The effect of probiotics on immunogenicity of spermatozoa in couples suffering from recurrent spontaneous abortion.

BACKGROUND: Impaired spermatozoa immunogenicity can result in pregnancy complications such as recurrent spontaneous abortion (RSA). Given that spermatozoa contact with microbiota, it is possible that inappropriate microbiota composition in the reproductive tract could result in the alteration of spermatozoa antigenicity. Probiotics, as a representative of microbiota, may therefore have a beneficial effect on this altered immunogenicity. The objective of this study was to determine the effect of probiotics on spermatozoa immunogenicity. METHODS: Twenty-five fertile couples and twenty-five RSA couples were included in this study. Spermatozoa were purified and treated with probiotics. Untreated and probiotic treated spermatozoa were evaluated for human leukocyte antigen (HLA) class I & II expression by flow cytometry. Untreated and probiotic treated spermatozoa were also cocultured with the wife's peripheral blood mononuclear cells (PBMC) for 12 days. Then, the supernatant was assessed for IgG and APCA by enzyme-linked immunosorbent assay (ELISA) and complement-dependent cytotoxicity (CDC) assay respectively. RESULTS: Probiotic treatment of spermatozoa leads to an increase of HLA class I & II expression in both the fertile and RSA groups. The probiotic treatment resulted in a decrease in both IgG and APCA in the fertile group, but an increase in both IgG and APCA in the RSA group. CONCLUSIONS: The results of this study suggest that a supplementary probiotic treatment may be useful in couples suffering from RSA with an immunologic cause, because it improves disturbed HLA expression on spermatozoa and improves disturbed APCA and IgG production in the presence of spermatozoa.

CD33 as a leukocyte-associated marker expressed on human spermatozoa.

OBJECTIVE: Sialic acid-binding immunoglobulin-type lectins (Siglecs) are commonly present on immune cells and often mediate cell-to-cell interactions and signaling. Studies have shown the presence of Siglecs 1, 2, 5, 6, 10 and 14 on human spermatozoa. To the best of our knowledge, the expression of CD33 on spermatozoa has not yet been studied. Semen samples were collected from 25 healthy men with normal semen status. CD33 expression on purified spermatozoa was evaluated by flow cytometry methods. RESULTS: The results demonstrate the expression of CD33 on the surface of purified spermatozoa. The mean (± SD) of MFI (mean fluorescence intensity) was 12.85 (± 1.33) and the mean percentage of spermatozoa that express CD33 was 73.75 (± 3.75). Results were obtained showing that spermatozoa express CD33 (or Siglec-3) on their surface. The physiological role of these molecules on spermatozoa remains to be determined. It is recommended that further research be undertaken regarding the role of Siglecs (such as CD33) on spermatozoa apoptosis.

The effect of probiotic bacteria on toll-like receptor-2 and -4 expression by spermatozoa in couples with unexplained recurrent spontaneous abortion.

The disturbance of microbiota composition in the female reproductive tract (FRT) can result in several reproductive disorders. Spermatozoa express toll like receptors (TLRs) and may encounter many types of microbiota in the FRT, however no study has been performed regarding the interaction between spermatozoa TLRs and FRT microbiota in unexplained recurrent spontaneous abortion (URSA) and fertile couples. In this study, we investigate the interaction of vaginal lactobacillus casei probiotic as a representative of FRT microbiota with TLR2 and 4 on spermatozoa. Ten fertile couples and ten URSA couples were involved in this study. Untreated and lactobacillus casei probiotic treated purified spermatozoa were evaluated for TLR2 and 4 expression by flow cytometry. Vaginal lactobacillus casei probiotic treatment of spermatozoa led to increased expression of TLR4 and decreased expression of TLR2 on spermatozoa in both URSA and fertile couples. Vaginal lactobacillus casei probiotic led to an increase in TLR4 expression and a decrease in TLR2 expression on spermatozoa in fertile and URSA groups. However, the disturbed expression of TLR2 and 4 was not completely correct, and further studies with other types of vaginal lactobacilli are needed. In contrast to our expectation, vaginal lactobacillus casei probiotic could not improve the disturbed expression of TLR2 and TLR4 in the RSA group. This could be due to small sample size and the use of one type of lactobacillus. Therefore, further study needs to be performed with other types of lactobacilli to determine the effect of microbiota and probiotics on spermatozoa function such as motility, acrosome reaction, sperm capacitation, sperm and egg fusion and spermatozoa motility and apoptosis and etc. Nevertheless, this study can provide a first step to investigate the effectiveness of vaginal microbiota on spermatozoa, and consequently design new strategies for RSA couples.

Rescue effect of curcumin against copper toxicity.

Turmeric has long been used not only as an indispensable part of Asian cuisine but as a medicinal herb for dressing wounds, bites, burns, treating eye infections and acne. Curcuminoids are the active substances and their synthetic derivatives (i.e. diacetylcurcumin (DAC) and metal-curcumin complexes) possess an incredibly wide range of medicinal properties that encompass chelation capacity for multiple heavy metals, antioxidant activity, anti-inflammatory properties, cytotoxicity against cancerous cells, antiviral and antibacterial effects, antihypertensive and insulin sensitizing role, and regulatory role on apoptosis. The aforementioned properties have put curcumin on spotlight as a potential treatment for ailments such as, hepatic diseases, neurodegenerative diseases, metabolic syndrome, dyslipidemia, cardiovascular disease, auto-immune diseases, malignancies and conditions associated with metal overload. Copper is essential for major biological functions, however, an excess causes chronic ailments including neurodegenerative disorders. The fascinating approach of curcumin could alleviate such effect by forming a complex. Thus, this review aims to present available data on the effect of copper-curcumin interaction in various in vitro, ex-vivo in vivo, and clinical studies.

Blood IgMs from healthy donors and patients with systemic lupus erythematosus reduce the inflammatory properties of platelets from healthy donors.

Uncontrolled inflammation is the underlining mechanism of many human diseases and the increasing prevalence of such diseases mandate to develop new anti-inflammatory treatments. Utilizing the anti-inflammatory properties as well as other protective/beneficial features of natural IgMs (nIgMs) for treatment of human disorders seems as an easily accessible goal by the use of blood-purified IgMs as an alternative for polyclonal nIgMs. Despite the other blood cells, the functions of platelets have not been inspected under the influence of blood IgMs adequately. However, platelets, the second most numerous blood cells, are involved in the pathology of many inflammatory disorders through the production/expression of many inflammatory molecules. Thus, in the present study, we purified IgMs from serum of healthy donors and plasma of patients with systemic lupus erythematosus (SLE). Subsequently, we carried out comparative analysis of the inflammatory functions of normal platelets (P-selectin expression, GPIIb/IIIa activation, and secretion of soluble CD40L and TNF-α) that were stimulated by SLE microparticles (as key endogenous inflammation-drivers) in the presence or absence of the two IgM preparations; one with normal level of nIgMs (healthy blood IgMs) and the other with likely altered nIgM content (SLE blood IgMs). Both blood IgM preparations could suppress the elevated activation parameters of platelets in response to SLE microparticles. Additionally, the impact of SLE blood IgMs on the platelets was not superior to that of normal blood IgMs. The anti-inflammatory effects of blood IgMs on the activated platelets have been shown for the first time in the present study. Thus, this study provides evidence in favor of use of healthy blood IgMs as an anti-inflammatory therapy in clinical settings.

50-bp insertion/deletion polymorphism of the superoxide dismutase-1 is associated with bladder cancer risk in an Iranian population.

Bladder cancer (BC) is considered the sixth prevalent malignancy in men and the ninth leading cause of malignancy-related worldwide. Superoxide dismutase (SOD) is an antioxidant enzyme in the defense system against oxidative stress. Hence, we aimed to investigate whether the 50 bp Insertion/Deletion(Ins/Del) polymorphism of the SOD1 associated with the risk of BC. The study was conducted on 158 BC patients and 153 age-matched healthy subjects. Genomic DNA from all individuals was screened for the 50-bp SOD1 promoter deletion using PCR assay. Our results demonstrated an association between SOD1 Ins/Del (45% vs. 32%) genotype and risk of BC and this genotype elevated the susceptibility to BC (OR = 1.80, 95% CI: (1.10-2.90), P = 0.01). In addition, the Del allele of the SOD1 variation was detected to be more prevalent in the BC patients with the frequency of 28% and 20% in cases and healthy groups, correspondingly (OR = 1.61, 95% CI: (1.10-2.36), P = 0.01). It seems that SOD1 50-bp Ins/Del genotype, as well as Del, allele, is associated with an increased risk of BC in an Iranian population. However, further investigations in more diverse populations are necessary to assess the value of the novel biomarkers as a risk stratification biomarker for BC.

Nrf2 signaling pathway in trace metal carcinogenesis: A cross-talk between oxidative stress and angiogenesis.

A large number of people worldwide are affected by chronic metal exposure, which is known to be associated with different type of malignancies. The mechanisms of metal carcinogenicity are complex in nature, and excessive reactive oxygen species (ROS) generation induced by chronic metal exposure, among the other factors, has been proposed as one of the major mechanisms involved in that process. In tumor cells, ROS buildup may lead to cell death through intrinsic and extrinsic signaling pathways. Furthermore, ROS-mediated redox signaling has a crucial role in angiogenesis, which is recognized as an essential step in tumor progression. There are several redox-modulating pathways and among them, the nuclear factor erythroid2-related factor2 (Nrf2), as a sensor of oxidative or electrophilic stress, has introduced as a master regulator of cellular response against environmental stresses. Activation of Nrf2 signaling induces expression of wide variety of antioxidant and detoxification enzymes genes. Thus, this transcription factor has recently received much attention as a target for cancer chemoprevention. But meanwhile, constitutive Nrf2 activation in cancerous cells may promote cancer progression and resistance to chemotherapy. The current review describes the major underlying mechanisms involved in carcinogenesis of trace metals: copper, silver, and cadmium, with a special focus on the Nrf2 signaling pathway as a crossroad between oxidative stress and angiogenesis.

Spermatozoa Induce Maternal Mononuclear Cells for Production of Antibody with Cytotoxic Activity on Paternal Blood Mononuclear Cells.

OBJECTIVE: The maternal immune response to paternal antigens is induced at insemination. We believe that pregnancy protective alloantibodies, such as anti-paternal cytotoxic antibody (APCA), may be produced against the paternal antigens in the context of stimulated immunity at insemination and that they increase during pregnancy. APCA is necessary for pregnancy. It is directed towards paternal human leucocyte antigens (HLAs) and has cytotoxic activity against paternal leucocytes. The present study aims to determine whether APCA is produced by the maternal peripheral blood mononuclear cells (PBMCs) in contact with the husband's spermatozoa and to evaluate the relation of APCA production with HLA class I and II expressions by spermatozoa in fertile couples. MATERIALS AND METHODS: This cross-sectional study included 30 fertile couples with at least one child. The maternal PBMCs were co-cultured with the husband's spermatozoa and the supernatant was assessed for the presence of IgG by ELISA. Cytotoxic activity of the supernatant on the husband's PBMCs was assessed by the complement-dependent cytotoxicity (CDC) assay. RESULTS: IgG was produced in all co-cultures, and the mean level of supernatant IgG was 669 ng/ml. The cytotoxic activity of the supernatant was observed in all the supernatant obtained from the co-cultures. The mean percentage of APCA in supernatant was 73.93%. CONCLUSION: Based on the results of this study it can be concluded that APCA may be a natural anti-sperm antibody (ASA), which can be produced during exposure to spermatozoa and may have some influence before pregnancy. Further research is required to determine the role of APCA before pregnancy.

Progesterone-induced blocking factor (PIBF) influences the expression of membrane progesterone receptors (mPRs) on peripheral CD4+ T lymphocyte cells in normal fertile females.

PURPOSE: Progesterone-induced blocking factor (PIBF) is a protein secreted by lymphocytes exposed to progesterone (P4). P4 and PIBF have immunomodulatory effects on peripheral CD4+ T cells during normal pregnancy. Membrane progesterone receptors (mPRs) may correlate with the immunomodulatory properties of P4 on T cells. Variation in expression of mPRs may influence P4 regulatory performance during pregnancy. On the other hand, PIBF increases in pregnant normal women compared to women who have experienced abortion. The present study aimed to determine whether PIBF, in addition to having a direct influence on the immune system, can affect P4 performance through its effect on mPR expression. Such novel research findings demonstrate the importance of PIBF in the maintenance of pregnancy. METHODS: Isolated peripheral blood mononuclear cells (PBMCs) from 30 healthy women were stimulated with the mitogen phytohemagglutinin (PHA). Cells were either exposed to various concentrations of PIBF or had no exposure at all in a culture medium at 37 °C for 3 days. The mean fluorescence intensity (MFI) of mPRα and mPRß was evaluated using polyclonal and monoclonal antibodies on CD4+ T cells. RESULTS: PIBF was able to significantly increase mPR expression on the surface of peripheral CD4+ T cells (p ≤ 0.05). CONCLUSION: This study characterized the effects of PIBF on mPR expression on peripheral CD4+ T cells of healthy fertile women. Thus, a decrease in PIBF concentration during abnormal pregnancy can modulate mPR expression and regulatory performance of P4 on T cells. Future research into this issue is likely to open up a new understanding of the etiology of abortion.

Human Amniotic Epithelial Cells Affect the Functions of Neutrophils.

BACKGROUND AND OBJECTIVES: As a stem cell group, Human amniotic epithelial cells (HAECs) have numerous advantages over their embryonic and adult counterparts for therapeutic utility. They are closer to clinical applications compared to other stem cell types. Additionally, the anti-inflammatory and immunoregulatory properties of HAECs toward several immune cells have been shown previously. Nevertheless, despite the ever-increasing importance of neutrophils in the immune and non-immune processes, a few studies investigated the interaction of neutrophils and HAECs. To increase the current knowledge of HAECs immunology which is necessary for optimizing their future clinical applications, here we explored the effect of HAECs on two chief neutrophil functions; respiratory burst and phagocytosis. METHODS AND RESULTS: Freshly isolated human blood neutrophils were co-cultured with different number of HAECs for about 24 or 48 hours, then the oxidative burst and phagocytosis of stimulated neutrophils were assessed and compared. The results demonstrated a substantial elevation in the phagocytosis percentage, conversely a significant reduction in the oxidative burst of HAECs-cocultured neutrophils. These effects were dose-dependent, but did not show similar patterns. Likewise, the elongation of coculture period inversely influenced the HAECs-induced effects on the two neutrophil functions. CONCLUSIONS: The present study, for the first time, investigated the HAECs-mediated effects on the two main neutrophil functions. The findings suggest that HAECs by enhancement of phagocytic ability and simultaneously, attenuation of oxidative burst capacity of neutrophils protect the fetus from both microbial treats and oxidative stress and their consequent inflammation; thus corroborate the current anti-inflammatory vision of HAECs.

Can the Decreased Expression of Human Leukocyte Antigen Class â and â ¡ by Spermatozoa Lead to Recurrent Spontaneous Abortion?

BACKGROUND & OBJECTIVE: Unexplained recurrent spontaneous abortion (URSA) is defined as an unknown cause of occurrence of three or more clinically detectable pregnancy losses before 20 weeks of gestation, but it occurs presumably as a result of the immune system dysfunctions. We supposed that the disruption of semen or spermatozoa might be responsible for the dysfunction of the immune system in women with URSA. Semen and spermatozoa (as antigens) induce female reproductive tract (FRT) immunity. This stimulated immunity is necessary for pregnancy occurrence. The disruption of semen or spermatozoa can be a result of altering a variety of surface molecules on spermatozoa, especially polymorphic human leukocyte antigen (HLA) molecules or antigens. Despite the importance of HLA antigens in reproduction, to the best of our knowledge, no one has studied the relation of HLA expression between spermatozoa and URSA. Therefore, this paper aims to assess this relation. METHODS: Semen samples were collected from 15 URSA couples and 20 normal couples. After purification of normal spermatozoa, the HLA class I and II expressions were evaluated by flow cytometry methods. RESULTS: Results showed that the expression of both HLA class I and II by spermatozoa, in URSA couples, was significantly less than the control couples. CONCLUSION: The decreased expression of polymorphic HLA class Ⅰ and Ⅱ by spermatozoa can be related to URSA occurrence.

The expression of human leukocyte antigen by human ejaculated spermatozoa.

BACKGROUND: After coitus and insemination, an inflammatory response is evident in the female reproductive tract (FRT). Semen contains a variety of immune-activating components that have a major role in the induction of an immune response in the FRT. One of the most important is (human leukocyte antigen) HLA molecules which are present in soluble form in seminal plasma and in membrane form on the surface of cells (such as epithelial and leukocytes) existing in semen. Nevertheless, there is considerable debate over the expression of HLA antigens by human spermatozoa. Considering the critical role of HLA molecules in reproduction and the induction of an immune response, it is very important to clearly define HLA expression by spermatozoa and the role of these molecules in sperm morphology, motility, and strength to fertilize an egg. Therefore, the objective of this study was to determine HLA expression by ejaculated spermatozoa. The results of this study will facilitate the design of future studies. METHOD: Semen samples were collected from 50 healthy men with normal semen status by masturbation after 2-3 days of sexual abstinence. After purification of normal spermatozoa, HLA class I & II expression was evaluated by quantitative real-time PCR and flow cytometry methods. RESULTS: The results showed the expression of both HLA class I & class II by spermatozoa. The results also showed that the expression of HLA class Ⅱ was significantly more than HLA class Ⅰ. CONCLUSION: Spermatozoa express both HLA class I & class II molecules.

Decreased Toll-like Receptor (TLR) 2 and 4 Expression in Spermatozoa in Couples with Unexplained Recurrent Spontaneous Abortion (URSA).

Studies have shown that toll-like receptors (TLRs) play some important roles in reproductive processes such as ovulation, spermatogenesis, sperm capacitation, fertilization, and pregnancy to the best of our knowledge, no study has evaluated the expression and role of these molecules and their impairment in spermatozoa; accompanied by pregnancy complications such as recurrent spontaneous abortion (RSA). Therefore, this study investigates the alteration of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) expression in spermatozoa in men whose spouse have unexplained RSA. Fifteen fertile couples and fifteen couples with unexplained recurrent spontaneous abortion (URSA) were included in this study. The level of TLR2 and TLR4 expression in untreated and lipopolysaccharide (LPS) or PAM3CYS in treated spermatozoa were examined by flow cytometry. The results showed reduced expression of TLR4 in untreated spermatozoa and decreased LPS or PAM3CYS levels in treated spermatozoa in the URSA group compared to the control group. No significant differences were found in TLR2 expression of untreated spermatozoa in RSA and control groups. After the treatment of spermatozoa with LPS, the TLR2 expression was decreased in both groups. After the treatment of spermatozoa with PAM3CYS, the level of TLR2 expression was significantly increased in the URSA group; while no significant differences were shown in the control group in comparison to untreated spermatozoa. We have concluded that decreased TLR4 expression and a differently increased TLR2 expression in response to ligand treatment in spermatozoa is associated with URSA.

Indoleamine 2,3-Dioxygenase Is Dispensable for The Immunomodulatory Function of Stem Cells from Human Exfoliated Deciduous Teeth.

OBJECTIVE: In this study, we sought to better understand the immunoregulatory function of stem cells derived from human exfoliated deciduous teeth (SHED). We studied the role of the interferon gamma (IFN-γ)-indoleamine 2,3-dioxygenase (IDO)-axis in immunoregulation of SHED compared to bone marrow derived mesenchymal stem cells (BMMSCs) under the same conditions. MATERIALS AND METHODS: In this cross-sectional study, recently isolated human T cells were stimulated either by mitogen or inactivated allogeneic peripheral blood mononuclear cells (PBMCs). These T cells were subsequently co-cultured with, either SHED or BMMSCs in the presence or absence of 1-methyl-tryptophan (1-MT) or neutralizing anti- human-IFN-γ antibodies. In all co-cultures we evaluated lymphocyte activation as well as IDO activity. RESULTS: SHED, similar to conventional BMMSCs, had anti-proliferative effects on stimulated T cells and reduced their cytokine production. This property of SHED and BMMSCs was changed by IFN-γ neutralization. We detected IDO in the immunosuppressive supernatant of all co-cultures. Removal of IDO decreased the immunosuppression of BMMSCs. CONCLUSION: SHED, like BMMSCs, produced the IDO enzyme. Although IFN-γ is one of inducer of IDO production in SHED, these cells were not affected by IFN-γ in the same manner as BMMSCs. Unlike BMMSCs, the IDO enzyme did not contribute to their immunosuppression and might have other cell-type specific roles.

Variations in T-helper 17 and Regulatory T Cells during The Menstrual Cycle in Peripheral Blood of Women with Recurrent Spontaneous Abortion.

BACKGROUND: Disorders in immune system regulation may result in pregnancy abnormalities such as recurrent spontaneous abortion (RSA). This study aims to determine the ratio of regulatory T (Treg) and T helper (Th) 17 cells in unexplained RSA (URSA) women during proliferative and secretory phases of their menstrual cycles compared to healthy non-pregnant women. MATERIALS AND METHODS: In this case control study, 25 women with URSA and 35 healthy, non-pregnant women were enrolled. The percentage of Th17 and Treg cells in participants peripheral blood were determined by flow cytometry. RESULTS: The percentage of Th17 cells and their related cytokines in serum (IL-17A) were higher in the proliferative and secretory phases of the menstrual cycles of URSA women compared to the control women. However, a lower percentage of Treg cells and their related cytokines in serum, transforming growth factor (TGF) ß1 and interleukin (IL)-10 were detected in the proliferative but not the secretory phase of the URSA group. The ratio of Th17/CD4+ Treg was higher in the URSA group than the control group. We observed an increased ratio of Th17/CD4+ Treg during the proliferative and secretory phases in URSA women. CONCLUSION: The imbalance between Th17 and Treg cells during the proliferative phase of menstrual cycles in the URSA group may be considered a cause for spontaneous abortion.

Comparing the immunoregulatory effects of stem cells from human exfoliated deciduous teeth and bone marrow-derived mesenchymal stem cells.

Stem cells from human exfoliated deciduous teeth (SHED) have been introduced recently and possess characteristics similar to mesenchymal stem cells (MSCs). Because of their convenient accessibility and safety of harvest, SHED can be a preferable source for the ever-increasing MSCs' applications  While they are new, their immunoproperties have not been adequately studied. In this study, we aimed to explore the effect of SHED on T lymphocytes and compare it to conventional MSCs (BMMSCs).At first the isolated T lymphocytes were activated specifically/nonspecifically in vitro and cocultured with SHED or BMMSCs under the same conditions, subsequently their proliferation and cytokine secretion (IL-2 and IFN-γ) were measured.In our experiment, BMMSCs and SHED inhibit the proliferation and cytokine production of both PHA and alloantigen stimulated T lymphocytes in a dose-dependent manner. In direct and indirect contact to T lymphocytes, the inhibition of BMMSCs (but not of SHED) was significantly different The cytokine production from activated T cells was affected differently by two types of MSCs. The inhibition decreased by the separation of lymphocytes and MSCs by a semipermeable membrane, but it was not abolished.This study showed that SHED suppress the activation of human T lymphocytes in vitro like other MSCs. Compared to BMMSCs, this suppression was alleviated. In the equal conditions, the pattern of immune-modulation of BMMSCs and SHED was different, suggesting that SHED do not exert the exact mechanisms of BMMSCs' immunosuppression., This finding should be verified by further studies focused on the detailed mechanisms  of the immunomodulation of SHED and also BMMSCs.