Secretory leukocyte protease inhibitor and progranulin as possible regulators of cervical remodeling in pregnancy.

Secretory leukocyte protease inhibitor (SLPI) and progranulin (PGRN) are secretory proteins with an anti-inflammatory property. Their involvement in cervical remodeling in pregnant uterus is not yet elucidated. Thus, this study aimed to explore the significance of SLPI and PGRN in the maintenance of pregnancy by investigating the factors associated with their expression levels at the cervix. Concentrations of SLPI and PGRN proteins were measured in cervical mucus samples collected from asymptomatic pregnant women at 24-26 weeks of gestation (n = 166). The concentrations of those molecules were analyzed with clinical parameters related to risk for preterm delivery (PD). In pregnant mice, we evaluated the effect of lipopolysaccharide-induced inflammation and progesterone effect modulation on cervical mRNA expression of SLPI and PGRN. The cervical PGRN level was significantly lower in women with short cervix (<35 mm) and with a history of threatened PD. In women with short cervix, cervical SLPI concentrations were positively correlated with inflammatory cytokines, interleukin-6 (R2 = 0.75) and interleukin-8 (R2 = 0.71). In pregnant mice, cervical mRNA expressions of PGRN and SLPI were increased in response to progesterone supplementation and were suppressed by a progesterone antagonist, mifepristone. Lipopolysaccharide-induced inflammation caused remarkable upregulation in cervical SLPI mRNA level but not in PGRN. Progesterone and local inflammation are the factors controlling expression levels of PGRN and SLPI at the cervix. The observed relationship of PGRN and SLPI levels in the cervical mucus with PD-related clinical parameters supports that those anti-inflammatory molecules possibly play a significant role in appropriate regulation of cervical remodeling.

Activation of the Complement System in the Lower Genital Tract During Pregnancy and Delivery.

Background: Human pregnancy alters profoundly the immune system. The local involvement and mechanisms of activation of the complement system in the cervicovaginal milieu during pregnancy and delivery remain unexplored. Objectives: To determine whether normal pregnancy and delivery are associated with local activation of complement or changes in the immunoglobulin profile in the cervix. Study Design: This study was designed to assess IgA, IgG, and complement activation in the cervicovaginal area in three groups of patients: i) 49 pregnant women (week 41+3-42+0) not in active labor, ii) 24 women in active labor (38+4-42+2), and iii) a control group of nonpregnant women (n=23) at child-bearing age. We collected mucosal samples from the lateral fornix of the vagina and external cervix during routine visits and delivery. The Western blot technique was used to detect complement C3 and its activation products. For semiquantitative analysis, the bands of the electrophoresed proteins in gels were digitized on a flatbed photo scanner and analyzed. IgA and IgG were analyzed by Western blotting and quantified by ELISA. One-way ANOVA and Tukey's Multiple Comparison tests were used for statistical comparisons. Results: A higher abundance but lower activation level of C3 in both the external cervix (P<0.001) and lateral fornix of the vagina (P<0.001) was observed during delivery (58 ± 22, n= 24) in comparison to the groups of nonpregnant (72 ± 13%; mean ± SD, n=23) and pregnant women (78 ± 22%, n=49). Complement activating IgG was detected in higher abundance than IgA in the cervicovaginal secretions of pregnant women. In a small proportion samples also C3-IgG complexes were detected. Conclusions: Our results reveal an unexpectedly strong activation of the complement system and the presence IgG immunoglobulins in the cervicovaginal area during pregnancy, active labor, and among nonpregnant women. In contrast to the higher amounts of C3 in the cervicovaginal secretions during labor, its activation level was lower. Complement activating IgG was detected in higher concentrations than IgA in the mucosal secretions during pregnancy and labor. Taken together our results imply the presence a locally operating humoral immune system in the cervicovaginal mucosa.

Antibody-Mediated Immobilization of Virions in Mucus.

Antibodies have been shown to hinder the movement of herpes simplex virus virions in cervicovaginal mucus, as well as other viruses in other mucus secretions. However, it has not been possible to directly observe the mechanisms underlying this phenomenon, so the nature of virion-antibody-mucin interactions remain poorly understood. In this work, we analyzed thousands of virion traces from single particle tracking experiments to explicate how antibodies must cooperate to immobilize virions for relatively long time periods. First, using a clustering analysis, we observed a clear separation between two classes of virion behavior: freely diffusing and immobilized. While the proportion of freely diffusing virions decreased with antibody concentration, the magnitude of their diffusivity did not, implying an all-or-nothing dichotomy in the pathwise effect of the antibodies. Proceeding under the assumption that all binding events are reversible, we used a novel switch-point detection method to conclude that there are very few, if any, state switches on the experimental timescale of 20 s. To understand this slow state switching, we analyzed a recently proposed continuous-time Markov chain model for binding kinetics and virion movement. Model analysis implied that virion immobilization requires cooperation by multiple antibodies that are simultaneously bound to the virion and mucin matrix and that there is an entanglement phenomenon that accelerates antibody-mucin binding when a virion is immobilized. In addition to developing a widely applicable framework for analyzing multistate particle behavior, this work substantially enhances our mechanistic understanding of how antibodies can reinforce a mucus barrier against passive invasive species.

Cervical Local Immune Response for High-Risk Human Papillomavirus Infection: Involvement With Cervical Mucus SLPI Proteins.

PURPOSE: To evaluate cervical mucus secretory leukocyte protease inhibitor (SLPI) concentrations in patients with high-risk human papillomavirus (hrHPV) 16 or 18 positive and low-grade squamous intraepithelial lesions (LGSIL) or high-grade squamous intraepithelial lesions (HGSIL). METHOD: Patients with HPV 16 or 18 positive from 30 to 45 years of age whose cervical cancer screening results reported cytologically LGSIL or HGSIL were included in the study. In the control group, we included participants in the same age with cytology negative and HPV-negative healthy women. All cytological LGSIL or HGSIL results were histopathologically confirmed with colposcopic biopsy specimens. Finally, the study consisted of a total of 3 groups each containing 25 participants as follows: (1) Pap smear and HPV-negative control group, (2) HPV 16 or HPV 18 and LGSIL-positive participants, and (3) HPV 16 or 18 and HGSIL-positive participants. Cervical mucus SLPI levels were analyzed using the enzyme-linked immunosorbent assay method. RESULTS: The mean cervical mucus SLPI levels were 32.94 ng/mL (range: 23-41.29 ng/mL) in the hrHPV + LGSIL group, 29.40 ng/mL (range: 21.03-38.95 ng/mL) in the hrHPV + HGSIL, and 18.75 ng/mL (range: 13.58-29.24 ng/mL) in the healthy control group. Cervical mucus SLPI levels were found to be significantly higher in the hrHPV + LGSIL and hrHPV + HGSIL groups compared to the control group ( P < .001). CONCLUSIONS: The data from the present study indicate that SLPI seems to be one of the important immunomodulatory proteins that provide local immune response in cervical mucosa.

Cervico-vaginal mucus (CVM) - an accessible source of immunologically informative biomolecules.

Cervico-vaginal mucus (CVM), the product of epithelial cells lining the uterus, cervix and vagina, is secreted to facilitate uterine lubrication and microbial clearance. Predominantly composed of water and mucins, CVM also contains high levels of immuno-active proteins such as immunoglobulin A (IgA), lactoferrin and lysozyme which protect against infection by blocking adhesion and mediating microbial killing. The repertoire of cytokines, chemokines and antimicrobial peptides is predominantly generated by the secretions of endometrial epithelial cells into the uterine lumen and concentrated in the CVM. The quantity and relative proportions of these inflammatory biomarkers are affected by diverse factors including the estrus cycle and health status of the animal and therefore potentially provide important diagnostic and prognostic indicators. We propose that measuring molecular signatures in bovine CVM could be a useful approach to identifying and monitoring genital tract pathologies in beef and dairy cows.

Herpes simplex virus-binding IgG traps HSV in human cervicovaginal mucus across the menstrual cycle and diverse vaginal microbial composition.

IgG possesses an important yet little recognized effector function in mucus. IgG bound to viral surface can immobilize otherwise readily diffusive viruses to the mucin matrix, excluding them from contacting target cells and facilitating their elimination by natural mucus clearance mechanisms. Cervicovaginal mucus (CVM) is populated by a microbial community, and its viscoelastic and barrier properties can vary substantially not only across the menstrual cycle, but also in women with distinct microbiota. How these variations impact the "muco-trapping" effector function of IgGs remains poorly understood. Here we obtained multiple fresh, undiluted CVM specimens (n = 82 unique specimens) from six women over time, and employed high-resolution multiple particle tracking to quantify the mobility of fluorescent Herpes Simplex Viruses (HSV-1) in CVM treated with different HSV-1-binding IgG. The IgG trapping potency was then correlated to the menstrual cycle, and the vaginal microbial composition was determined by 16 s rRNA. In the specimens studied, both polyclonal and monoclonal HSV-1-binding IgG appeared to consistently and effectively trap HSV-1 in CVM obtained at different times of the menstrual cycle and containing a diverse spectrum of commensals, including G. vaginalis-dominant microbiota. Our findings underscore the potential broad utility of this "muco-trapping" effector function of IgG to reinforce the vaginal mucosal defense, and motivates further investigation of passive immunization of the vagina as a strategy to protect against vaginally transmitted infections.

Cervical mucus and contraception: what we know and what we don't.

OBJECTIVES: Cervical mucus varies in response to both natural and artificial hormonal changes. It is commonly believed that cervical mucus thinning is associated with normal fertility and that progestogen-induced thickening is an essential contraceptive mechanism. This review aims to broadly summarize our current knowledge about cervical mucus from both a clinical and basic research perspective. STUDY DESIGN: We reviewed published literature pertinent to cervical mucus and contraception across scientific disciplines. We first present the most current understanding of the composition of cervical mucus, how it is hormonally regulated, and examine the role of mucus as an immune barrier. We then critically assess the current clinical tests used as surrogate markers for a contraceptive effect. Finally, we review contraceptive studies that have specifically focused on cervical mucus changes. RESULTS: Existing research suggests that cervical mucus has potential to be a contraceptive target with unique, multipurpose characteristics. However, methodologic limitations associated with clinical assessments of cervical mucus complicate our understanding of contraceptive treatment effects. Key pathways involved in cervical mucus production with potential as novel nonhormonal contraceptive targets have been identified. CONCLUSIONS: More research is needed to clarify the role of cervical mucus in current hormonal contraceptives and to support the development of novel nonhormonal cervix-based methods.

Diffusion of Immunoglobulin G in Shed Vaginal Epithelial Cells and in Cell-Free Regions of Human Cervicovaginal Mucus.

Human cervicovaginal mucus (CVM) is a viscoelastic gel containing a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens. Here, to investigate the affinity between IgG and different mucus constituents, we used Fluorescence Recovery After Photobleaching (FRAP) to measure the diffusion of IgG in fresh, minimally modified CVM. We found that CVM exhibits substantial spatial variations that necessitate careful selection of the regions in which to perform FRAP. In portions of CVM devoid of cells, FRAP measurements using different IgG antibodies and labeling methods consistently demonstrate that both exogenous and endogenous IgG undergo rapid diffusion, almost as fast as in saline, in good agreement with the rapid diffusion of IgG in mid-cycle endocervical mucus that is largely devoid of cells. This rapid diffusion indicates the interactions between secreted mucins and IgG must be very weak and transient. IgG also accumulated in cellular debris and shed epithelial cells that had become permeable to IgG, which may allow shed epithelial cells to serve as reservoirs of secreted IgG. Interestingly, in contrast to cell-free regions of CVM, the diffusion of cell-associated IgG was markedly slowed, suggesting greater affinity between IgG and cellular constituents. Our findings contribute to an improved understanding of the role of IgG in mucosal protection against infectious diseases, and may also provide a framework for using FRAP to study molecular interactions in mucus and other complex biological environments.

The role of the cervix in fertility: is it time for a reappraisal?

Knowledge regarding the role of the cervix in fertility has expanded considerably over the past 20 years and in this article, we propose that it is now time for its function to be reappraised. First, we review the anatomy of the cervix and the vaginal ecosystem that it inhabits. Then, we examine the physiology and the role of the cervical mucus. The ongoing mystery of the exact mechanism of the sperm-cervical mucus interaction is reviewed and the key players that may unlock this mystery in the future are discussed. The soluble and cellular biomarkers of the lower female genital tract which are slowly being defined by contemporary research are reviewed. Attempts to standardize these markers, in this milieu, are hindered by the changes that may be attributed to endogenous or exogenous factors such as: age, hormonal changes during the menstrual cycle, ectropion, infection, smoking and exposure to semen during sexual intercourse. We review what is known about the immunology of the cervix. With the widespread use of large loop excision of the transformation zone (LLETZ) for treatment of cervical intraepithelial neoplasia, the anatomy of the cervix is changing for many women. While LLETZ surgery has had very positive effects in the fight against cervical cancer, we debate the impact it could have on a woman's fertility.

IgG in cervicovaginal mucus traps HSV and prevents vaginal herpes infections.

IgG is the predominant immunoglobulin in cervicovaginal mucus (CVM), yet how immunoglobulin G (IgG) in mucus can protect against infections is not fully understood. IgG diffuses rapidly through cervical mucus, slowed only slightly by transient adhesive interactions with mucins. We hypothesize that this almost unhindered diffusion allows IgG to accumulate rapidly on pathogen surfaces, and the resulting IgG array forms multiple weak adhesive crosslinks to mucus gel that effectively trap (immobilize) pathogens, preventing them from initiating infections. Here, we report that herpes simplex virus serotype 1 (HSV-1) readily penetrated fresh, pH-neutralized ex vivo samples of CVM with low or no detectable levels of anti-HSV-1 IgG but was trapped in samples with even modest levels of anti-HSV-1 IgG. In samples with little or no endogenous anti-HSV-1 IgG, addition of exogenous anti-HSV-1 IgG, affinity-purified from intravenous immunoglobulin, trapped virions at concentrations below those needed for neutralization and with similar potency as endogenous IgG. Deglycosylating purified anti-HSV-1 IgG, or removing its Fc component, markedly reduced trapping potency. Finally, a non-neutralizing IgG against HSV-gG significantly protected mice against vaginal infection, and removing vaginal mucus by gentle lavage abolished protection. These observations suggest that IgG-Fc has a glycan-dependent "muco-trapping" effector function that may provide exceptionally potent protection at mucosal surfaces.

Differential binding of IgG and IgA to mucus of the female reproductive tract.

Cells of the endocervix are responsible for the secretion of mucins, which provide an additional layer of protection to the female reproductive tract (FRT). This barrier is likely fortified with IgA as has previously been shown in the gastrointestinal tract and lungs of mice. Mucus associated IgA can facilitate clearance of bacteria. While a similar function for IgG has been proposed, an association with mucus has not yet been demonstrated. Here we find that IgA and IgG are differentially associated with the different types of mucus of the FRT. We observed that while both IgA and IgG are stably associated with cervical mucus, only IgG is associated with cervicovaginal mucus. These findings reveal that antibodies can bind tightly to mucus, where they can play a significant role in the fortification of the mucus barriers of the FRT. It may be possible to harness this interaction in the development of vaccines designed to protect the FRT mucosal barriers from sexually transmitted diseases such as HIV.

Human cervicovaginal mucus contains an activity that hinders HIV-1 movement.

Cervical and vaginal epithelia are primary barriers against HIV type I (HIV-1) entry during male-to-female transmission. Cervical mucus (CM) is produced by the endocervix and forms a layer locally as well as in the vaginal compartment in the form of cervicovaginal mucus (CVM). To study the potential barrier function of each mucus type during HIV-1 transmission, we quantified HIV-1 mobility in CM and CVM ex vivo using fluorescent microscopy. Virions and 200-nm PEGylated beads were digitally tracked and mean-squared displacement was calculated. The mobility of beads increased significantly in CVM compared with CM, consistent with the known decreased mucin concentration of CVM. Unexpectedly, HIV-1 diffusion was significantly hindered in the same CVM samples in which bead diffusion was unhindered. Inhibition of virus transport was envelope-independent. Our results reveal a previously unknown activity in CVM that is capable of impeding HIV-1 mobility to enhance mucosal barrier function.

[Cytokine status of cervical mucus in women with transitory and persistent course of papillomavirus infection].

AIM: Study the levels of cytokines in cervical mucus of women with transitory and persistent course of papillomavirus infection (PVI). MATERIALS AND METHODS: Material from 122 women was studied: 32 patients in control group, 68 patients with transitory course of PVI and 52 women with persistent course of PVI. The presence or absence of high risk HPV DNA was confirmed by real time PCR. High risk HPV DNA was determined in scrapes of epithelial cells of cervical canal. Quantitative determination of cytokines (IFN-alpha, IFN-gamma, IL-1beta, receptor antagonist IL-1, IL-2, IL-4, IL-8 and IL-10, TNFalpha in cervical mucus was performed by enzyme immunoassay. RESULTS: Transitory course of PVI was characterized by an increase of IFN-alpha and IFN-gamma, persistent course of PIV--by a decrease of IFN-alpha, IL-2 level was increased in both groups of patients with PVI. CONCLUSION: The outcome of PVI is controlled by interferon component of the immunity. Because of this during observation of patients with PVI the determination of IFN-alpha, IFN-gamma content in cervical mucus is an appropriate diagnostic procedure in clinical practice for the prognosis of risk of development of high risk PIV persistency.

Changes in the soluble mucosal immune environment during genital herpes outbreaks.

BACKGROUND: Genital tract secretions provide variable inhibitory activity against herpes simplex virus (HSV) ex vivo. We hypothesize that the anti-HSV activity may prevent the spread of virus from the more commonly affected sites, such as the external genitalia, to the upper genital tract. METHODS: The antimicrobial activity of cervicovaginal lavage (CVL) and concentrations of mucosal immune mediators were measured in 10 HIV-seronegative women with an active external herpetic lesion and compared with 10 HIV-seronegative women who were HSV-1 and HSV-2 seronegative. Samples were obtained at the time of a symptomatic external lesion (day 0), after 1 week of oral acyclovir (day 7), and 1 week after completing treatment (day 14). Controls were evaluated at parallel intervals. RESULTS: The anti-HSV activity was higher in CVL obtained from cases compared to controls at presentation (day 0) (54.3% vs. 28%), fell to similar levels on day 7, and then rebounded on day 14 (69% vs. 25%). The anti-HSV activity correlated positively and significantly with the concentrations of several inflammatory proteins; the concentrations of these proteins tended to be higher in cases compared with controls and followed a similar temporal pattern. CONCLUSIONS: Increases in inflammatory immune mediators and anti-HSV activity were detected in CVL at the time of clinical outbreaks and after completion of a short course of acyclovir. These mucosal responses may protect against HSV spread but could facilitate HIV infection and contribute to the clinical observation that, independent of clinical lesions, HSV-2 is a risk factor for HIV acquisition.

HIV-neutralizing activity of cationic polypeptides in cervicovaginal secretions of women in HIV-serodiscordant relationships.

BACKGROUND: HIV exposed seronegative (HESN) women represent the population most in need of a prophylactic antiviral strategy. Mucosal cationic polypeptides can potentially be regulated for this purpose and we here aimed to determine their endogenous expression and HIV neutralizing activity in genital secretions of women at risk of HIV infection. METHODOLOGY/PRINCIPAL FINDINGS: Cervicovaginal secretions (CVS) of Kenyan women in HIV-serodiscordant relationships (HESN, n = 164; HIV seropositive, n = 60) and low-risk controls (n = 72) were assessed for the cationic polypeptides HNP1-3, LL-37 and SLPI by ELISA and for HIV neutralizing activity by a PBMC-based assay using an HIV primary isolate. Median levels of HNP1-3 and LL-37 in CVS were similar across study groups. Neither HSV-2 serostatus, nor presence of bacterial vaginosis, correlated with levels of HNP1-3 or LL-37 in the HESN women. However, an association with their partner's viral load was observed. High viral load (>10,000 HIV RNA copies/ml plasma) correlated with higher levels of HNP1-3 and LL-37 (p = 0.04 and 0.03, respectively). SLPI was most abundant in the low-risk group and did not correlate with male partner's viral load in the HESN women. HIV neutralizing activity was found in CVS of all study groups. In experimental studies, selective depletion of cationic polypeptides from CVS rendered the remaining CVS fraction non-neutralizing, whereas the cationic polypeptide fraction retained the activity. Furthermore, recombinant HNP1-3 and LL-37 could induce neutralizing activity when added to CVS lacking intrinsic activity. CONCLUSIONS/SIGNIFICANCE: These findings show that CVS from HESN, low-risk, and HIV seropositive women contain HIV neutralizing activity. Although several innate immune proteins, including HNP1-3 and LL-37, contribute to this activity these molecules can also have inflammatory properties. This balance is influenced by hormonal and environmental factors and in the present HIV serodiscordant couple cohort study we show that a partner's viral load is associated with levels of such molecules.

Measurement of mucosal biomarkers in a phase 1 trial of intravaginal 3% StarPharma LTD 7013 gel (VivaGel) to assess expanded safety.

OBJECTIVE: The aim of this study was to examine the effect of the 3% StarPharma LTD 7013 gel (VivaGel) on mucosal immune markers hypothesized to be associated with HIV-1 acquisition. DESIGN: Phase 1, placebo-controlled, randomized, double-blind clinical trial was performed in 54 young women in the United States and Kenya. Participants used carbopol gel with and without (placebo) StarPharma LTD 7013 twice daily over 14 days. Cervical specimens were collected for cytokines, chemokines, T cells, and dendritic cells at days 0, 7, 14, and 21. A negative binomial regression model was used to assess differences between study arms. RESULTS: Several mucosal immune parameters were increased in the VivaGel arm compared with placebo. For cytokines D7, IL-6 (P = 0.05); D 14, interferon gamma (P = 0.03), IL-2 (P = 0.04), IL-5 (P = 0.003), and IL-10 (P = 0.001) were increased. On D7, CD8+/CD69+ T cells tended to be increased (P < 0.08); limiting analysis to visits without blood or bacterial vaginosis, these findings were stronger as follows: at D7, CD8+/CD69+ T cells were increased in the VivaGel arm (P < 0.005), as were CD4+/CD69+ cells (P = 0.001) and CD4+/CCR5+ T cells (P = 0.01). The changes described for D7 and 14 were no longer seen at D21. CONCLUSIONS: Markers associated with inflammation and epithelial damage were reversibly elevated in the VivaGel arm compared with the placebo arm after 7-14 days of twice daily product use.

Influence of lactoferrin in preventing preterm delivery: a pilot study.

Lactoferrin (Lf) is an approximately 80-kDa iron-binding glycoprotein, belonging to the transferrin family, with well-known bacteriostatic and bactericidal properties. It is produced and stored in specific (secondary) neutrophil granules and released during neutrophil activation and degranulation. Nowadays, Lf has a well-known therapeutic indication for combating iron deficiency anemia (IDA) in pregnant women. Studies suggest that Lf plays an important role against cervicovaginal infections by decreasing cytokines levels, such as interleukin (IL)-6, in cervicovaginal fluid. The aim of this preliminary trial was to evaluate the effectiveness of Lf in preventing preterm delivery caused by cervical infections and ripening. From November 2009 to August 2010, 21 pregnant women (26-32 weeks pregnant), aged between 22 and 36 years, suffering from IDA, at risk of preterm delivery, were prospectively enrolled in the study. One group (N=14) received 100 mg of recombinant human lactoferrin (bLf) [corrected] (lattoferrina; AG-pharma) twice a day before meals, for one month. The other group (N=7) received 520 mg of ferrous sulfate (Ferro-Grad; Abbott Laboratories, USA) once a day. The patients underwent transvaginal ultrasound to evaluate cervical length and funneling, and vaginal swabs were used to detect infections and cervicovaginal fluid sample collection to determine IL-6 levels. The results showed a correlation between the oral administration of 200 mg of bLf [corrected] with both the normalization of vaginal flora (vaginal infection disappearance) and the decrease in IL-6 cervicovaginal fluid levels in women at risk of preterm delivery.

Immunomodulatory factors in cervicovaginal secretions from pregnant and non-pregnant women: a cross-sectional study.

BACKGROUND: Pregnant women are at an increased risk for HIV infection due to unknown biological causes. Given the strong effect of sex-hormones on the expression of immunomuodulatory factors, the central role of mucosal immunity in HIV pathogenesis and the lack of previous studies, we here tested for differences in immunomuodulatory factors in cervico-vaginal secretions between pregnant and non-pregnant women. METHODS: We compared concentrations of 39 immunomodulatory factors in cervicovaginal lavages (CVL) from 21 pregnant women to those of 24 non-pregnant healthy women from the US. We used Bonferroni correction to correct for multiple testing and linear regression modeling to adjust for possible confounding by plasma cytokine concentration, cervical ectopy, total protein concentration, and other possible confounders. Cervical ectopy was determined by planimetry. Concentration of immunomodulatory factors were measured by a multiplex assay, protein concentration by the Bradford Method. RESULTS: Twenty six (66%) of the 39 measured immunomodulatory factors were detectable in at least half of the CVL samples included in the study. Pregnant women had threefold lower CVL concentration of CCL22 (geometric mean: 29.6 pg/ml versus 89.7 pg/ml, p = 0.0011) than non-pregnant women. CVL CCL22 concentration additionally correlated negatively with gestational age (Spearman correlation coefficient [RS]: -0.49, p = 0.0006). These associations remained significant when corrected for multiple testing. CCL22 concentration in CVL was positively correlated with age and negatively correlated with time since last coitus and the size of cervical ectopy. However, none of these associations could explain the difference of CCL22 concentration between pregnant and non-pregnant women in this study, which remained significant in adjusted analysis. CONCLUSIONS: In this study population, pregnancy is associated with reduced concentrations of CCL22 in cervicovaginal secretions. The role of CCL22 on HIV transmission should now be investigated in prospective studies.

Protein profiling underscores immunological functions of uterine cervical mucus plug in human pregnancy.

The cervical mucus plug (CMP) differs from the cervical secretions of non-pregnant women, and is the ultimate sealant of the uterine cavity during pregnancy. Although several studies have analyzed biochemical properties of large glycoproteins in the CMP, comprehensive information about its protein composition is yet unavailable. We hypothesized that protein profiling of the CMP could provide key clues to its physiological functions in pregnancy. For this purpose, five CMPs obtained from women in labor at term were analyzed by LC-MS/MS. Out of 291 total proteins identified, 137 were detected in two or more samples, which included S100A8, S100A9, and complement proteins (C3, C4a, C4b, C6, and C8g). Several proteins, which have not been described in the cervical mucus of non-pregnant women or in cervicovaginal fluids, such as CD81 antigen and pregnancy zone protein, were also identified. Gene ontology analysis of identified proteins showed significant enrichment of 28 biological processes such as 'activation of plasma proteins involved in acute inflammatory response' and 'positive regulation of cholesterol esterification'. We report the proteome of CMPs from pregnant women at term for the first time, and the overall findings strongly suggest an important role for the CMP in the maintenance of pregnancy and parturition.