Fertile Non-pregnant Women Research Articles

CD56 and Tim-3 molecule expression in different monocyte subsets in physiological pregnancy

Monocytes play an important role in the systemic immune defense against pathogens and maintaining physiological pregnancy. During pregnancy peripheral monocytes migrate into the decidua and form the pool of decidual macrophages which participate in the formation and development of placental tissues. The population of peripheral blood monocytes is phenotypically and functionally heterogeneous. In humans, there are different monocyte subsets depending on the expression of CD14 and CD16. CD56-positive monocytes are found in healthy women. Their number is positively correlated with body mass index, body fat. Tim-3 (T cell Ig and mucin domain-containing protein 3) expression is observed in peripheral monocytes during pregnancy. It is known that peripheral monocyte functions effectively change at pregnancy to form the immune tolerance at the maternal-fetal interface and the systemic immune defense against pathogens. However, the monocyte phenotype shift during pregnancy remain poorly understood. Therefore, the aim of the study was to evaluate the CD56 and Tim-3 expressions in monocyte subsets in human pregnancy. Peripheral blood mononuclear cells were isolated from peripheral blood of pregnant women (gestational age 29 weeks (28-31) by density gradient centrifugation and analyzed by flow cytometry. Peripheral blood of healthy non-pregnant fertile women (in follicular phase of the menstrual cycle) aged 21-29 years was studied as control. Pregnant women had a lower percentage of classical CD14hi/CD16- monocytes in comparison with non-pregnant. The percentages of intermediate (CD14hi/CD16+) and non-classical (CD14low/CD16+) monocytes did not change. The CD56 molecule expression was observed in all monocyte subsets in pregnant and non-pregnant women. Pregnant women had a higher percentage of CD56-positive classical (CD14hiCD16-) and non-classical (CD14lowCD16+) monocytes than non-pregnant. The percentage of CD56-positive intermediate (CD14hiCD16+) monocytes did not change. The percentages of double-positive CD56+Tim-3+ classical (CD14hiCD16-) and non-classical (CD14lowCD16+) monocytes were increased in pregnant women. The numbers of double-positive CD56+Tim-3+intermediate (CD14hiCD16+) monocytes did not change. Thus, the CD56 and Tim-3 expressions in different monocyte subsets were changed in human pregnancy.

Galectin-9 influences the Tim-3 molecule expression in natural killer different subpopulations

Galectin-9 is a b-galactoside binding lectin with expressed immunoregulatory activity. During pregnancy galectin-9 is produced by trophoblast cells and regulates the function of natural killer (NK) cells at the maternal-fetal interface via binding to Tim-3 (T-cell Ig and mucin domain-containing protein 3) molecules. Natural killer (NK) lymphocytes belong to the innate lymphoid cells, which have a cytotoxic effect on target cells and are capable of producing a large number of regulatory factors (cytokines, chemokines). Decidual NK have a tolerant phenotype and play a leading role in the regulation of invasive trophoblast growth and provide peripheral immune tolerance in the area of uteroplacental contact. Peripheral NK cells express Tim-3 molecules. Galectin-9 concentration is increased in peripheral blood during physiologic pregnancy. At pregnancy phenotype and functions of peripheral NK cells are changed to maintain the maternal–fetal immune tolerance. Peripheral NK cells migrate to the maternal-fetal interface and are transformed into a decidual NK-like phenotype cells. Galectin-9 concentration is decreased in women with a complicated pregnancy and miscarriage. However the galectin-9 effects on different NK cell subpopulations of peripheral blood are not investigated. Therefore, we studied the galectin-9 influence on phenotype transformation and Tim-3 expression of NK cells isolated from peripheral blood of healthy non-pregnant fertile women. CD56+NK cells were obtained by immunomagnetic separation and cultivated in vitro during 72 hours with cytokines (IL-2 and IL-15). Galectin-9 (5 ng/mL) and anti-Tim-3 (10 mg) antibodies were added to the NK cultures. Galectin-9 concentration is corresponded to its level during first trimester of physiologic pregnancy. The number of regulatory NK (CD16-CD56bright), cytotoxic NK (CD16+CD56dim/-) cells and Tim-3 expression on different NK subpopulations were assessed by flow cytometry. It was found that Tim-3 was expressed on all subpopulations of peripheral blood NK cells (CD16-CD56brightNK, CD16+CD56dimNK, CD16+CD56-NK). Incubation with galectin-9 increased the expression of Tim-3 on regulatory CD16-CD56brightNK cells and did not change on cytotoxic CD16+CD56dim/-NK cells. Galectin-9 reduced the percentage of cytotoxic CD16+CD56dimNK in culture, but did not influence the number of regulatory CD16-CD56bright NK and cytotoxic CD16+CD56-NK cells. Thus, galectin-9 regulates Tim-3 molecule expression and NK cell subpopulation distributions in vitro culture.

Expression of M2-associated molecules in circulating monocyte subsets in fertile non-pregnant women and pregnant women with uncomplicated pregnancy

In humans circulating monocytes include classical (CD14++CD16- ), intermediate (CD14++CD16+) and non-classical/alternative (CD14+CD16++) monocytes, which in turn can be activated via the classical or alternative pathway. Pregnancy is accompanied by significant changes in the monocyte compartment, which is manifested by an increase in the number of circulating monocytes, including the proportion of intermediate monocytes, and a change in their function. However, the functional properties of monocyte subsets during gestation remain largely unexplored. We hypothesized that circulating monocytes may be activated in an alternative pattern and acquire features of M2 polarization (anti-inflammatory / immunosuppressive properties). The aim of the investigation was to study M2-associated markers that characterize the anti-inflammatory and immunosuppressive potential of myeloid cells in subpopulations of circulating monocytes in fertile nonpregnant women and women with uncomplicated pregnancy in the 2nd trimester. It was shown that in fertile non-pregnant women intermediate and non-classical monocytes are characterized by a higher expression of M2-associated markers (CD206, Arginase 1, MerTK) compared to classical monocytes. In the 2nd trimester of pregnancy, the expression of these molecules on monocytes increases significantly, which is manifested by 1) an increase in the proportion of CD206+ cells in subpopulations of classical and intermediate monocytes, 2) an increase in the mean fluorescence intensity of Arginase 1 in all monocyte subsets, 3) an increase in the proportion of MerTK+ cells in subpopulations of classical and intermediate monocytes and mean fluorescence intensity across all monocyte subsets. The highest content of CD206+ and MerTK+ cells in pregnant women is detected in the subpopulation of intermediate monocytes, and the highest values of the mean fluorescence intensity of Arginase 1 and MerTK – in the subpopulations of intermediate and non-classical monocytes. The data obtained demonstrate that monocytes of pregnant women in the 2nd trimester of pregnancy are characterized by signs of M2 polarization. This is confirmed not only by an increase in the expression of the M2-associated mannose receptor CD206, but also by an increase in the expression of Arginase 1 and MerTK, which mediate the immunosuppressive activity of myeloid cells and, in particular, macrophages of the M2 phenotype. Further studies of M2-associated markers in monocyte subpopulations during gestation will allow a more detailed characterization of the regulatory role of circulating myeloid cells during pregnancy.

Expression of inhibitory receptors PD-1, CTLA-4, and Tim-3 by peripheral T cells during pregnancy

Background. Inhibitory receptors and their ligands (also called checkpoint molecules) are important feedback regulators of the immune response. However, their role in immunological adaptation during pregnancy remains poorly understood.The aim of the study was to evaluate the level of checkpoint molecule (PD-1, CTLA-4, Tim-3) expression in peripheral T cells in pregnant women compared with fertile non-pregnant women.Materials and methods. The study included 36 women in the second half of pregnancy without pregnancy complications, 12 of whom had extragenital pathology. The control group consisted of 28 age-matched fertile non-pregnant women. The proportion of CD8+PD-1+, CD8+TIM-3+, CD8+PD-1+TIM-3+, CD4+PD-1+, CD4+TIM-3+, and CD4+PD-1+TIM-3+ was evaluated by flow cytometry using the corresponding monoclonal antibodies (BD Biosciences, USA).Results. The proportion of CD4+Tim-3+ and CD8+PD-1+ Т cells and CD4+ and CD8+ Т lymphocytes co-expressing PD-1 and Tim-3 in the peripheral blood of pregnant women was statistically significantly higher than in non-pregnant women. An increase in CD4+Tim-3+ and CD8+PD-1+ T cells was observed both in pregnant women with and without extragenital pathology. However, pregnant women with extragenital pathology were characterized by a higher CD8+PD-1+ count and a smaller number of CD8+Tim-3+ cells, as well as by a lack of an increase in PD-1+Tim-3+ T cells typical of pregnant women. The number of comorbidities was directly correlated with the proportion of CD8+PD-1+ lymphocytes and inversely correlated with the proportion of CD8+Tim-3+ and CD4+ PD-1+Tim-3+ cells. In addition, the expression of checkpoint molecules was associated with gestational age (a direct correlation was found with the proportion of CD8+Tim-3+, CD4+PD-1+Tim-3+, and CD8+PD-1+Tim-3+ cells) and to a lesser extent – with the age of pregnant women (an inverse relationship was found with the proportion of CD8+Tim-3+ cells).Conclusion. Pregnant women in the second half of pregnancy are characterized by increased expression of PD-1 and Tim-3 molecules in peripheral T cells. At the same time, concomitant extragenital pathology affects the expression of these molecules.

P-421 Increased myeloid-derived suppressor cells in women with recurrent miscarriage and recurrent implantation failure

Abstract Study question We sought to explore the levels of myeloid-derived suppressor cells (MDSCs) in women experiencing recurrent miscarriage (RM) or recurrent implantation failure (RIF) Summary answer MDSCs were significantly expanded in RM patients with respect to fertile healthy women (p = 0.03, 8.25 vs 7.00). What is known already RM and RIF are two distinct disorders that can be associated with a baseline pro-inflammatory state. Different surrogate inflammatory biomarkers have been associated with these disorders in a subgroup of patients, including expanded cytotoxic NK cells, increased pro-inflammatory cytokines or reduced regulatory T cells (TReg). MDSCs are a heterogeneous population of leukocytes, known for their capacity to modulate immune responses. The immunosuppressive function of MDSC in cancer has been well established, while a controversial role in autoimmune diseases has been reported. Typically, there are two major groups of MDSCs in humans: granulocytic/polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Study design, size, duration A cross-sectional study to evaluate the levels of MDSCs in women with RM and RIF consecutively referred to our Reproductive Immunology Unit. 55 patients and 23 healthy women were evaluated from 2021 to 2022. RM was defined as the loss of two or more pregnancies and not necessarily consecutive. RIF was defined as the failure to achieve a clinical pregnancy after transferring at least four good-quality embryos. Participants/materials, setting, methods A full fertility screening was performed for all women and their partners as part of routine care. A group of healthy non-pregnant women up to 45 years old with two or more children (proven fertility) and no spontaneous miscarriage was used as control group. Freshly collected blood samples were analyzed by multiparametric flow cytometry using a BD FACS CANTO II. Peripheral blood M-MDSCs were characterized based on HLA-DR, CD14, CD33 and CD45 expression. Main results and the role of chance In this study, we evaluated 55 women, 35 that fulfilled RM criteria and 20 with RIF. In addition, a group of 23 non-pregnant fertile women was included as a control group. We characterized MDSCs as CD14+CD33+CD45+HLA-DR-/LOW, according to previous reports. We observed a significant expansion of peripheral blood MDSCs in RM group compared to the control group (8.25% [6.62-11.9] vs 7.05% [6-7.8], p = 0.03) and RIF group showed a similar increasing trend compared to controls (8.6% [5.65-12.35] vs 7.05% [6-7.8], p = 0.09). We performed ROC curves analysis to evaluate the predictive power. We established an optimal cut-off level of 8.3% in RM group, with a sensitivity of 50% and a specificity of 86.96%. Moreover, women with more than 8.3% had a higher probability of presenting RM compared to the control group (OR: 6.66; CI: 1.68-26.46). MDSCs are characterized by their ability to exert a potent immunosuppressive function. However, recent investigations have shown that MDSCs could act as pro-inflammatory cells under certain inflammatory conditions, such as autoimmune diseases. In this context, MDSCs could worsen the pro-inflammatory state in women, leading to miscarriage or implantation failure. Limitations, reasons for caution In order to validate these results, we should extend our cohort and control group. Moreover, we need to perform a functional evaluation of this subset so we could elucidate their role in these conditions. Wider implications of the findings MDSCs might be a good predictor for RM and RIF patients and with others (as expanded NK cells) may classify a subgroup of patients with a pro-inflammatory profile that can benefit from personalized therapies. Trial registration number Not aplicable

Post-hoc evaluation of peripheral blood natural killer cell cytotoxicity in predicting the risk of recurrent pregnancy losses and repeated implantation failures

Peripheral blood NK cytotoxicity assay (NKC) is one of the commonly utilized diagnostic tools for recurrent pregnancy losses (RPL) and repeated implantation failures (RIF). In this retrospective cohort study, we aimed to assess the cutoff values of NKC for RPL and RIF. A total of 883 women were included in this study; 24 nonpregnant fertile women, 604 nonpregnant women with three or more RPL, 163 nonpregnant women with two or more of RIF, 48 normal pregnant women, and 44 pregnant women with a history of RPL. Peripheral blood NKC assay was performed by flow cytometry. The differences between groups were analyzed using Student’s t-test, a logistic regression analysis, and the area under the receiver operating characteristic curve analysis. Both nonpregnant fertile and normal pregnant women had significantly lower NKC at an effector to target cell ratio (E:T) of 50:1 (13.5 ± 1.1% and 12.9 ± 1.0%, respectively) when compared to women with RPL and RIF, and pregnant women with a history of RPL (23.6 ± 0.3%, 23.9 ± 0.5%, and 23.7 ± 1.0%, P < 0.0001 respectively). In addition, the area under the receiver operating characteristics curve for RPL and RIF using pre-conception NKC was 0.863 (P < 0.0001) and 0.879 (P < 0.0001), respectively, and for RPL using post-conception NKC was 0.736 (P = 0.001). These findings suggest that NKC significantly distinguishes nonpregnant women with RPL and RIF from fertile controls and pregnant RPLwomen from normal pregnant controls.

Vitamin D levels and C-reactive protein/albumin ratio in pregnant women with cerebral venous sinus thrombosis.

Changes in coagulation system during pregnancy have been put forth as risk factors for cerebral venous sinus thrombosis (CVT), yet we still have limited knowledge on markers for predicting the risk of CVT in pregnant women. Therefore, we aimed to investigate the significance of vitamin D (VD) levels and C-reactive protein (CRP)/albumin ratio (CAR), an inflammation marker, as risk factors for CVT in pregnant women. 23 pregnant women who were followed up for CVT, 26 healthy pregnant women who had no pregnancy complications, and 31 non-pregnant fertile women were included in the study. CAR and VD levels were compared between groups. CAR was significantly higher in the pregnant CVT group compared to the other two groups (p < 0.001). CAR was also significantly higher in the healthy pregnant group than the non-pregnant fertile group (p < 0.001). VD levels were determined to be statistically significantly lower in the pregnant CVT group compared to the other two groups (p < 0.001). However, VD levels did not significantly differ between healthy pregnant group and non-pregnant fertile group (p > 0.05). We found no significant correlation between CAR and VD levels in any of the three groups. Pregnant women with CVT were found to have a high rate of severe VD deficiency. Low VD levels and high CAR levels in pregnant women may be associated with an increased risk of CVT.

Primary Women Infertility and Thyroid Disorders

The study was conducted to explore the prevalence of thyroid autoimmune disorders among primaryinfertile women. Hormonal and immunological serum markers were tested including: TSH, T3, T4, TPO Ab,prolactin and Interleukin-6. Thyroid abnormalities were observed in 38.5% of the primary infertile women.Thyroid autoimmunity was more prevalent among primary infertile women (9.6%) than pregnant (4.0%)and non-pregnant (4.3%) fertile women, but with no significant difference. Presence of thyroid hormonesrelated clinical manifestation in infertile women was higher (26.9%) in comparison to pregnant fertilewomen and non-pregnant fertile groups. Abnormalities in menstrual cycles were much higher in infertilewomen (90.4%) than non-pregnant fertile women (27.7%). Serum level of IL-6 was with no significantdifferences among all study groups. In conclusion, AITD has a minor participation in the thyroid disorder–associated infertility and their menstrual abnormalities in comparison with the larger scale of the non-AITDparticipation. No role for the IL-6 in the thyroid associated primary infertility in women was detected.

NK and trophoblast cells interaction: cytotoxic activity on recurrent pregnancy loss

The trial objective was to determine the peripheral blood NK cells cytotoxic activity effect on trophoblast cells at recurrent pregnancy loss (RPL). The investigation involved non-pregnant women with PRL in proliferating and secretory menstrual cycle phases (PMCPh and SMCPh, respectively); women of 6–7 weeks pregnancy with RPL in past medical history; healthy fertile non-pregnant women in PMCPh and SMCPh, women of 6–7 weeks physiological pregnancy, nulliparity healthy women with regular menstrual function in PMCPh and SMCPh. NK cells cytotoxic activity was determined using peripheral blood mononuclear cells. The target cells were JEG-3 line trophoblasts. It has been established that NK cells cytotoxic activity effect on trophoblasts is lower in SMCPh than in PMCPh in non-pregnant fertile women. The NK cells cytotoxic activity was higher in SMCPh than in PMCPh in non-pregnant women with PRL and also higher than the same value in SMCPh in non-pregnant fertile women. The increased NK cells cytotoxic activity values in SMCPh in women with RPL may be the reason for miscarriage.

Changes in expression of Ki-67, CD16 and CD56 by natural killer cells from peripheral blood mononuclear cells in the setting of recurrent miscarriage after in vitro culturing in the presence of trophoblast cells and IL-2.

The aim of this research was to assess the proliferative activity of Natural Killer Cells (NK cells) from Peripheral Blood Mononuclear Cells (PBMCs) in the presence of trophoblast cells in women with a history of recurrent miscarriages. We examined the peripheral blood of women with recurrent miscarriage in the proliferative (n = 12) or secretory (n = 13) phase of their menstrual cycle, and pregnant women with a history of recurrent miscarriage at 6-7weeks of their current pregnancy (n = 14). Controls were fertile non-pregnant women in the proliferative (n = 11) or secretory (n = 13) phase of their menstrual cycle, and pregnant women at 6-7weeks of a physiologically normal pregnancy (n = 20). We used IL-2 as a factor maintaining PBMCs viability during long-term culturing. We established that culturing in the presence of IL-2 contributed to an increase in the number of CD56+CD16- NK cells and to a decrease in the number of CD56+CD16+ NK cells from PBMCs compared with these numbers before culturing in both healthy women and in women with recurrent miscarriage. After culturing of PBMCs in the presence of trophoblast cells and IL-2 (compared with culturing without trophoblast cells), the intensity of Ki-67 expression by NK cells was reduced in the whole NK cell population (CD3-CD56+), and in the CD56+CD16- and CD56+CD16+ populations of NK cells in women with recurrent miscarriage and in healthy controls. The intensity of CD56 expression was reduced in the presence of trophoblast cells and IL-2 in non-pregnant women with recurrent miscarriage in the secretory versus the proliferative phase of the menstrual cycle.

Human pregnancy is accompanied by modifications in B cell development and immunoglobulin profile

Though human pregnancy success has been classically linked with a shift into a Th2 immunoglobulin producing cell response, a clear picture concerning B cell development and immunoglobulin profile during human pregnancy is missing. We analyzed in this work the dynamic of different B cell populations in peripheral blood of pregnant women on the first, second and third trimester of pregnancy. As control, age-matched non-pregnant fertile women were included. Additionally, we quantified the levels of immunoglobulin (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE) in the serum of pregnant and non-pregnant women. We observed a significant decrease in the percentages of transitional B cells in peripheral blood of pregnant women as compared to non-pregnant control women. Besides, percentages of naïve as well as switched and non-switched memory B cells in peripheral blood of pregnant women were similar to those in non-pregnant control women. Interestingly, although we did not observe differences in the activation status of B cells as well as in the percentages of plasma cells between pregnant and non-pregnant women, we observed significantly higher levels of IgM, IgA, IgG3, more likely natural antibodies, as well IgG4 in serum of pregnant women compared to non-pregnant age matched control women.

Factors Associated with Contraceptive Use among Women of Reproductive Age in Rural Districts of Burkina Faso.

Given the current low contraceptive use and corresponding high levels of unwanted pregnancies leading to induced abortions and poor maternal health outcomes among rural populations, a detailed understanding of the factors that limit contraceptive use is essential. Our study investigated household and health facility factors that influence contraceptive use decisions among rural women in rural Burkina Faso. We collected data on fertile non-pregnant women in 24 rural districts in 2014. Of 8,657 women, 1,098 used a modern contraceptive. Women having a living son, a child younger than one year, and household wealth were more likely to use modern contraceptives. Women in polygamous marriages and women living at least 5 kilometers from a health facility were less likely to use contraception. We conclude that modern contraceptive use remains weak, hence, programs aiming to encourage contraceptive use must address barriers at both the health facility and the household level.

Changes in cerebral autoregulation in the second half of pregnancy and compared to non-pregnant controls.

The mechanism by which pregnancy affects the cerebral circulation is unknown, but it has a central role in the development of neurological complications in preeclampsia, which is believed to be related to impaired autoregulation. We evaluated the cerebral autoregulation in the second half of pregnancy, and compared this with a control group of healthy, fertile non-pregnant women. In a prospective cohort analysis, cerebral blood flow velocity of the middle cerebral artery (determined by transcranial Doppler), blood pressure (noninvasive arterial volume clamping), and end-tidal carbon dioxide (EtCO2) were simultaneously collected for 7min. The autoregulation index (ARI) was calculated. ARI values of 0 and 9 indicated absent and perfect autoregulation, respectively. ANOVA and Pearson's correlation coefficient were used, with p<0.05 considered significant. A total of 76 pregnant and 18 non-pregnant women were included. The ARI did not change during pregnancy, but pregnant women had a significantly higher ARI than non-pregnant controls (ARI 6.7±0.9 vs. 5.3±1.4, p<0.001). This remained significant after adjusting for EtCO2 (p<0.001). Cerebral autoregulation functionality is enhanced in the second half of pregnancy, when compared to non-pregnant fertile women, even after controlling for EtCO2. The autoregulation does not change with advancing gestational age.

Anti-Mullerian-Hormone during pregnancy and peripartum using the new Beckman Coulter AMH Gen II Assay.

BackgroundAMH levels determined by the conventional AMH assay declined during pregnancy and postpartum. A new Beckman Coulter AMH Gen II assay removes the potentially assay-interfering complement which is activated in pregnancy. The aim of this study was to evaluate if the decline of AMH levels in the serum of pregnant women during the course of pregnancy and peripartum was assay-dependent and thus artificial.MethodsIn this cross-sectional study prepartal blood samples were collected from 62 patients (median age 30.6 years [interquartile range: 25.6 - 34.5]) in the third trimester of pregnancy and again 1–4 days after delivery between 2011 and 2012. In another cohort of 11 patients (median age 34.1 years [interquartile range: 32.6 - 37.8]) blood samples were taken in different trimesters of pregnancy between 1995 and 2001. The conventional and the modified AMH assay were performed in the same patient serum samples. We used the conventional and the modified AMH-Gen-II ELISA (Beckman Coulter, Immunotech, Webster, USA) for the assessment of AMH levels. The Wilcoxon signed rank test was used for determining differences between AMH levels pre- and postpartum. The method of Bland and Altman was applied for analyzing the agreement of both methods for determining AMH levels.ResultsAMH values peripartum were lower than those expected in fertile non-pregnant women of comparable age. An overall mean difference of 0.44 ng/ml was observed between the conventional and the modified assay. Measurements with the modified assay showed a significant decline of postpartal levels compared with prepartal levels which is consistent with values obtained using the conventional assay (both p < 0.00001). Compared to the longitudinal measurements of AMH levels determined using the conventional assay, AMH levels obtained using the modified assay suggest a steeper decline of values during the course of pregnancy.ConclusionBy comparing the conventional assay for AMH determination with the modified assay the present study confirmed that AMH levels decline during the course of pregnancy and early after delivery.

Day-specific probabilities of conception in fertile cycles resulting in spontaneous pregnancies

When, within the female cycle, does conception occur in spontaneously fertile cycles? This study provides reference values of day-specific probabilities of date of conception in ongoing pregnancies. The maximum probability of being within a 5-day fertile window was reached on Day 12 following the last menstrual period (LMP). The true date of conception is not observable and may only be estimated. Accuracy of these estimates impacts on obstetric management of ongoing pregnancies. Timing of ovulation and fertility has been extensively studied in prospective studies of non-pregnant fertile women using error-prone proxies, such as hormonal changes, body-basal temperature and ultrasound, yielding day-specific probabilities of conception and fertile windows. In pregnant women, date of conception may be retrospectively estimated from early pregnancy fetal measurement by ultrasound. Retrospective analysis of consecutive pregnancies in women referred for routine first-trimester screening, over a 3-year period (2009-2011) in a single ultrasound center (n = 6323). Within the overall population, 5830 cases with a certain date of last menses were selected for analysis. The date of conception was estimated using a crown-rump length biometry and an equation derived from IVF/ICSI pregnancies. Day-specific probabilities of conception were estimated across several covariates, including age, cycle characteristics and ethnicity, using deconvolution methods to account for measurement error. Overall, the day-specific probability of conception sharply rises at 7 days after the LMP, reaching its maximum at 15 days and returning to zero by 25 days. Older women tend to conceive earlier within their cycle, as did women with regular cycles and white and black women compared with Asian ethnicity. The probability of being within the fertile window was 2% probability at Day 4, a maximum probability of 58% at Day 12 and a 5% probability by Day 21 of the cycle. Although conception is believed to occur within hours following ovulation, a discrepancy is theoretically possible. However, when comparing our results to those of prospective studies, no such difference was found. The equation used for estimating the date of pregnancy was estimated in IVF/ICSI pregnancies, which could lead to potential bias in spontaneous pregnancies. However, in our population, the observed bias was negligible. Non-fertile cycles and early pregnancy losses are necessarily overlooked because of the nature of our data. Because of the wider access to retrospective data and the potential bias in prospective studies of ovulation monitoring, this study should broaden the perspectives of future epidemiologic research in fertility and pregnancy monitoring. None.

The expression of IL-6Rα and Gp130 in fallopian tubes bearing an ectopic pregnancy

Women with tubal ectopic pregnancies have high levels of circulating interleukin 6 (IL-6). IL-6 treatment in vitro significantly reduces the ciliary activity of tubal epithelium. The effects of IL-6 on target cells occur via the formation of a high-affinity complex with its receptors IL-6Rα and glycoprotein 130 (Gp130). IL-6Rα is specifically expressed in the cilia of the epithelial cells. In this study, we performed a quantitative reverse transcriptase polymerase chain reaction to determine the mRNA expression of IL-6Rα and Gp130 in the fallopian tubes obtained from 12 women with ectopic pregnancies, 12 women with normal pregnancies, and 12 healthy nonpregnant women in the luteal phase of their menstrual cycle. Fallopian tubes were evaluated from specimens taken during tubal ligation in normal pregnancies and nonpregnant fertile women or during tubal surgery in ectopic pregnancies. We observed that IL-6Rα mRNA expression in fallopian tubes was increased in ectopic pregnancy compared with that in the midluteal phase. We also found that the Gp130 mRNA expression was significantly lower in fallopian tubes from ectopic pregnancies than in those from nonpregnant women during the midluteal phase of their menstrual cycle, although its expression was noticeably high in fallopian tubes in the midluteal phase, which suggests that high Gp130 levels may possibly contribute to embryo transport into the uterus.

Chemerin Regulates NK Cell Accumulation and Endothelial Cell Morphogenesis in the Decidua during Early Pregnancy

Although decidual natural killer (NK) cell accumulation and vascular remodeling are critical steps to ensure successful pregnancy, the molecular mechanisms controlling these events are poorly defined. Herein we analyzed whether chemerin, a recently identified chemoattractant involved in many pathophysiological processes, could be expressed in the uterine compartment and could regulate events relevant for the good outcome of pregnancy. Chemerin expression in human primary culture of stromal (ST) cells, extravillous trophoblast cells, and decidual endothelial cells (DEC) was analyzed by RT-PCR, ELISA, and Western blot. Migration through ST or DEC of peripheral blood and decidual (d) NK cells from pregnant women was performed using a transwell assay. A DEC capillary-like tube formation assay was used to evaluate endothelial morphogenesis. Chemerin is differentially expressed by decidual cells during early pregnancy being present at high levels in ST and extravillous trophoblast cells but not in DEC. Notably, ST cells from pregnant women exhibit and release higher levels of chemerin as compared with ST cells from menopausal or fertile nonpregnant women. Chemerin can support peripheral blood NK cell migration through both DEC and ST cells. Although dNK cells exhibit lower chemerin receptor (CMKLR1) expression than their blood counterpart, CMKLR1 engagement on dNK cells resulted in both ERK activation and migration through decidual ST cells. Interestingly, DEC also express CMKLR1 and undergo ERK activation and capillary-like tube structure formation upon exposure to chemerin. Our data indicate that chemerin is up-regulated during decidualization and might contribute to NK cell accumulation and vascular remodeling during early pregnancy.

Retrobulbar Hemodynamics During Healthy and Preeclamptic Pregnancy

Objective: We aimed to evaluate retrobulbar hemodynamics during healthy and preeclamptic pregnancy. Methods: We studied retrobulbar hemodynamics in 21 healthy pregnant, 20 preeclamptic and 21 non-pregnant fertile women. The peak systolic velocity, end-diastolic velocity and resistivity index (RI) of the ophthalmic artery (OA), central retinal artery (CRA) and posterior ciliary artery (PCA) were measured by color Doppler imaging. Results: The systolic and diastolic blood pressures of the preeclamptic women (155±8.5 and 105±7.5 mmHg, respectively) were significantly higher than those of healthy pregnant women (110±5.5 and 70±7.5 mmHg, respectively, p=0.008). The RI values of OA, CRA and PCA in healthy pregnant women were significantly lower than those of the non-pregnant women. The RI of the CRA and PCA in preeclamptic women were significantly higher than those of healthy pregnant women. Although there was no statistical significant difference, the RI of the OA was lower in preeclamptic women than healthy pregnant women. Conclusion: Resistance of all retrobulbar arteries decreased during healthy pregnancy. On the other hand, increased resistance was detected in PCA and CRA, besides the nonsignificant decrease in OA during preeclamptic pregnancy. Different auto regulator control mechanisms in response to hypertension seem to be responsible for the different conducts of different sized arteries of orbita during preeclamptic pregnancy. (Gazi Med J 2012; 23: 55-8)

Polymorphisms in antithrombin and in tissue factor pathway inhibitor genes are associated with recurrent pregnancy loss

Recurrent pregnancy loss (RPL) is a multifactorial condition. The effect of antithrombin (SERPINC1), protein C (PROC), thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI) single nucleotide polymorphisms (SNPs) on the risk of RPL is thus far unknown. Our objective was to determine the association of SNPs in the above mentioned genes with RPL. We included 117 non-pregnant women with three or more consecutive losses prior to 20 weeks of pregnancy without a previous history of carrying a fetus to viability, and 264 healthy fertile non-pregnant women who had at least two term deliveries and no known pregnancy losses. The PROC (rs1799809 and rs1799808), SERPINC1 (rs2227589), THBD (rs1042579) and TFPI (rs10931292, rs8176592 and rs10153820) SNPs were analysed by Real Time PCR. Genotype frequencies for PROC 2418A>G, PROC 2405C>T, THBD 1418C>T, TFPI (T-33C and TFPI C-399T) SNPs were similar in cases and controls. The carriers of SERPINC1 786A allele (GA + AA genotypes) had an increased risk for RPL (odds ratio [OR]: 1.77, 95% confidence interval [CI]: 1.05-3.00, p= 0.034) while women carrying the TFPI -287C allele (TC + CC genotypes) had a protection effect on having RPL (OR: 0.46, 95% CI: 0.26-0.83, p= 0.009). The TCC haplotype for TFPI T-33C/ TFPI T-287C/ TFPI C-399T SNPs was less frequent in cases (5.7%) than in controls (11.6%) (OR: 0.45, 95% CI: 0.23-0.90, p= 0.025). In conclusion, our data indicate that SERPINC1 786G>A variant increases the risk for RPL, while TFPI T-287C variant is protective; however, further studies are required to confirm our findings.

Polymorphisms in Antitrombin (SERPINC1) and Tissue Factor Pathway Inhibitor (TFPI) Genes Are Associated with Recurrent Pregnancy Loss

Abstract 711 Background:Recurrent pregnancy loss (RPL) is a multifactorial condition. The effects of antithrombin (SERPINC1), protein C (PROC), thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI) gene polymorphisms in the risk of having RPL are unknown. Aims:To determine the effects of SERPINC1, PROC, THBD and TFPI gene polymorphisms in the risk of having RPL. Methods:One hundred and fifteen non-pregnant women with three consecutive losses prior to 20 weeks of pregnancy without carrying a fetus to viability (primary RPL) and 253 healthy fertile non-pregnant women who had at least two children and no known pregnancy losses were studied. The PROC (rs1799809 and rs1799808), SERPINC1 (rs2227589), THBD (rs1042579) and TFPI (rs10931292 and rs8176592) gene polymorphisms were detected respectively by Real Time PCR. Results:No difference was observed among the genotypes frequencies for PROC 2418A>G, PROC 2405C>T, THBD 1418C>T and TFPI T-33C polymorphisms in two groups (Table 1, P>0.05). However, the carriers of 140T allele (CT + TT genotypes) for SERPINC1 C+140T polymorphism had 1.73 (95% CI: 1.00 – 2.99) times risk of having RPL. Furthermore, the carriers of TC + CC genotypes for TFPI T-287C polymorphism had a protection effect of having RPL (OR: 0.48, 95% CI 0.27– 0.86). Conclusions:The variant of SERPINC1 C+140T polymorphism is associated with higher risk of having RPL while the variant of TFPI T-287C polymorphism is associated with a protection effect.Table 1 –Genotypes distributions of investigated SNPsPolymorphismSNP IDChrChr positionGenotypesRPL groupControl groupOR (CI 95%)P valorPROC 2418A>Grs17998092128175875AA37 (32.5)88 (34.8)1.00AG + GG77 (67.5)165 (65.2)1.11 (0.69–1.78)0.663PROC 2405C>Trs17998082128175862CC42 (36.8)87 (34.4)1.00CT + TT72 (63.2)166 (65.6)0.90 (0.57–1.42)0.649SERPINC1 C+140Trs22275891173886216CC86 (75.4)213 (84.2)1.00CT + TT28 (24.6)40 (15.8)1.73 (1.00–2.99)0.047THBD c.1418C>Trs10425792023028724CC87 (76.3)195 (77.1)1.00CT + TT27 (23.7)58 (22.9)1.04 (0.62–1.76)0.873TFPI T-287Crs109312922188419479TT97 (85.1)185 (73.1)1.00TC + CC17 (14.9)68 (26.9)0.48 (0.27–0.86)0.013TFPI T-33Crs81765922188332692TT51 (44.7)114 (45.1)1.00TC + CC63 (55.3)139 (54.9)1.01 (0.65–1.58)0.954Chr: chromosome; SNP: single nucleotide polymorphisms; RPL: recurrent pregnancy loss.The data are absolute (relative) frequencies.Financing: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2007/57605-0) Disclosures:No relevant conflicts of interest to declare.