Kuniaki Ota

Vitamin D deficiency may be a risk factor for recurrent pregnancy losses by increasing cellular immunity and autoimmunity

STUDY QUESTION Do women with recurrent pregnancy losses (RPL) and low vitamin D have increased prevalence of auto- and cellular immune abnormalities when compared with women with RPL who have normal vitamin D, and does vitamin D have any effect on cellular immunity in vitro? SUMMARY ANSWER A high proportion of women with RPL have vitamin D deficiency and the risk of auto- and cellular immune abnormalities is increased in women with RPL and vitamin D deficiency. WHAT IS KNOWN ALREADY Vitamin D deficiency in pregnant women is associated with increased risk of obstetrical complications such as pre-eclampsia, bacterial vaginosis associated preterm delivery, gestational diabetes mellitus and small-for-gestational age births. STUDY DESIGN, SIZE, DURATION A retrospective cross-sectional study of 133 women with RPL who were enrolled in a 2-year period, together with laboratory experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS Women with three or more consecutive spontaneous abortions prior to 20 weeks of gestation who were enrolled at the University clinic. Serum vitamin D level, cellular activity and autoimmune parameters in vivo and in vitro were measured. MAIN RESULTS AND THE ROLE OF CHANCE Sixty-three out of 133 women (47.4%) had low vitamin D (<30 ng/ml). The prevalence of antiphospholipid antibody (APA) was significantly higher in low vitamin D group (VDlow) (39.7%) than in the normal vitamin D group (VDnl) (22.9%) (P< 0.05) and the adjusted odds ratio (OR) for APA in VDlow was 2.22 with the 95% confidence interval (CI) of 1.0-4.7. The prevalence of antinuclear antigen antibody (VDlow versus VDnl; 23.8% versus 10.0%, OR 2.81, 95% CI 1.1-7.4), anti-ssDNA (19.0% versus 5.7%, OR 3.76, 95% CI 1.1-12.4) and thyroperoxidase antibody (33.3% versus 15.7%, OR 2.68, 95% CI 1.2-6.1) was significantly higher in VDlow than those of VDnl (P < 0.05 each). Peripheral blood CD19(+) B and CD56(+) NK cell levels and NK cytotoxicity at effector to target cell (E:T) ratio of 25:1 were significantly higher in VDlow when compared with those of VDnl (P < 0.05 each). Reduction (%) of NK cytotoxicity (at E:T ratio of 50:1 and 25:1) by IgG (12.5 mg/dl) was significantly lower in VDlow than those of VDnl (P < 0.05, P < 0.01, respectively). There were no differences in Th1/Th2 ratios between VDlow and VDnl. When vitamin D3 was added in NK cytotoxicity assay in vitro, NK cytotoxicity at E:T ratio of 50:1 was significantly suppressed with 10 nMol/L (nM) (11.9 ± 3.3%) and 100 nM (10.9 ± 3.7%) of vitamin D3 when compared with controls (15.3 ± 4.7%) (P < 0.01 each). TNF-α/IL-10 expressing CD3(+)/4(+) cell ratios were significantly decreased with 100 nM of vitamin D3 (31.3 ± 9.4, P < 0.05) when compared with controls (40.4 ± 11.3) in vitro. Additionally, INF-γ/IL-10 expressing CD3(+)/4(+) cell ratio was significantly decreased with 100 nM of vitamin D3 (12.1 ± 4.0, P < 0.05) when compared with controls (14.8 ± 4.6). IFN-γ and TNF-α secretion from NK cells were significantly decreased (P < 0.01 each), and IL-10, IL-1β, vascular endothelial growth factor and granulocyte colony stimulating factor levels were significantly increased (P < 0.01 each) with vitamin D3 100 nM when compared with those of controls. LIMITATIONS, REASONS FOR CAUTION The prevalence of vitamin D deficiency in women with RPL in this study is open to a possible type I error since women with vitamin D supplementation were excluded from this study. WIDER IMPLICATIONS OF THE FINDINGS Assessment of vitamin D level is recommended in women with RPL. Vitamin D supplementation should be explored further as a possible therapeutic option for RPL. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the intramural funding from Department of Microbiology and Immunology, Chicago Medical School at Rosalind Franklin University of Medicine and Science. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

Management of Women with Recurrent Pregnancy Losses and Antiphospholipid Antibody Syndrome

Antiphospholipid antibodies (aPL) have been associated with recurrent pregnancy losses (RPL) and other obstetrical complications. The diagnostic criteria for the classical antiphospholipid antibody syndrome (APS) have been utilized for the detection of obstetrical APS in women with RPL. However, laboratory findings and immunopathology of obstetrical APS are significantly different from those of classical APS. In addition, many women with RPL who have positive aPL do not have symptoms consistent with the current APS criteria. The induction of a proinflammatory immune response from trophoblasts and complement activation by aPL rather than thromboembolic changes has been reported as a major immunopathological feature of obstetrical APS. Heparin treatment has been reported to be effective in prevention of early pregnancy loss with APS but not for the late pregnancy loss or complications. The complex effects of heparin may explain the limited efficacy of heparin treatment in RPL. New diagnostic criteria for obstetrical APS are needed urgently, and new therapeutic approaches should be explored further.

1,25-Dihydroxy-vitamin D3 regulates NK-cell cytotoxicity, cytokine secretion, and degranulation in women with recurrent pregnancy losses

Vitamin D has a pivotal role in regulating immune responses by promoting Th2 immune responses and suppressing Th1 responses. Propensities to a Th1 immune response and increased NK‐cell levels and cytotoxicity have been reported in women with recurrent pregnancy losses (RPL). In women with RPL, vitamin D deficiency is prevalent; however, the effect of vitamin D on NK cells is largely unknown. In this study, we demonstrated that CD69+ activating receptor expression on NK cells was significantly decreased by incubation with 1,25(OH)2D3 in a dose‐dependent manner, while CD158a and CD158b inhibitory receptor expression was upregulated. The degranulation marker CD107a was significantly downregulated on NK cells following incubation with 1,25(OH)2D3. NK‐cell conjugation with K562 target cells was not affected by 1,25(OH)2D3; however, depolarization of perforin granules in conjugated NK cells was significantly increased. TLR4 expression on NK cells was significantly decreased and TNF‐α and IFN‐γ production was significantly reduced by 1,25(OH)2D3 through interference with NF‐κB. Our results suggest 1,25(OH)2D3 has immune regulatory effects on NK cell cytotoxicity, cytokine secretion and degranulation process as well as TLR4 expression. Potential therapeutic application of 1,25(OH)2D3 for dysregulated NK‐cell immunity should be explored in the future.

Humoral and cellular autoimmunity in women with recurrent pregnancy losses and repeated implantation failures: A possible role of vitamin D.

Women with recurrent pregnancy losses (RPL) and repeated implantation failures (RIF) have auto- and cellular immune abnormalities. Approximately, 20% of women with RPL have autoimmune abnormalities, particularly antiphospholipid antibodies (APA). In addition, these women have a higher prevalence of antinuclear antibody, anti-thyroperoxidase and anti-thyroglobulin antibodies, and other non-organ-specific autoantibodies. In women with RPL, the presence of autoimmunity is often associated with cellular immune abnormalities, such as increased NK cell levels and Th1/Th2 cell ratios. Vitamin D (VD) plays a major role in regulation of auto- and cellular immune abnormalities. VD deficiency is prevalent in women with RPL, and women with VD deficiency have increased auto- and cellular immune abnormalities as compared with women with normal VD levels. VD has immune regulatory effects on various immune effectors including T, B and NK cells. Potential therapeutic application of VD for RPL and RIF with auto- and cellular immune abnormalities should be explored.

Expression of a2 Vacuolar ATPase in Spermatozoa is Associated with Semen Quality and Chemokine-Cytokine Profiles in Infertile Men

Background A number of laboratory tests have been developed to determine properties of spermatozoa quality but few have been adopted into routine clinical use in place of the WHO semen analysis. We investigated whether Atp6v0a2 (a2 isoform of vacuolar ATPase) is associated with abnormal semen quality and changes in chemokine-cytokine profiles in infertile men. Patients and Methods Semen samples were collected from 35 healthy donors and 35 infertile men at the Andrology laboratory from August 2011 to June 2012. The levels of Atp6v0a2 mRNA and protein, and its localization in spermatozoa were determined. a2NTD (the N-terminal portion of Atp6v0a2) and secreted chemokine-cytokine profiles in seminal fluid were measured. Results Atp6v0a2 protein (P<0.05) and mRNA (P<0.05) in spermatozoa from infertile men were significantly lower than those from fertile men. Fluorescent microscopy revealed that Atp6v0a2 is mainly expressed in the acrosomal region. Infertile men’s seminal fluid had significantly lower G-CSF (P<0.01), GM-CSF (P<0.01), MCP-1 (P<0.05), MIP-1α (P<0.01) and TGF-β1 (P<0.01) levels when compared to the seminal fluid from fertile men. Seminal fluid a2NTD levels were significantly correlated with G-CSF (P<0.01), GM-CSF (P<0.01), MCP-1 (P<0.05), MIP-1α (P<0.01) and TGF-β1 (P<0.01) which are key molecules during the onset of pregnancy. Conclusion These results suggested that a critical level of Atp6v0a2 is required for the fertile spermatozoa and its decreased level in spermatozoa could be used to predict male infertility. This study provides a possibility that Atp6v0a2 could be potentially used as a diagnostic marker for the evaluation of male infertility.

Elevation of soluble form of receptor for advanced glycation end products (sRAGE) in recurrent pregnancy losses (RPL): possible participation of RAGE in RPL

OBJECTIVE To determine whether the soluble receptor for advanced glycation end products (sRAGE) and immune inflammatory markers are associated with recurrent pregnancy losses (RPL). DESIGN Prospective case-control study. SETTING University clinic. PATIENT(S) A total of 93 women (age 35.8±4.6 years) were enrolled including 63 women with three or more recurrent pregnancy losses (RPL), and age-matched fertile controls with a history of at least one live birth and no history of pregnancy losses (n=30). INTERVENTION(S) Peripheral blood collection. MAIN OUTCOME MEASURE(S) Assessment of anthropometric, metabolic, and inflammatory immune variables. RESULT(S) Levels of sRAGE were statistically significantly higher in RPL patients than in control patients (1,528.9±704.5 vs. 1,149.9±447.4 pg/mL). In the multivariate analysis, the levels of insulin, plasminogen activator inhibitor-1, the resistance index of the uterine radial artery, and the ratio of tumor necrosis factor-α/interleukin-10 producing T helper cells were statistically significantly associated with the serum sRAGE level. CONCLUSION(S) Elevated levels of serum sRAGE are associated with RPL. The soluble receptor for advanced glycation end products might contribute to RPL by reducing uterine blood flow and subsequently causing ischemia in the fetus via inflammatory and thrombotic reactions.

Persistent high levels of IgM antiphospholipid antibodies in a patient with recurrent pregnancy losses and rheumatoid arthritis

Recurrent pregnancy losses (RPL) and unexplained infertility (UI) are often associated with the presence of antiphospholipid antibodies (APA). We report one case with RPL, UI, and persistent IgM APA without B‐cell isotype switch.

Diabetes and Female Sterility/Infertility

AGEs (advanced glycation end products) are pro-inflammatory molecules that trigger a state of intracellular oxidative stress and inflammation after binding to their cell membrane receptors, such as RAGE. The activation of the AGE-RAGE axis has been well known to play a role in type 2 diabetes mellitus (T2DM), obesity, metabolic syndrome (MetS), cardiovascular disease (CVD), aging, inflammation, and neurodegenerative disorders. AGEs might contribute to the etiology of polycystic ovary syndrome and infertility. This article explains for the relationship between the AGE-RAGE system and infertility as well as ovarian reserve in women of reproductive age.

Giant Polyp of Uterine Cervix: A Case Report and Brief Literature Review

Cervical polyps are most commonly seen in the female with uterine bleeding. On the other hand, giant cervical polyps with a size greater than 4 cm are rare and until now only several cases have been described in literature. The size and the clinical presentation can mimic a cervical neoplasia. The management is surgical and can be conservative regarding to the benign pathological feature of this entity. We report the case of a giant cervical polyp of 12.0 cm which was protruding from the external cervical os and that developed spontaneously in a multiparous 48-year-old woman who clinically presented vaginal bleeding. Colposcopic examination showed a mass originating from posterior lip of external cervical os. On MRI, there was the appearance of multicystic lesion with an inner solid component. We performed that the lesion was resected by the ultrasonic harmonic scalpel. The histological examination showed benign polyp. At 10 months’ follow-up, there was no recurrence seen after surgery. Although carcinomatous change occurs in 1.7% of cervical polyps, malignant degeneration did not occur in the previous reported cases. The diagnosis, management and outcome of this rare entity had been reviewed according to the literature.