and bactericidal activity of sera ex vivo. RESULTS: Ureaplasma was cultured from 50% of 95-day gestation lamb cord blood samples compared to 10% of 125-day gestation lambs. Bactericidal activity increased with increased gestational age, and a direct correlation between functional complement activity and bactericidal activity (R2 =.86; P<.001) was found for 95-day gestational lambs. CONCLUSIONS: Ureaplasma bacteraemia in vivo was confined to early preterm lambs with low complement function, but Ureaplasma infection itself did not diminish complement levels.

Intra-amniotic Ureaplasma parvum-Induced Maternal and Fetal Inflammation and Immune Responses in Rhesus Macaques.

BACKGROUND: Although Ureaplasma species are the most common organisms associated with prematurity, their effects on the maternal and fetal immune system remain poorly characterized. METHODS: Rhesus macaque dams at approximately 80% gestation were injected intra-amniotically with 107 colony-forming units of Ureaplasma parvum or saline (control). Fetuses were delivered surgically 3 or 7 days later. We performed comprehensive assessments of inflammation and immune effects in multiple fetal and maternal tissues. RESULTS: Although U. parvum grew well in amniotic fluid, there was minimal chorioamnionitis. U. parvum colonized the fetal lung, but fetal systemic microbial invasion was limited. Fetal lung inflammation was mild, with elevations in CXCL8, tumor necrosis factor (TNF) α, and CCL2 levels in alveolar washes at day 7. Inflammation was not detected in the fetal brain. Significantly, U. parvum decreased regulatory T cells (Tregs) and activated interferon γ production in these Tregs in the fetus. It was detected in uterine tissue by day 7 and induced mild inflammation and increased expression of connexin 43, a gap junction protein involved with labor. CONCLUSIONS: U. parvum colonized the amniotic fluid and caused uterine inflammation, but without overt chorioamnionitis. It caused mild fetal lung inflammation but had a more profound effect on the fetal immune system, decreasing Tregs and polarizing them toward a T-helper 1 phenotype.

T cell cytokine responses to stimulation with Ureaplasma parvum in pregnancy.

UNLABELLED: Ureaplasma spp. are a common vaginal microorganism causally linked to inflammation-driven preterm birth (PTB). The nature of the immune response to Ureaplasma spp. may influence PTB risk. This study sought to define maternal T cell cytokine responses to in vitro stimulation with Ureaplasma parvum serovar 3 (UpSV3) in vaginally colonised (UP+) and non-colonised (UP-) pregnant women. Whole blood flow cytometry demonstrated an increase (p=0.027) in the baseline frequency of IFNγ-positive CD3(+)CD4(-)(CD8(+)) T cells in UP+ women. UpSV3 stimulation resulted in a significant and specific increase (p=0.001) in the frequency of IFNγ-positive CD3(+)CD4(-)(CD8(+)) T cells, regardless of vaginal colonisation status. UpSV3 stimulation also increased the frequency of IFNγ-positive CD3(+)CD4(+) T cells, particularly in the UP+ group (p=0.003). This is the first published study to examine T cell responses to Ureaplasma spp. EXPOSURE: Future appropriately-powered studies are needed to assess whether insufficient priming or a loss of tolerance to Ureaplasma spp. is occurring in UP+ women at risk of PTB.

Differential recognition of the multiple banded antigen isoforms across Ureaplasma parvum and Ureaplasma urealyticum species by monoclonal antibodies.

Two separate species of Ureaplasma have been identified that infect humans: Ureaplasma parvum and Ureaplasma urealyticum. Most notably, these bacteria lack a cell wall and are the leading infectious organism associated with infection-related induction of preterm birth. Fourteen separate representative prototype bacterial strains, called serovars, are largely differentiated by the sequence of repeating units in the C-terminus of the major surface protein: multiple-banded antigen (MBA). Monoclonal antibodies that recognise single or small groups of serovars have been previously reported, but these reagents remain sequestered in individual research laboratories. Here we characterise a panel of commercially available monoclonal antibodies raised against the MBA and describe the first monoclonal antibody that cross-reacts by immunoblot with all serovars of U. parvum and U. urealyticum species. We also describe a recombinant MBA expressed by Escherichia coli which facilitated further characterisation by immunoblot and demonstrate immunohistochemistry of paraffin-embedded antigens. Immunoblot reactivity was validated against well characterised previously published monoclonal antibodies and individual commercial antibodies were found to recognise all U. parvum strains, only serovars 3 and 14 or only serovars 1 and 6, or all strains belonging to U. parvum and U. urealyticum. MBA mass was highly variable between strains, consistent with variation in the number of C-terminal repeats between strains. Antibody characterisation will enable future investigations to correlate severity of pathogenicity to MBA isoform number or mass, in addition to development of antibody-based diagnostics that will detect infection by all Ureaplasma species or alternately be able to differentiate between U. parvum, U. urealyticum or mixed infections.

Effects of Ureaplasma urealyticum Infection on Sperm Quality and Concentrations of Nitric Oxide and Cytokine in the Semen of Infertile Males.

PROBLEM: The relationship between genital infection and two sperm parameters, namely, concentrations of nitric oxide (NO) and cytokines [e.g., interleukin (IL)-17 and IL-18], in the semen of infertile males remains undetermined. METHOD OF STUDY: Semen samples from 81 infertile (with or without Ureaplasma urealyticum infection) and normal males were subjected to semen analysis. RESULTS: Nitric oxide concentration in the semen of infertile males with genital infection was higher than those of infertile males without genital infection and of normal males (P < 0.05). Sperm density, pH, percentage of forward, movement of sperm, sperm activation rate, sperm survival rate, and normal rate of the sperm morphology of infertile males with U. urealyticum infection were significantly lower than those of infertile males without genital infection and of normal males (P < 0.05). NO concentrations were also positively correlated with IL-17 and IL-18 concentrations in the semen of infertile males. CONCLUSION: Increased NO concentration and abnormal IL-17 and IL-18 expression were induced by genital infection and induced damage to male reproductive capacity, thereby causing male infertility.

Autoimmune central diabetes insipidus in a patient with ureaplasma urealyticum infection and review on new triggers of immune response

Diabetes insipidus is a disease in which large volumes of dilute urine (polyuria) are excreted due to vasopressin (AVP) deficiency [central diabetes insipidus (CDI)] or to AVP resistance (nephrogenic diabetes insipidus). In the majority of patients, the occurrence of CDI is related to the destruction or degeneration of neurons of the hypothalamic supraoptic and paraventricular nuclei. The most common and well recognized causes include local inflammatory or autoimmune diseases, vascular disorders, Langerhans cell histiocytosis (LCH), sarcoidosis, tumors such as germinoma/craniopharyngioma or metastases, traumatic brain injuries, intracranial surgery, and midline cerebral and cranial malformations. Here we have the opportunity to describe an unusual case of female patient who developed autoimmune CDI following ureaplasma urealyticum infection and to review the literature on this uncommon feature. Moreover, we also discussed the potential mechanisms by which ureaplasma urealyticum might favor the development of autoimmune CDI.

Role of Ureaplasma Respiratory Tract Colonization in Bronchopulmonary Dysplasia Pathogenesis: Current Concepts and Update.

Respiratory tract colonization with the genital mycoplasma species Ureaplasma parvum and Ureaplasma urealyticum in preterm infants is a significant risk factor for bronchopulmonary dysplasia (BPD). Recent studies of the ureaplasmal genome, animal infection models, and human infants have provided a better understanding of specific virulence factors, pathogen-host interactions, and variability in genetic susceptibility that contribute to chronic infection, inflammation, and altered lung development. This review provides an update on the current evidence supporting a causal role of ureaplasma infection in BPD pathogenesis. The current status of antibiotic trials to prevent BPD in Ureaplasma-infected preterm infants is also reviewed.

Autoimmune central diabetes insipidus in a patient with ureaplasma urealyticum infection and review on new triggers of immune response.

Diabetes insipidus is a disease in which large volumes of dilute urine (polyuria) are excreted due to vasopressin (AVP) deficiency [central diabetes insipidus (CDI)] or to AVP resistance (nephrogenic diabetes insipidus). In the majority of patients, the occurrence of CDI is related to the destruction or degeneration of neurons of the hypothalamic supraoptic and paraventricular nuclei. The most common and well recognized causes include local inflammatory or autoimmune diseases, vascular disorders, Langerhans cell histiocytosis (LCH), sarcoidosis, tumors such as germinoma/craniopharyngioma or metastases, traumatic brain injuries, intracranial surgery, and midline cerebral and cranial malformations. Here we have the opportunity to describe an unusual case of female patient who developed autoimmune CDI following ureaplasma urealyticum infection and to review the literature on this uncommon feature. Moreover, we also discussed the potential mechanisms by which ureaplasma urealyticum might favor the development of autoimmune CDI.

Neonatal CNS infection and inflammation caused by Ureaplasma species: rare or relevant?

Colonization with Ureaplasma species has been associated with adverse pregnancy outcome, and perinatal transmission has been implicated in the development of bronchopulmonary dysplasia in preterm neonates. Little is known about Ureaplasma-mediated infection and inflammation of the CNS in neonates. Controversy remains concerning its incidence and implication in the pathogenesis of neonatal brain injury. In vivo and in vitro data are limited. Despite improving care options for extremely immature preterm infants, relevant complications remain. Systematic knowledge of ureaplasmal infection may be of great benefit. This review aims to summarize pathogenic mechanisms, clinical data and diagnostic pitfalls. Studies in preterm and term neonates are critically discussed with regard to their limitations. Clinical questions concerning therapy or prophylaxis are posed. We conclude that ureaplasmas may be true pathogens, especially in preterm neonates, and may cause CNS inflammation in a complex interplay of host susceptibility, serovar pathogenicity and gestational age-dependent CNS vulnerability.

Immune profiling of BALB/C and C57BL/6 mice reveals a correlation between Ureaplasma parvum-Induced fetal inflammatory response syndrome-like pathology and increased placental expression of TLR2 and CD14.

PROBLEM: Both BALB/c and C57BL/6 mice are susceptible to intrauterine infection with Ureaplasma parvum, but only protypical TH2/M2 BALB/c mice develop severe chorioamnionitis, fetal infection, and fetal inflammatory response syndrome-like (FIRS) pathology. METHOD OF STUDY: Microscopy, gene expression analysis, and ELISA were used to identify placental innate immune responses relevant to macrophage polarity, severe chorioamnionitis, and fetal infection. RESULTS: Both mouse strains exhibited a pro-M2 cytokine profile at the maternal/fetal interface. In BALB/c mice, expression of CD14 and TLRs 1, 2, 6 was increased in infected placentas; TLR2 and CD14 were localized to neutrophils. Increased TLR2/CD14 was also observed in BALB/c syncytiotrophoblasts in tissues with pathological evidence of FIRS. In contrast, expression in C57BL/6 placentas was either unchanged or down-regulated. CONCLUSION: Our findings show a link between increased syncytiotrophoblast expression of CD14/TLR2 and FIRS-like pathology in BALB/c mice. Functional studies are required to determine if CD14 is contributing to fetal morbidity during chorioamnionitis.

Effects of Ureaplasma parvum lipoprotein multiple-banded antigen on pregnancy outcome in mice.

Ureaplasma spp. are members of the family Mycoplasmataceae and have been considered to be associated with chorioamnionitis and preterm delivery. However, it is unclear whether Ureaplasma spp. have virulence factors related to these manifestations. The purpose of the present study was to determine whether the immunogenic protein multiple-banded antigen (MBA) from Ureaplasma parvum is a virulence factor for preterm delivery. We partially purified MBA from a type strain and clinical isolates of U. parvum, and also synthesized a diacylated lipopeptide derived from U. parvum, UPM-1. Using luciferase assays, both MBA-rich fraction MRF and UPM-1 activated the NF-κB pathway via TLR2. UPM-1 upregulated IL-1ß, IL-6, IL-12p35, TNF-α, MIP2, LIX, and iNOS in mouse peritoneal macrophage. MRF or UPM-1 was injected into uteri on day 15 of gestation on pregnant C3H/HeN mice. The intrauterine MRF injection group had a significantly higher incidence of intrauterine fetal death (IUFD; 38.5%) than the control group (14.0%). Interestingly, intrauterine injection of UPM-1 caused preterm deliveries at high concentration (80.0%). In contrast, a low concentration of UPM-1 induced a significantly higher rate of fetal deaths (55.2%) than the control group (14.0%). The placentas of the UPM-1 injection group showed neutrophil infiltration and increased iNOS protein expression. Our data indicate that MBA from the clinical isolate of U. parvum is a potential virulence factor for IUFD and preterm delivery in mice and that the N-terminal diacylated lipopeptide is essential for the initiation of inflammation.

Effects of Ureaplasma urealyticum lipid-associated membrane proteins on rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: As an infectious agent might play a role in rheumatoid arthritis (RA) development, this study investigated effects of Ureaplasma urealyticum lipid-associated membrane proteins (UuLAMPs) on RA synovial fibroblast (RASF) proliferation, and tumour necrosis factor (TNF)-α and interleukin (IL)-1ß production by THP-1 macrophages. Possible immunogenic proteins in UuLAMPs were identified. METHODS: RASFs were cultured from synovial tissue from donors with RA. Serum samples from donors with/without RA and with/without U. urealyticum infection were used for immunogenicity analyses. THP-1 macrophages served as a model for synovial macrophages. TNF-α and IL-1ß mRNA levels were assessed using reverse transcription-polymerase chain reaction; protein levels were estimated using enzyme-linked immunosorbent assay. UuLAMPs underwent separation and Western blot analyses. RESULTS: UuLAMPs (0.025-0.4 µg/ml) stimulated RASF proliferation in a dose- and time-dependent manner, and increased TNF-α and IL-1ß levels in THP-1 macrophages. Several immunogenic UuLAMPs were identified, but antibodies to a 25 kDa protein were only found in RA patients with U. urealyticum infection. CONCLUSIONS: UuLAMPs might induce RASF proliferation and proinflammatory cytokine secretion in synovium from RA patients. A 25 kDa U. urealyticum protein might act as a cross-reactive antigen.

Viral infection of the pregnant cervix predisposes to ascending bacterial infection.

Preterm birth is the major cause of neonatal mortality and morbidity, and bacterial infections that ascend from the lower female reproductive tract are the most common route of uterine infection leading to preterm birth. The uterus and growing fetus are protected from ascending infection by the cervix, which controls and limits microbial access by the production of mucus, cytokines, and antimicrobial peptides. If this barrier is compromised, bacteria may enter the uterine cavity, leading to preterm birth. Using a mouse model, we demonstrate, to our knowledge for the first time, that viral infection of the cervix during pregnancy reduces the capacity of the female reproductive tract to prevent bacterial infection of the uterus. This is due to differences in susceptibility of the cervix to infection by virus during pregnancy and the associated changes in TLR and antimicrobial peptide expression and function. We suggest that preterm labor is a polymicrobial disease, which requires a multifactorial approach for its prevention and treatment.