Menstrual blood contains immune cells with inflammatory and anti-inflammatory properties.

AIM: Successful pregnancy requires balanced regulation of immune cells at the feto-maternal interface. Systemic monitoring of the immune system cannot precisely outline local immune status in the uterus. In this survey, endometrial immune milieu was investigated using a non-invasive method of analysis of menstrual blood (MB). The results were compared with peripheral blood (PB). METHOD: PB and MB of healthy fertile women (n = 15) were collected simultaneously on the second day of their menstrual cycle. T and natural killer T cell subpopulations were immunophenotyped by flow cytometry. RESULTS: Among examined cell populations, the frequency of CD4 + Foxp3+, CD4 + Foxp3 + CD25-, CD4 + Foxp3 + CD25+ and IL17+ T cells (P = 0.022, 0.028, 0.017 and 0.005, respectively) and TCRαß+, CD45RO+, CD16-, IFNγ + and IL17+ NKT (CD56 + CD3+) cells (P = 0.010, 0.037, 0.038, 0.015 and 0.021, respectively) were significantly higher in MB compared with PB. Conversely, PB contained a higher percentage of CD16+ T cells (P = 0.025) in comparison with MB. CONCLUSION: MB contains cells of an inflammatory and anti-inflammatory nature, implying the existence of finely tuned cell homeostasis during menstruation. Our results imply that MB could be viewed as an easy-to access specimen for monitoring endometrial immune cells, especially those that have preferential endometrial localization.

Menstrual blood closely resembles the uterine immune micro-environment and is clearly distinct from peripheral blood.

STUDY QUESTION: Is menstrual blood a suitable source of endometrial derived lymphocytes? SUMMARY ANSWER: Mononuclear cells isolated from menstrual samples (menstrual blood mononuclear cells (MMC)) are clearly distinct from peripheral blood mononuclear cells (PBMC) and show a strong resemblance with biopsy-derived endometrial mononuclear cells. WHAT IS KNOWN ALREADY: A critical event in the onset of pregnancy is the implantation of the embryo in the uterine wall. The immune cell composition in the endometrium at the time of implantation is considered pivotal for success. Despite advancing knowledge on the composition of the immune cell population in the uterus, the role of endometrial immune cells in reproductive disorders is still not fully resolved, mainly due to the fact that this type of research requires invasive techniques. Here, we collected menstrual fluid and validated this unique non-invasive technique to obtain and study the endometrium-derived immune cells which would be present around the time of implantation. STUDY DESIGN, SIZE, DURATION: Five healthy non-pregnant females with regular menstruation cycles and not using oral contraceptives collected their menstrual blood using a menstrual cup in five consecutive cycles. Sampling took place over the first 3 days of menses, with 12 h intervals. Peripheral blood samples, taken before and after each menstruation, were obtained for comparative analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: MMC and PBMC samples were characterized for the different lymphocyte subsets by flow cytometry, with emphasis on NK cells and T cells. Next, the functional capacity of the MMC-derived NK cells was determined by measuring intracellular production of IFN-γ, granzyme B and perforin after culture in the presence of IL-2 and IL-15. MAIN RESULTS AND THE ROLE OF CHANCE: In support of their endometrial origin, MMC samples contained the typical composition of mononuclear cells expected of endometrial tissue, were phenotypically similar to the reported phenotype for biopsy-derived endometrial cells, and were distinct from PBMC. Increased percentages of NK cells and decreased percentages of T cells were found in MMC when compared with PBMC from the same female. The MMC-derived NK cells were pre-dominantly CD56(bright)/CD16(-), in contrast to the primarily CD56(dim)/CD16(+) peripheral blood NK cells. MMC-derived NK cells expressed CD103, indicating their mucosal origin. In addition, the pattern of natural cytotoxicity receptor (NCR) expression in MMC-derived NK cells was comparable with that in endometrial biopsy-derived NK cells. Compared with PBMC, the NKp30 expression was decreased, while the percentage of NKp44 positive cells was increased in MMC samples. CXCR3 and CXCR4 were hardly expressed by MMC-derived NK cells, indicating that these cells are not of PBMC origin. NK cells from MMC samples were functional as shown by their capacity to produce IFN-γ, granzyme B and perforin, upon stimulation with IL-2 and IL-15. MMC-derived T cells revealed an increased expression of CD103, CD69 and CXCR4 compared with PBMC-derived T cells. Importantly, MMC collection using a menstrual cup proved highly reliable and reproducible between women and between cycles. LIMITATIONS, REASONS FOR CAUTION: Based on the parameters we studied, MMC appear similar to biopsy-derived endometrial mononuclear cells. However, sampling is not done at the exact same time in the menstrual cycle, and thus we cannot exclude some, as yet undetected, differences. Also, it should be considered that for some women, the use of the menstrual cup may be unpleasant. WIDER IMPLICATIONS OF THE FINDINGS: Menstrual blood may be a source of endometrial cells and may create new opportunities to study uterine immunological cells in fertility issues. STUDY FUNDING/COMPETING INTEREST(S): No external funding was obtained for the present study. None of the authors have any conflict of interest to declare. TRIAL REGISTRATION NUMBER: NA.

Effect of the menstrual cycle on inflammatory cytokines in the periodontium.

BACKGROUND AND OBJECTIVE: The effects of different levels of steroid hormones, as experienced during puberty, pregnancy and menopause, on the periodontium have been demonstrated, but changes in sex hormone levels during the menstrual cycle, and the influence of these changes on the periodontium, remain unresolved. The aim of this study was to investigate the effect of the menstrual cycle on the levels of interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) in gingival crevicular fluid and on periodontal clinical parameters, including the gingival bleeding index (GBI) and the modified gingival index (MGI), in periodontally healthy women. MATERIAL AND METHODS: Twenty-seven periodontally healthy women with a regular menstrual cycle were included in the study. Clinical parameters, including the GBI, the MGI and the simplified oral health index, were recorded during menstruation, ovulation and premenstruation phases (e.g. on days 1-2, 12-14 and 22-24, respectively) of the menstrual cycle. Gingival crevicular fluid and unstimulated saliva were collected, at each study phase, for assessment of IL-1ß, TNF-α, estrogen and progesterone. RESULTS: Both the GBI and the MGI increased significantly during the menstrual cycle, and were significantly higher during ovulation than during menstruation or premenstruation (p < 0.001). No significant change in the simplified oral health index was observed during the menstrual cycle ( p = 0.18). The levels of IL-1ß and TNF-α increased during the different phases of the menstrual cycle, but only the change in the TNF-α concentration was significant ( p < 0.05). CONCLUSION: This study indicated that changes occurring during the menstrual cycle influence the periodontium and induce inflammatory conditions.

Induction of endometriosis alters the peripheral and endometrial regulatory T cell population in the non-human primate.

BACKGROUND: Endometriosis is a gynecological condition that is characterized by extreme abdominal pain and also decreased fertility. Regulatory T cells (Tregs) have immunosuppressive activity critical for embryonic implantation and likewise the acceptance of tissue engraftment. Utilizing the induced non-human primate (Papio anubis) model of endometriosis, we hypothesize that endometriosis decreases the peripheral and endomet rial Treg profile, whereas ectopic lesions have increased Treg localization. METHODS: Peripheral blood and endometrium were obtained throughout the menstrual cycle prior to and after induction of disease. Animals were randomly assigned to control (n = 7) or diseased (n = 16) treatment groups. Endometriosis was induced by i.p. injection of autologous menstrual tissue for 2 consecutive months during menses. Peripheral blood and endometrial tissue were collected at d9-11PO at 1, 3, 6, 9, 12 and 15 months post-induction of disease for fluorescence-activated cell sorting, quantitative RT-PCR and immunohistochemistry. Ectopic lesions were excised at 1 and 6 months post-inoculation and also harvested at necropsy (15 months) and processed for RNA of IHC. Identification of Tregs through analysis of FOXP3 expression was conducted utlilizing several methodologies. Differences were determined by non-parametric statistical analysis between all treatment groups and time points. RESULTS: In control animals, the proportion of peripheral natural Tregs (nTregs) was reduced (P < 0.05) during the mid- and late secretory stages of the menstrual cycle compared with menses. The induction of disease decreased peripheral Treg expression at early time points (P < 0.05) and this remained low throughout the time course, compared with the pre-inoculatory level of an individual. FOXP3 gene expression and Treg populations were also decreased in the eutopic endometrium (P < 0.05) compared with control animals, whereas these parameters were increased in ectopic lesions (P < 0.05), compared with the eutopic endometrium. CONCLUSIONS: Our data suggest that a reduction in peripheral Tregs may be a causative factor for endometriosis-associated infertility, while the increase in ectopic Treg expression may aid lesion development. Furthermore, endometriosis appears to disrupt Treg recruitment in both eutopic and ectopic endometrium.

Inflammation, leukocytes and menstruation.

Menstruation has many of the features of an inflammatory process. The complexity and sequence of inflammatory-type events leading to the final tissue breakdown and bleeding are slowly being unravelled. Progesterone has anti-inflammatory properties, and its rapidly declining levels (along with those of estrogen) in the late secretory phase of each non-conception cycle, initiates a sequence of interdependent events of an inflammatory nature involving local inter-cellular interactions within the endometrium. Intracellular responses to loss of progesterone (in decidualized stromal, vascular and epithelial cells) lead to decreased prostaglandin metabolism and loss of protection from reactive oxygen species (ROS). Increased ROS results in release of NFκB from suppression with activation of target gene transcription and increased synthesis of pro-inflammatory prostaglandins, cytokines, chemokines and matrix metalloproteinases (MMP). The resultant leukocyte recruitment, with changing phenotypes and activation, provide further degradative enzymes and MMP activators, which together with a hypoxic environment induced by prostaglandin actions, lead to the tissue breakdown and bleeding characteristic of menstruation. In parallel, at sites where shedding is complete, microenvironmentally-induced changes in phenotypes of neutrophils and macrophages from pro- to anti-inflammatory, in addition to induction of growth factors, contribute to the very rapid re-epithelialization and restoration of tissue integrity.

Relation of perimenstrual asthma with disease severity and other allergic co-morbidities--the first report of perimenstrual asthma prevalence in Saudi Arabia.

BACKGROUND: Perimenstrual asthma (PMA) has been documented in 30-40% of asthmatic women; however, there have been few epidemiological investigations of PMA in practice. OBJECTIVES: Determination of the prevalence of perimenstrual asthma in a sample of female Saudi asthmatic patients, and to study the relation of PMA to asthma severity, aspirin-induced asthma and to other allergic co-morbidities. STUDY DESIGN: Data were analysed from all female asthmatic patients followed up in the unit from January 2008 to May 2009 who were not pregnant, not on oral contraceptive pills, not menopausal, nor had had a hysterectomy. They were asked about exacerbation of their asthma state regarding worsening of symptoms, need for more rescue medications and even visits to emergency room just before or in the first days of menstruation, or both. Moreover, relation with aspirin-induced asthma and other associated allergic co-morbidities recorded in their medical files with prevalence of PMA was reported. RESULTS: The prevalence was found to be 8.2%. Asthma severity was found to be significantly related to PMA (p<0.0001). Aspirin-induced asthma and allergic co-morbidities were more prone to occur in cases with PMA than other studied asthmatics. CONCLUSION: We have found a low prevalence of PMA in Saudi women of fertile age compared to other studies published. Study findings support the hypothesis that PMA is related to asthma severity.

Ovarian hormones-autophagy-immunity axis in menstruation and endometriosis.

Menstruation occurs in few species and involves a cyclic process of proliferation, breakdown and regeneration under the control of ovarian hormones. Knowledge of normal endometrial physiology, as it pertains to the regulation of menstruation, is essential to understand disorders of menstruation. Accumulating evidence indicates that autophagy in the endometrium, under the regulation of ovarian hormones, can result in the infiltration of immune cells, which plays an indispensable role in the endometrium shedding, tissue repair and prevention of infections during menstruation. In addition, abnormal autophagy levels, together with resulting dysregulated immune system function, are associated with the pathogenesis and progression of endometriosis. Considering its potential value of autophagy as a target for the treatment of menstrual-related and endometrium-related disorders, we review the activity and function of autophagy during menstrual cycles. The role of the estrogen/progesterone-autophagy-immunity axis in endometriosis are also discussed.

Does intercourse during menses increase the risk for sexually transmitted disease?

Intercourse during menses is an acceptable sexual behavior that is practiced by 3-30% of sexually active women. This pattern of sexual behavior may contribute to transmission of some sexually transmitted disease. Yet, intercourse during menses is probably not associated with increased risk for pelvic inflammatory disease. The above-mentioned susceptibility for increased risk for sexually transmitted disease in women engaged in sexual intercourse during menses could be explained by the sexual behavior itself and/or by hampered response of innate immune system during menses. Still, while counseling couples about intercourse during menses, its potential advantages should be weighed against possible disadvantages.

Diminished Frequency of Menstrual and Peripheral Blood NKT-Like Cells in Patients With Unexplained Recurrent Spontaneous Abortion and Infertile Women.

Systemic monitoring of immune system may not precisely outline the local immune status in the uterus. This survey is a continuation of our previous studies on potential usefulness of menstrual blood (MB) immunophenotyping as a tool for investigation of immunological disturbances in pregnancy-related disorders. Peripheral blood (PB) and MB from healthy fertile (n = 15), unexplained recurrent spontaneous abortion (URSA; n = 15), and unexplained infertile women (n = 8) were collected simultaneously in the second day of their menstrual cycle and frequency of natural killer T (NKT)-like cell subpopulations were assessed by flow cytometry. Menstrual blood of all experimental groups contained higher percentage of TCRαß+, CD45RO+, and CD16- NKT-like cells compared to corresponding PB. Frequency of MB NKT-like cells in unexplained infertile participants was lower than fertile and URSA groups. Compared to normal participants, patients with URSA had lower frequency of PB TCRαß+ and higher CD16+, while in infertile woman frequencies of PB CD45RO+, CD45RO-, CD16-, IL17+, and MB CD45RO+ NKT-like cells were lower. Although, PB and MB seemingly have the same histological nature, our results showed that MB contained different composition of NKT-like subsets with different cytokine profiles and could be viewed as one potential biological sample for evaluation of patients with infertility and URSA.

Gender difference, sex hormones, and immediate type hypersensitivity reactions.

Gender differences in the development and prevalence of human diseases have long been recognized. Immense interest grows in the understanding of the role of sex hormones in the homeostasis of immunity. Asthma predominates in boys before puberty and this gender preference reverses after puberty and in adulthood, when adult women tend to have a more severe disease, often recalcitrant to treatment. Atopic eczema in preschool children shows insignificant gender difference or male preponderance in different studies, with more adult females suffering from atopic eczema. The limited data on the prevalence of immediate hypersensitivity to hymenoptera venom show controversial results. Discrepancy exists regarding the gender difference in food allergy, with females reporting significantly more allergic reactions in questionnaire studies. In general, adverse reactions to nonionic iodinated radiocontrast media are more commonly observed in females. The course of allergic diseases varies unpredictably during pregnancy, whereas hormone replacement therapy in postmenopausal women usually has a favorable influence on the course of asthma. Experiments in rodents confirm an effect of estrogens on mast cell activation and allergic sensitization, while progesterone is shown to suppress histamine release but potentiate IgE induction. Dehydroepiandrosterone may antagonize the production of Th2 cytokines but the effect of testosterone and the other androgens remains less defined. Actual data from human studies are lacking.

Individual differences in self-regulatory failure and menstrual dysfunction predict upper respiratory infection symptoms and antibody response to flu immunization.

Prior research indicates that cognitive priming manipulations that activate personal goals acutely increase or decrease natural killer cell cytotoxicity depending on whether individuals see themselves as making or failing to make progress toward their goals. Those findings in a laboratory setting revealed a psychobiological pathway whereby experiences of failure can influence health, but did not assess the impact of chronic perceived success/failure in goal pursuit on actual health outcomes. Three new studies investigated whether individual differences in perceived failure to attain personal goals influenced the self-reported symptoms of upper respiratory infections (URIs) as well as antibody response to flu immunization. Based on pilot data in young women, it also was hypothesized that the occurrence of menstrual dysfunction might interact with goal pursuit failure to more specifically predict cold and flu symptoms and optimal responses to vaccination. Perceived failure to attain goals did predict the reporting of URI symptoms as well as antibody levels post-immunization, both alone and in combination with menstrual dysfunction.

Acute systemic immune activation following vaginal exposure to staphylococcal enterotoxin B--implications for menstrual shock.

Menstrual toxic shock syndrome (mTSS) is an acute systemic inflammatory disease associated with the superantigenic exotoxin, toxic shock syndrome toxin (TSST)-1, produced by Staphylococcus aureus and the use of high absorbency tampons. Even though S. aureus is capable of elaborating several other superantigenic exotoxins, only TSST-1 has been implicated in the pathogenesis of mTSS possibly because most other superantigenic exotoxins are known enterotoxins. Nonetheless, we have shown recently that one of the enterotoxigenic staphylococcal superantigens, staphylococcal enterotoxin B (SEB), can cause robust systemic immune activation following exposure through non-enteric mucosa, including nasal or conjunctival routes. In a similar manner, we show here that vaginal administration of SEB in HLA class II transgenic mice can cause robust systemic immune activation characterized by profound elevation of proinflammatory cytokines in the serum, activation and expansion of SEB-reactive CD4(+) and CD8(+) T cells in peripheral lymphoid organs and SEB-induced deletion of immature thymocytes. Vaginal administration of SEB also caused leukocytic infiltration in major organs, such as liver and lung, reminiscent of human toxic shock syndrome. Systemic immune activation following vaginal superantigen delivery was independent of the stage of the estrus cycle in the mouse. Using HLA class II transgenic mice, we have shown that exposure to SEB through the vaginal canal can cause robust systemic immune activation. SEB could thus play a role in the pathogenesis of mTSS.

Atypical presentation and management of fibrodysplasia ossificans progressiva.

We report a case of an 18-year-old woman, with bilateral acute inflammatory pain on the hip area, during the premenstrual period, and progressive increase in volume and rigidity of both hips. Bilateral exuberant soft tissue calcifications were present on the radiographic exams, and the patient also presented with bilateral short-length hallux valgus. A heterozygous mutation in the protein kinase domain of ACVR1 gene was found, allowing the diagnosis of fibrodysplasia ossificans progressive. Due to the relation between the disease flares and the premenstrual period, the patient was put into a chemically induced amenorrhea, with no new inflammatory crises since.This case illustrates the importance of an accurate diagnosis to prevent unnecessary diagnostic procedures, as well as the need to develop specific treatment strategies to address each patient's particular needs.

A shift in the balance of T17 and Treg cells in menstrual blood of women with unexplained recurrent spontaneous abortion.

To determine usefulness of menstrual blood for assessment of differential frequency of T cell subsets, peripheral blood (PB) and menstrual blood (MB) from healthy fertile (n=15), unexplained recurrent spontaneous abortion (URSA) women (n=15) and unexplained infertile women (n=8) were collected in the second day of menstrual bleeding. Frequency of T cell subsets was measured by flow cytometry. URSA and unexplained infertile patients had higher frequency of TCRαß(+)CD3(+)CD56(-) and CD45RO(+) T cells as well as CD45RO(+)/CD45RO(-) ratio in PB as compared to MB. Frequency of CD3(+)TCRγδ(+) cells in PB of unexplained infertile and MB of URSA patients were significantly lower than that in fertile group. In all groups MB contained higher percentage of CD4(+)CD25(+)Foxp3(+) regulatory and lower percentages of CD16(+) T cells compared to PB. T17 cells (CD3(+)CD56(-)IL-17(+)) was found to be significantly higher in MB of only fertile and unexplained infertile subjects and not URSA patients in comparison with PB. Compared to PB, only fertile women had higher T17:Treg ratio in their MB. URSA women had significantly lower MB T17 cells and T17:Treg ratio compared to those in fertile and unexplained infertile women. Based on the findings presented here, we speculate that MB has its own unique immune milieu which is not solely continuation of the immune environment of secretory endometrium. Indeed, immunophenotyping of MB immune cells could potentially be a useful tool for investigation of immunological disturbances in pregnancy-related disorders.

Pathophysiology and Immune Dysfunction in Endometriosis.

Endometriosis is an estrogen-dependent, chronic, proinflammatory disease prevalent in 10% of women of reproductive age worldwide. Characterized by the growth of endometrium-like tissue in aberrant locations outside of the uterus, it is responsible for symptoms including chronic pelvic pain, dysmenorrhea, and subfertility that degrade quality of life of women significantly. In Canada, direct and indirect economic cost of endometriosis amounts to 1.8 billion dollars, and this is elevated to 20 billion dollars in the United States. Despite decades of research, the etiology and pathophysiology of endometriosis still remain to be elucidated. This review aims to bring together the current understanding regarding the pathogenesis of endometriosis with specific focus on mechanisms behind vascularization of the lesions and the contribution of immune factors in facilitating lesion establishment and development. The role of hormones, immune cells, and cytokine signaling is highlighted, in addition to discussing the current pharmaceutical options available for management of pain symptoms in women with endometriosis.