Transorbital electrical stimulation (tES) has been studied as a new noninvasive method for treating intractable eye diseases by delivering weak electrical current to the eye through a pair of electrodes attached to the skin around the eye. Studies have reported that the therapeutic effect of tES is determined by the effective stimulation of retinal cells that are densely distributed in the posterior part of the retina. However, in conventional tES with a pair of electrodes, a greater portion of the electric field is delivered to the anterior part of the retina. In this study, to address this issue, a new electrode montage with multiple electrodes was proposed for the effective delivery of electric fields to the posterior retina. Electric field analysis based on the finite element method was performed with a realistic human head model, and optimal injection currents were determined using constrained convex optimization. The resultant electric field distributions showed that the proposed multi-channel tES enables a more effective stimulation of the posterior retina than the conventional tES with a pair of electrodes. There are two well-known methods that deliver the electric current to the eye noninvasively. One is transcorneal electrical stimulation that delivers the currents via a contact-lens-type electrode attached right above the cornea3. Previous studies reported that transcorneal electrical stimulation has beneficial effects on the improvement of visual functions in patients with optic neuropathy2 and retinitis pigmentosa (RP)6. According to the studies that used animal models with eye diseases, improvement of visual functions resulting from transcorneal electrical stimulation was closely associated with the survival of the retinal ganglion cells (RGCs) and photoreceptors preserved from the degeneration, suggesting that the neuroprotective effect on retinal cells determines the outcome of the transcorneal electrical stimulation7. Additionally, it was found that the increase in the survival of RGCs after transcorneal electrical stimulation is related to an increase in the insulin-like growth factor 1 (IGF-1), brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic factor (CNF), which are released from Müller cells in the retina9.Noninvasive electrical stimulation of the eyes has been studied as a promising therapeutic tool to recover visual functions in patients suffering from various eye diseases10. Compared to transcorneal electrical stimulation, tES is less invasive with no side effects such as dry-eye and punctate keratitis and easier to apply11. Repetitive tES, applied to patients with optic nerve damage, has been reported to improve visual field size, visual acuity, and detection ability13. Repetitive tES has also reportedly strengthened the alpha-band functional connectivity in patients with chronic prechiasmatic visual system damage14. Another study has demonstrated that the tES-treated group showed a significant improvement in visual fields and reaction times during the visual-field-related task compared to the sham stimulation group15. Furthermore, tES has also been effective in improving visual function in patients with RP16. A previous study reported that the effectiveness of tES was related to the synchronization of cortical activities after retinal cells were stimulated10. Another study insisted that improvement of visual functions together with changes in the spectral EEG alpha band power and connectivity in the occipital lobe after tES might be caused by a retinofugal entrainment through firing of RGCs15. Indeed, a previous in vivo experimental study with rats also demonstrated that electrically evoked, tES-induced responses stemmed from the retina17. These series of findings suggest that a stronger electric field should be delivered to the cells in the retina to increase the therapeutic effect of tES.The other method is transorbital electrical stimulation (tES) that delivers weak electrical current to the eye via electrodes attached to the skin around the eye. The stimulation parameters such as electrode configurations, current waveforms, injection current intensities differed among studies. Generally, square pulses in bursts with the frequency range of 5–30 Hz were applied for tES18. According to a numerical simulation study with the conventional electrode montage, most electric fields were delivered to the anterior part of the eye15. Therefore, the conventional tES dominantly stimulated the anterior part of the retina despite a large number of retinal cells, including RGCs and Müller cells, being densely distributed in the posterior part of the retina, particularly around the fovea19. Therefore, considering the abovementioned action mechanisms of both tES, a stronger electric field should be delivered to the posterior part of the retina to increase the effectiveness of tES. In the conventional tES, however, electric field delivered to the peripheral side of the retina (anterior retina) reaches an individual phosphene threshold20, which represents the maximally allowable injection current in tES that does not evoke phosphenes in an individual, before a sufficient amount of stimulation current is delivered to the posterior retina. Therefore, it is necessary to reduce the electric field delivered to the anterior retina in relative to that delivered to the posterior retina to maximize the overall therapeutic effects of tES.Generally, the conventional electrode configuration used for tES comprises two active electrodes attached to the skin near the orbital cavity and a single reference electrode placed on the occipital pole or extra-cephalic regions like the wrist and neck23, we employed tissue electrical conductivity values at DC frequency and solved a quasi-static Laplace equation because the frequency range used for tES (5 – 30 Hz) was low enough for the quasi–static approximation. Indeed, it was reported that there was no difference between the electric fields calculated assuming DC and AC with a relatively high frequency (~ 1 kHz)24. The optimal injection currents of the active electrodes were determined to maximize the electric field delivered to the posterior retina, near the fovea, by employing a constrained convex optimization approach. The efficacy of the new stimulation conditions was evaluated by comparing it with the conventional electrode montage.This study proposes a novel tES montage with eight active electrodes, with the diameter of 1 cm, attached around the eye (approximately 2 cm away from the center of the cornea) and a reference electrode on the occipital pole to reduce the difference in the electric field intensities delivered to the anterior and posterior retina. In other words, the study aims to maximize the electric field delivered to the posterior retina when that delivered to the anterior retina reaches the individual phosphene threshold. As aforementioned, short duration square pulses at a specific frequency are generally employed for tES. Although the electrical conductivity values of tissues are dependent on the frequency of injected current25. The right eye was segmented into six tissues with different electrical conductivities: the sclera, vitreous body, retina, lens, ciliary body with iris, and anterior chamber, using the ANSYS v18.2   human head model was constructed using T1-weighted magnetic resonance (MR) images of a young male subject (26 years old), which were acquired from a 3 T MAGNETOM Trio scanner  with a resolution of 1 × 1 × 1 mm. The subject was required to provide a written informed consent after he had been informed of the purpose of the experiment. He also agreed the publication of his head images in an online open-access publication by signing the written informed consent. The experimental protocol was approved by the Institutional Review Board (IRB) Committee of Hanyang University (HYI-17-180-5). All data acquisitions were performed in accordance with the guidelines and regulations set by the IRB of Hanyang University. The SimNIBS v2.0 was used to automatically segment head tissues, including those of the scalp, skull, cerebrospinal fluid (CSF), gray matter, and white matter29 was employed to determine each electrode’s optimal injection currents capable of delivering a maximum electric field to the ROI. The objective function, given by the following equation, was set to maximize the sum of the electric field intensity component along the normal to the retinal surface in the ROI.The optimization approach used in a previous transcranial direct current stimulation (tDCS) studyE(r) and d(r) represent the electric field intensity vector and the unit vector perpendicular to the retinal surface at location r, respectively. I, where I represents the L × 1 sized injection current vector, with L being the number of active electrodes. Two constraints, C1 and C2, were introduced to consider the safety of the system.Here tot and sind represent the limit of the total injection current amplitude and the maximum current amplitude allowed for each active electrode, respectively29.Here 30. The electric field in the entire analysis domain was calculated using the FEM formulated with the electrostatic Laplace equation given by V is the electrical potential, and σ is the electrical conductivity. Dirichlet boundary conditions were then applied: + 1 V on the upper side of the active electrode and − 1 V on the upper side of the reference electrode. The calculated electric field was scaled such that the 1 mA of current was flowing into the reference electrode. We assumed that all tissue compartments were homogeneous, and their electrical conductivity values were set according to a previous study18. This process was repeated until the electric field distributions for each of the active electrodes were calculated. The electric field of the m-th element of the FE head model, Em, generated by the injection current vector, I, could be calculated using the superposition sum of the electric field vectors, pre-evaluated assuming the 1 mA current injection via a single active electrode and the reference electrode. Then, Eq.  in (ind) in  in  and the (ind) in  were setmax, outside represents the maximum electric field formed outside the ROI on the retinal surface, and α denotes the threshold value for the electric field outside the ROI. It is well known that the stimulation focality and the mean electric field intensity in the ROI (referred to as Emean) have a tradeoff relationship33, where the focality can be defined as the ratio between Emax, outside, and Emean. As the value of α decreases, while the focality generally increases, the mean field intensity in the ROI decreases because the total injection current starts decreasing at a lower α value33. This study aimed to deliver the maximum electric field to the ROI and reduce the stimulation in the non-ROI regions, maintaining the total injection current at 1 mA. Accordingly, we empirically determined the value of α using an iterative CVX satisfying constraints C1, C2, and C3 by gradually reducing the value of α. After determining the optimal injection currents, the effectiveness of the proposed montage was validated by comparing it with three different conditions: the conventional montage, optimized suggested montage, and unoptimized suggested montage with equally distributed injection currents (injection current amplitude of 0.125 mA for one active electrode).Here Emax, ROI) and mean electric fields in the ROI , the Emax, outside, and the total injection current , with respect to different α values. The results show that a smaller electric field was delivered to the regions outside the ROI as the value of α decreased. However, the electric field intensity delivered to the ROI also decreased when α fell below 0.25 V/m, with the total injection current being smaller than 1 mA. Accordingly, we set the value of α to 0.25 V/m, which led to maximum focality while the total injection current remained at 1 mA. Figure It is important to note that we estimated the electric field in the direction normal to the surface of the retina, considering the direction of the arrangement of the RGC and photoreceptors, leading to both negative and positive electric field intensity values. Here, the negative values in the distribution represent the electric field coming out from the retinal surface. Figure 1. However, an electrode configuration, other than a conventional montage consisting of two active electrodes and a single reference electrode, to increase the therapeutic effect of tES is yet to be investigated. In this study, we propose a new tES electrode montage with eight active electrodes attached around the eye and a single reference electrode attached above the occipital area of the brain. We have optimized the injection current pattern that can effectively deliver a certain amount of electric field to the posterior retina while reducing the electric field being delivered to the anterior retina. For the proposed eight-channel montage, the optimized injection currents delivered a lower electric field to the anterior retina while preserving the electric field delivered to the posterior retina. This implies that the total injection current can be increased to deliver a stronger electric field to the posterior retina without evoking phosphene owing to the stimulation of the anterior retina.tES, a noninvasive oculo-modulation method of improving visual functions, has attracted increased attention as a promising alternative to a relatively more invasive epiretinal implant or transcorneal electrical stimulationBased on preliminary simulations, we determined the locations of eight active electrodes empirically. We first tried to employ 16 electrodes attached around the eye and one electrode attached over the occipital see Fig. a. FigureAdditionally, we drew streamlines of electric fields to explore how the resultant electric fields were formed with respect to three different electrode montage conditions see Fig. . The res34, which is possible even when a relatively small electric field is delivered to RGCs12. Indeed, it is known that alternating electric fields whose amplitude range from 0.1 to 0.2 V/m was sufficient to entrain endogenous neural oscillations38. Therefore, it is believed that the mean electric field of 0.16 V/m delivered to the posterior retina might have a sufficient possibility to induce rhythmic firing of the RGCs. Furthermore, there is still room for increasing the total injection current in the proposed electrode montage because the maximum electric field outside ROI (posterior retina) in the conventional montage was approximately four times larger than that in the proposed montage, as presented in Table As aforementioned, the therapeutic effect of tES results from the entrainment of rhythmic firing of the RGCs by tES12. As the electric field outside the ROI is four times larger than that in the ROI for the conventional montage, the phosphene threshold should be determined by the excitation of RGCs outside the ROI. Therefore, a sufficient stimulating current could not be delivered to the posterior retina, and the efficacy of the therapy was consequently degraded. A clinical tES study with RP patients demonstrated that using a stronger injection current than the individual phosphene threshold showed therapeutic effects, while the injection current determined based on the individual phosphene threshold did not exhibit any improvement in visual functions16. Schatz et al. reported that the visual field of RP patients was improved or remained unchanged by injecting the current equivalent to 150% of individual phosphene thresholds, whereas there was no marked tendency when injecting the current equivalent to 66% of individual phosphene threshold during transcorneal electrical stimulation4. Although the relationship between the strength of the injection current and the therapeutic effects of tES should be further investigated through a series of human trials, it seems evident that retinal stimulation with a stronger electric field allows for the elevation of the therapeutic effects of tES. Based on our simulation results, we strongly believe that the proposed montage should provide an opportunity to increase the total injection current while reducing the possibility of retinal phosphene, which would allow for better treatment opportunities for patients.For human trials with conventional montage, the injection current is determined based on the phosphene threshold that induces retinal phosphene39. It is to be noted that the maximum current amplitude injected through each active electrode was just 0.2 mA in our simulations. As the larger sized electrodes with a diameter of 1 cm were assumed in this study, we strongly believe that it would be safe to inject current much smaller than 3 mA in the proposed montage. In addition, a previous study reported that the maximal electric field amplitude that does not cause any tissue damage is 42 V/m18. As much smaller electric field is delivered to the retinal tissues in the suggested montage, the use of the suggested montage will not cause any damage to the retinal tissue. Nevertheless, these safety issues need to be further addressed through in-vivo experiments in the future studies.For the practical use of the suggested montage, safety issue needs to be addressed. According to a previous study on the safety of transcranial electrical stimulation, injection of 3 mA current through electrodes with a diameter of 0.8 cm did not cause any side effects, including skin burn, skin redness, and painmax, Emean, and Emax, outside values of the newly calculated field distribution were 0.18, 0.15, and 0.27, respectively, and those of the original distribution were 0.19, 0.16, and 0.25, respectively. These results suggest that slight displacements of active electrodes occurring during electrode attachment would not significantly influence the overall electric field distributions. Based on a previous study, the eye rotates with a rotation angle of approximately 10° during eyelid closure40. To demonstrate the effect of eye rotation, we additionally calculated the electric field distributions assuming that the eye was rotated either horizontally or vertically with a 10° rotation angle. For each simulation, the optimal injection currents obtained assuming no eye rotation was applied. Our simulation results showed that there was no significant change in the overall electric field distributions in the retina after rotating the eye (see Fig. max, outside slightly increased due to the eye rotation, the value was still much smaller than that in the conventional tES montage. Our results suggest that the eye rotation during eyelid closure would not significantly influence the overall electric fields when the suggested electrode montage is employed.In the practical application of the proposed multi-channel tES, the locations active electrodes can be slightly different from those assumed in the numerical simulations. To investigate the influence of slight electrode displacement on the resultant electric field distributions, we performed an additional simulation. We rotated eight active electrodes in the clockwise direction, as shown in Fig. 10. If the reference electrode is attached to a different area, the current pattern at each electrode might change as the location of the return or the reference electrodes influences the formation of electric fields41. As per our findings, we believe that the suggested montage with the optimal injection currents might produce the maximal focality for targeting the posterior retina regardless of the slight changes in the reference electrode’s location. However, there might be a possibility of improving the focal delivery of electric fields to the target by attaching the reference electrode to other areas, such as the neck and the arm, as considered in previous studies18. Thus, it would be an interesting topic for future research to employ a full human body model to verify the effectiveness of tES with the reference electrode attached to extra-cephalic regions.In the present study, we attached the reference electrode to the occipital pole based on previous studiesTo demonstrate the influence of the number of active electrodes on the effectiveness of multi-channel tES, additional simulations were conducted with different electrode montages with four and 12 active electrodes, when the same optimization process used in this study was employed. For the montage with 12 active electrodes, the active electrodes were attached approximately 2.7 cm away from the center of the eye, to keep the minimum distance between adjacent electrodes larger than 0.5 cm for preventing electrical short circuit in practical applications. It is to be noted that in the cases of the montages with four and eight active electrodes, the distance between the active electrodes and the center of the eye was assumed to be approximately 2 cm. After the optimization procedure, we compared electric field distributions in the retina obtained using three different montages with four, eight, and 12 active electrodes. The simulation results showed that the suggested montage with eight active electrodes was more beneficial to the focal stimulation of the posterior retina than the montages with four and 12 active electrodes, as presented in Fig. 45, it would be a promising future topic to investigate the distribution of the electric fields in the retina in in vivo experimental conditions and quantitatively compare the difference between the computed and measured electric fields.In addition, our simulation results were not validated via in vivo or ex vivo experiments. Although the optimization and simulation methods employed in this study have already been widely employed for the focal stimulation of cortical regions in transcranial electrical stimulationThis study suggests a novel electrode montage with multiple electrodes attached around the eye to stimulate the posterior retina, where retinal cells are densely distributed, for the first time. Our suggested montage, with the optimization of the injection current pattern, allows for more focal stimulation of the posterior retina while preventing the electric field from being delivered to other regions in the retina more effectively than the conventional montage and the same multi-electrode montage without the optimization of injection currents. Our simulation results demonstrated that the total injection current can be increased to deliver a much stronger electric field to the posterior retina without evoking retinal phosphene owing to the stimulation of the anterior retina by employing the new multi-channel electrode montage with optimized injection currents. Although additional in-vivo experimental studies are necessary, the proposed approach is thought to have a potential to become a better treatment option for patients who do not show therapeutic effects of tES.Supplementary Information 1.


We focused antepartally (M  = 34.3 ± 3.3) on sociodemographic and risk factors of psychopathology, such as prenatal distress , depressiveness , birth anxiety , childhood trauma (Childhood Trauma Questionnaire), and postpartally (M = 6.65 ± 2.6) on birth experience , as well as psychological adaption, such as postpartum depressive symptoms  and birth anxiety felt during birth . Women with fear of childbirth and the beginning of birth were likely to plan a hospital birth. In contrast, women with fear of touching and palpation by doctors and midwives, as well as women with childhood trauma, were more likely to plan an out-of-hospital birth. Furthermore, women with planned out-of-hospital births experienced a greater relief of their birth anxiety during the birth process than women with planned hospital birth. Our results especially show that women with previous mental illnesses, as well as traumatic experiences, seem to have special needs during childbirth, such as a safe environment and supportive care. The choice of birthplace may have an important impact on a woman’s health. In this longitudinal study, we investigated the psychopathological risk factors that drive women’s choice of birthplace, since their influence is currently not well understood. The research was conducted in 2011/12 and we analyzed data of 177 women (obstetric unit,  Current international guidelines emphasize the importance for low-risk women to have a choice for their intended birthplace. In Germany, women with low-risk pregnancies may choose any birth setting, including hospital obstetric units (OU), free-standing midwifery units (FMU) or at home . Over the last ten years, the proportion of out-of-hospital births in Germany remained persistently low, at 1.5% . FurtherWomen with pre-existing mental health problems and a history of traumatic experiences seem to have special needs during childbirth, such as a safe environment and supportive care . HoweverTo ensure high quality obstetric care, the evaluation of women’s decision-making processes and women’s needs are of great importance. The knowledge of factors that influence the choice of birthplace is a prerequisite to provide specific information to pregnant women and to address individual prenatal needs. In this study, we investigated the impact of psychopathological risk factors on the planned birthplace of pregnant women. Specifically, we focused on the influence of depressive symptoms and birth anxiety, as well as childhood trauma. Postpartally, we analyzed the effects of the chosen birthplace on birth experience and psychological adaption, since this can have important implications for psychosocial and maternal postpartum adjustment , as wellWhich psychopathological risk factors  were associated with the choice of birthplace?Does the choice of birthplace affect the birth experience of women?Does the choice of birthplace influence the maternal psychopathological adjustment postpartum ?In this study, we addressed the following research questions:n = 56 (31.6%) out of all 177). This bias by oversampling was accepted to provide special insight in this sample of women, and to make statistical comparisons feasible.The current investigation was designed as a prospective longitudinal study, carried out between March 2011 and March 2012. We contacted all obstetric units, freestanding midwifery units, and freelance midwives offering antenatal classes in and around the city of Dresden, Germany. The prenatal questionnaires were distributed in antenatal classes starting from the third trimester (29th week of pregnancy). To account for the fact that less than 1.5% of all German births take place outside the hospital, we specifically contacted midwives who offered care for out-of-hospital births. Targeted oversampling allowed us to recruit a comparatively high proportion of women planning out-of-hospital births , depressiveness , birth anxiety symptoms , and childhood trauma . Postpartally, the data collection (approximately 6 weeks postpartum) regarded birth experience  as well as psychological adaption, such as postpartal depressive symptoms  and birth anxiety felt during birth . M = 34.3  ±  3.3, range = 29–42 weeks; hereafter defined as t1) and approximately 6 weeks postpartum . The response rate at t2 was 88% which, in respect to the enormous changes during the postpartum adjustment process, can be considered as high. A drop-out analysis was conducted. Study participants completed questionnaires prenatally from the 29th week of gestation .n = 177 women were recruited and participated in both assessments. The women were allocated to three major groups according to their planned birthplace with n = 121 in OU, n = 42 in FMU, and n = 14 in OU. At t2, a “congruence” variable was defined by combining the planned and actual place of birth, with three possible outcome groups, in which the planned birthplace was congruent with the actual birthplace . The group of incongruent women, in which the planned birthplace (t1) differed from the actual birthplace (t2), were excluded from the postpartum analysis due to low sample size .Participation was voluntary and free of charge. Inclusion criteria included pregnancy in the third trimester (from 29th week) and sufficient knowledge of the German language. 1) provided the following content: sociodemographic variables , and standard questionnaires regarding potential psychopathological risk factors, in particular prenatal distress (PDQ), depressive symptoms (EPDS), birth anxiety symptoms (BAS), and childhood trauma (CTQ). The estimated processing time was 30 min. The postpartum questionnaire (t2) provided information on self-reported obstetric data as well as birth experience (SIL), depressive symptoms (EPDS) and birth anxiety felt during birth (BAS modified). The estimated processing time for the postpartum questionnaire was 25 min.The baseline questionnaire   measures the fears and worries of pregnant women regarding pregnancy and childbirth . The tweDepressive SymptomsThe Edinburgh Postnatal Depression Scale (EPDS)  is a widely used ten-item list for measuring depressiveness during the peripartum period with reference to the last 7 days ,24. AnswBirth Anxiety SymptomsThe Birth Anxiety Scale (BAS)  is a screening instrument to assess birth-related anxiety . The 77-To uncover underlying structures of the relatively large list of birth anxiety variables, an exploratory factor analysis was computed that yielded three significant factors. Based on the factor pattern, factor 1 was labeled as “fear of birth and beginning of birth”. Here, items loaded highly included “sudden onset of labor”, fear of pain, fear of physical effort and fear of complications. Factor 2 was labeled as “fear of touching and palpation by doctors and midwives”. Items loaded highly included a general fear of doctors, nurses and midwives, as well as a fear of vaginal examinations, the gynecologist’s chair, undressing in front of the doctor, or being touched by others during labor. Factor 3 was labeled as “fear of medical procedures and clinical environment”. Here, items loaded particularly highly described fear of the clinical environment, such as noises or smell in the delivery room, and fear of medical interventions, such as the induction of labor or cesarean section . Birth a2 (BAS modified) in order to retrospectively assess the actual level of birth anxiety felt during childbirth. Therefore, the individual aspects were not changed in their content.We modified the original BAS version for tChildhood TraumaThe short version of the Childhood Trauma Questionnaire (CTQ) developed by Bernstein et al. (2003)  is a retrospective measure of trauma in childhood and adolescence ,27. It cAdditionally, we questioned whether the study participants were asked by the gynecologist or midwife about their lifetime experience of violence. If this was the case, the study participants should indicate to what extent they considered this question to be distressing.Birth experienceThe German version of Salmon’s Item List (SIL) is a multidimensional assessment of birth experience . This quThe statistical analyses were performed as exploratory data analyses. In addition to descriptive data analyses, a chi-square test and Fisher´s exact test were used to test the significance of differences between the birthplace groups. When the assumptions for normality of the residuals and homoscedasticity for parametric tests were met, the univariate analysis of variance (ANOVA) was used to test the association between different birthplace choices and birth anxiety symptoms (BAS), depressive symptoms (EPDS), prenatal distress (PDQ) and birth experience (SIL). The non-parametric Kruskal–Wallis H-test was used when the assumptions for parametric testing were violated. An exploratory factor analysis with subsequent varimax rotation was performed to identify underlying structures, or so-called factors, using the relatively large number of variables of the birth anxiety questionnaire (BAS). Using a scree-plot, the number of three latent factors to keep were selected . AfterwaTo identify statistically significant differences between the birthplace groups, all post-hoc analyses were α-value-corrected for multiple testing, using the Bonferroni-post hoc-method. The Bonferroni method adjusts for the Family Wise Error Rate (FWER) for multiple testing. Controlling for FWER is a very conservative approach. Thus, the Bonferroni correction enables a much stronger interpretation when results surpass this more conservative threshold . p> 0.05). Planned hospital birth was the reference category for these comparisons.A multinomial logistic regression model was conducted to estimate the association of prenatal distress, depressive symptoms, birth anxiety symptoms and childhood trauma (separate independent variables) with planned place of birth as the dependent variable. The associations were adjusted for potentially confounding factors. All sociodemographic factors (covariate factors) were initially included, and the model was subsequently refined by removing those factors that were not significant predictors of the outcome measure /birth anxiety symptoms scores during pregnancy (BAS) with postpartal depressiveness scores (EPDS)/birth anxiety felt during birth (BAS), applying a two-way ANOVA with repeated measures. The ANOVA was adjusted for the effect of time of survey on the test results, as well as interactions between time of survey and birthplace. All statistical analyses were carried out using SPSS Statistics Version 24.0 .1). The drop-out analysis showed statistically significant differences between women who dropped-out and those who completed the study: women who dropped out were more often primipara , less educated  and had a lower socioeconomic status , compared with the women who completed the study  who were recruited at the study .1, the 177 study participants were 29 years old on average  (F(2) = 0.53; p = 0.59) and were, on average, in their 35th week of gestation (M  = 34.3  ±  3.3 weeks)  . Although OU showed a relatively higher proportion of working women (86%) than the two other groups, HB showed a relatively higher proportion of housewives (31%). At t3 weeks) . An amou< 0.001) . Furtherp = 0.001)  .p < 0.001), whereas FMU and HB had higher relative frequencies for “fear of touching and palpation by doctors and midwives” (H(2) = 8.53; p = 0.01) (p = 0.37) (Although no group differences were found for the birth anxiety scores (BAS), the three groups differed in their patterns of anxiety . The gro = 0.01) . All thr = 0.37) . p = 0.02) with significantly higher scores in HB than OU (p = 0.01) (p = 0.01) (p = 0.01). Prevalence of physical abuse was statistically significantly higher in HB (38.5%) than OU (6.8%) and FMU (11.9%)    and 16 women (9.0%) by their midwives  about previous experiences of violence. In group comparison, FMU and HB were statistically significantly more frequently asked by their midwives about traumatic experiences than OU. All women reported that they found this question “not stressful”  or “little stressful” . Childhood trauma scores (CTQ) differed between the birthplace groups (H(2) = 8.1;  = 0.01) . Moreove = 0.01) . The pre = 0.01) . Post-ho= 0.005) . Althoug = 0.19) . Overall2(10) = 58.32; p < 0.001), with an R2 of 0.36. The final model predicted 73.7% of the planned birthplaces correctly. The first set of coefficients represent the adjusted comparisons between OU and FMU. Prenatal distress  and parity  remained as statistically significant predictors (p < 0.05), indicating that women who had higher prenatal distress levels and who were primipara were more likely to choose a hospital birth  and choice of birthplace, after controlling for potential confounding factors. Planned hospital birth was the reference category for these comparisons. A significant regression equation was found , of which 119 gave birth in OU (97.5% of planned OU), 30 in FMU (73.8% of planned FMU) and 12 at home (84.6% of planned HB). Thus, for 16 women (12.5%), the actual birthplace differed from planned birthplace due to unplanned homebirth (n = 4), transfer to the hospital during birth (n = 9) and change to hospital prior to birth because of medical indications (n = 2).The majority of women gave birth at their planned birthplace . Although no group differences were found in the overall birth experience scores (SIL), the three birthplace groups differed in the dimension “good emotional adaption” (H(2) = 10.52; p = 0.005), which could be confirmed by a post-hoc-analysis between OUt1=t2:FMUt1=t2 (p = 0.04) and OUt1=t2:HBt1=t2 (p = 0.04) (p = 0.01 *), which could be identified to exist between OUt1=t2:HBt1=t2 (p = 0.02 *)  . Thus, t = 0.04) . Additio 0.02 *) . Thus, w1: H(2) = 5.37; p = 0.07; t2: H(2) = 1.28; p = 0.53) (p < 0.001). The interaction of time x birthplace was non-significant  = 1.58; p = 0.21). No differences between the congruent groups were detected for depressive symptoms (EPDS) before or after birth (t = 0.53) . A repea1), the congruent groups did not show any statistically significant difference (H(2) = 0.29; p = 0.86)  (p < 0.001), which persisted in the post-hoc analysis between OUt1=t2:FMUt1=t2 (p = 0.002) and OUt1=t2:HBt1=t2 (p = 0.014). A repeated measure two-way ANOVA with the factors of time (2 levels) and birthplace (3 levels) showed a significant main effect of time  = 51.34, p < 0.001). Accordingly, all three groups showed a statistically significant decrease in birth anxiety symptoms during the birth process. Additionally, the interaction of time x birthplace was significant  = 11.09; p < 0.001). Consequently, the out-of-hospital groups experienced a greater relief of their birth anxiety symptoms during birth process than the hospital group.When comparing prenatal birth anxiety (BAS at t = 0.86) . HBt1=t2S at t2) . The groThis study presents new insights on the impact of psychopathological risk factors on the choice of birthplace. We showed that traumatic experiences as well as certain factors of birth anxiety have an impact on the choice of birthplace. Furthermore, we detected associations between the choice of birthplace and birth experience, as well as subsequent maternal psychological adaption.. We identified parity as a statistically significant predictor in our regression model, indicating that women who were primiparous were more likely to choose a hospital birth. Similar results were reported in previous studies ,10,30. TThe influence of psychopathological risk factors on the planned place of birth is currently not well understood. Witteveen et al. (2016) showed that women planning to have a hospital birth were more likely to report symptoms of depression or anxiety disorders . In our Overall, about one quarter of all study participants reported childhood trauma. HB was especially associated with higher prevalence of early traumatic experiences. They reported more statistically significant occurrences of physical abuse and emotional neglect than the other two groups. A history of childhood trauma can have a variety of long-term effects; it increases vulnerability for low birthweight, as well as flashbacks due to the re-experience of traumatic memories, and increased anxiety and stress during labor ,18. PregAs mentioned above, care concepts of out-of-hospital births provide continuous care by the same person during pregnancy and the postpartum period, as well as one-to-one care during childbirth. This intensive care concept was identified as positive influencing factor on birth experience ,7,8. ConA possible systematic bias due to the recruitment of pregnant women via birth preparation courses can be assumed. It is possible that women with mental illnesses  tend to avoid participation in prenatal classes and may be underrepresented in this study. Furthermore, women who were less educated and had lower regular income were less likely to complete the study. In future studies, it would be desirable to encourage more of these women to participate. A selection bias was accepted by targeted oversampling of women with planned out-of-hospital birth, which ultimately resulted in neither an equal distribution of the groups nor a representative sample of the true population. Thus, a comparatively high proportion of women with planned out-of-hospital birth (31%) could be recruited, which enabled a special view on this group of women. The most important limitation of our research design is, however, the small subsample of 14 women in HB, since this hampers the extrapolation on a larger scale. The fact that the proportion of out-of-hospital births in Germany has remained persistently low at 1.5% demonstrates the difficulty to recruit women with planned homebirth. It should be noted that the data were collected in 2011/2012. However, the psychosomatic content of the German maternity guidelines  has not changed substantially since then, so it is reasonable to assume that a similar study would reach the same results today. Although the associations between the tested risk factors of psychopathology and planned place of birth were adjusted for potential confounding factors, it remains possible that there are additional confounding variables that were not included in the models. It can be assumed that previous birth experiences may have a decisive influence on the choice of birthplace, but this was not part of the survey. We suggest that future birthplace studies include previous birth experiences in multiparous women to be able to control for such potential confounding factors. To minimize the time effort for the women, we kept the questionnaire as short as possible. We had to accept a further possible bias in using self-reporting questionnaires to record women´s sociodemographic data. Additionally, it must be critically discussed that all evaluated data were subject to women´s self-assessment and thus could be influenced by subjective views. However, numerous validated psychometric questionnaires were used that showed good psychometric properties. Whereas the German version of the EPDS has been shown to have a good reliability , we stilFurthermore, we had no access to the participants’ medical records which could facilitate the identification of specific medical implications for a certain birthplace, especially for the OU group. Future research should address other mental health conditions such as anxiety disorders or PTSD, as well as other traumatic events such as intimate partner violence or traumatic birth experience, since these have not been under investigation so far. We will only be able to provide optimal care if we are aware about the special requirements of affected women for their planned place of birth. Our study demonstrated that psychopathological risk factors and traumatic experiences had an impact on the decision-making process of pregnant women for their choice of birthplace. We showed that factors such as birth anxiety and prenatal distress influenced the choice of birthplace, and that women with early traumatic experiences were significantly more likely to choose an out-of-hospital birth. Additionally, our study demonstrated that the choice of birthplace influenced the birth experience and maternal psychological adjustment. A deeper understanding of pregnant women’s needs will help us to individually adapt birth planning, birth talks, and care during childbirth. A routine screening on traumatic experiences should be included in prenatal care to provide affected women with optimal obstetric care, to prevent re-traumatization and to improve their childbirth experience. Regardless of the choice for a planned hospital or out-of-hospital birth, continuous care can help to improve for the emotional security of pregnant women in order to create a trusting, secure environment, which is an important prerequisite to prevent traumatic birth outcomes. Psychopathological risk factors and traumatic experiences should be adequately addressed in midwifery and gynecological routine examination for an informed decision-making process regarding the planned place of birth.


Both cardiovascular and reproductive complications may have origins in utero or in early life. Women in the Bogalusa Heart Study (n = 1401) had been linked to birth certificates for birthweight and gestational data, which were examined relative to childhood (ages 4–16) cardiometabolic indicators, indicated by mean levels overall and total risk factor burden as estimated by area under the curve (AUC) computed from longitudinal quadratic random-effects growth models. Women reported the birthweight and gestational age of each of their own pregnancies, and delivery medical records were linked to interview data where possible. Path analyses were conducted to examine the relationships among a woman’s own birth outcomes, childhood and preconception adult cardiovascular health, and birth outcomes. Mean blood pressure (systolic blood pressure (SBP) adjusted relative risk (aRR) per 1-SD increase, 1.27, 95% CI 1.04–1.57) and low-density lipoprotein  in childhood predicted preterm birth (PTB), while mean SBP  predicted term low birthweight. The AUC data suggested an association between blood pressure and PTB . Pre-pregnancy total cholesterol was negatively associated with gestational age. In path analyses, positive associations were found for each step between own birthweight, childhood BMI, pre-pregnancy BMI, and child’s birthweight. Childhood levels of some, though not all, cardiovascular risk factors may predict adverse birth outcomes . Cardiovascular and reproductive health are closely linked. Developmental origins of health and disease theory links preconception exposures and birthweight to adult cardiovascular health. For instance, analyses in the Bogalusa Heart Study have indicated relationships between birthweight and adult blood pressure , 2, arteCombining these lines of research, we hypothesized that a woman’s own birthweight and gestational age would also predict preconception and pregnancy health and that women with a higher burden of cardiovascular risk throughout childhood would have an increased risk of adverse birth outcomes   is a longitudinal study of cardiovascular risk founded by Dr. Gerald Berenson that began enrolling school children in the semi-rural community of Bogalusa, Louisiana in 1973 . ApproxiA follow-up study sought to determine the pregnancy histories and outcomes of women who had participated in the BHS (n = 5918). Three sources of information were potentially available: interview, medical records, and vital statistics. Potential participants were contacted via phone call, mailing or in person at the study field clinic. 1804 subjects were interviewed for 15–30 minutes regarding reproductive history, including the outcome of each pregnancy; the birthweight of all children; and whether each child was born early, late, or on time. For the purposes of this analysis, we limited the dataset to women who completed an interview in adulthood and had at least one BHS exam during their childhood (≤ age 16). The dataset was further restricted to women whose first birth was a singleton and occurred after age 16 to preserve temporality. The final sample included 1401 women with any childhood study visit, and 1052 with 2 or more visits during childhood .Two additional sources of information were available. Participants were asked for permission to access medical records, and those consenting signed release forms for prenatal, labor and delivery records for each pregnancy. Almost all participants gave permission for forms to be accessed, although in many cases they had been destroyed as over 10 years old . While 97% of women included in this analysis consented to release, medical records were obtained for only 10% of the sample . Vital records data from Louisiana, Texas, and Mississippi were also linked to BHS data for 55% (n = 3260) of all women ever seen in the larger study, and an estimated 65% of all women expected to have given birth . The strAdverse birth outcomes assessed included low gestational age  and birthweight. A measure of small-for-gestational-age (SGA) produced an unlikely proportion, most likely due to approximation of gestational age at term, so term low birthweight  was examined as an indicator of reduced fetal growth.Outcomes were taken from woman’s self-report. Where available, data on participant’s reproductive history as reported during the interview was compared with vital records and obstetric medical records in order to correct for misclassification of self-reports. Agreement between vital records and interview was good for participants who had data from both sources. After control for clustering within women, the mean discrepancy between the birth certificate and the woman’s report of birthweight was 2 g; for gestational age, the mean difference was 0.01 weeks . The fir2), systolic and diastolic blood pressure , low-density lipoprotein cholesterol , high-density lipoprotein cholesterol , total cholesterol , triglycerides, (mg/dL), insulin (uU/mL), and glucose (mg/dL). Serum cholesterol and triglycerides levels were assayed enzymatically on the Hitachi 902 Automatic Analyzer . Plasma glucose level was obtained as part of a multiple chemistry profile (SMA20) with the multichannel Olympus Au-5000 analyzer . A radioimmunoassay kit was used to measure plasma insulin .Exposures of interest included biological markers of cardiometabolic functioning in childhood (before age 16): body mass index , age at delivery (continuous), educational attainment  or some college, college graduate), marital status (married/not married) and smoking during pregnancy (yes/no). Models including non-BMI predictors were also adjusted for BMI at the visit prior to pregnancy. Related pregnancy complications assessed included preeclampsia, pregnancy-induced hypertension (PIH), and gestational diabetes (GDM). Although recall has been shown to be highly specific (>90%) for hypertensive disorders  and accuAnalysis proceeded in stages to test the conceptual model . We consNext, we computed the area under the curve (AUC) from longitudinal growth curve models, applying the methodology described in detail by Cook et al., in previous analysis of Bogalusa Heart Study data . The AUCWe used the AUC for each biologic indicator to examine the association between childhood mean cardiometabolic profiles and risk of PTB and TLBW at first pregnancy. Chi-square and t-tests compared crude differences in women with an adverse birth outcome and those without across categorical and continuous covariates, respectively. Separate adjusted log-Poisson models estimated the relative risk associated with each cardiometabolic indicator controlling for adult social, biological, and behavioral factors, as indicated in our model . All indicators were scaled to estimate relative risks associated with an interquartile range (IQR) increase in units. The group in the top 10% of the AUC was also compared to the others. Models were then examined with those with pregnancy complications removed, as well as adjusting for pregnancy complications, which addressed the possible associations with both pregnancy health and birth outcomes.Finally, we considered the entire conceptual model. By conducting a path analysis, we are able to examine the complete life course of intercorrelations among own birth characteristics, childhood and preconception cardiovascular health, and offspring birth outcomes. Before path analysis, multivariable linear regression models were conducted to examine the associations at each step by regressing risk factors at current step on the risk factors at the previous step in the path. For each outcome under study, each childhood AUC measure was examined for association with maternal own birthweight outcomes and gestational age; childhood AUC measures were examined for associations with each cardiometabolic risk factor in pre-pregnancy; pre-pregnancy factors were examined for association with weight gain during pregnancy; and both pre-pregnancy and weight gain were examined for association with the outcome. Variable selections were conducted at each step, and factors associated with each other at each step at p<0.1 were further explored in a formal path analysis under structural equation modeling using either the likelihood method or Bayes method (when likelihood methods did not converge), with additional control for age, race, and maternal education. All variables meeting the criteria were included in the path analysis, and the results examined for which statistical associations remained. The path analysis was conducted using Mplus Version 7 .This study was approved by the Institutional Review Board of Tulane University and all subjects provided written informed consent.The study sample was 59% white, 41% black . 17% repThe associations between childhood risk factors and adverse birth outcomes were mixed . AssociaThe path analysis is presented in Figs We examined the association of overall childhood and adolescent burden of cardiovascular risk with birth outcomes. Overall, higher childhood blood pressure was associated with a higher risk of PTB. Higher LDL cholesterol was associated with higher risk of PTB and lower risk of tLBW. Other risk factors had no or inconclusive associations.We therefore found a degree of support, but not full confirmation, for our conceptual model. Central to this analysis was examining the associations between preconception  cardiometabolic health and birth outcomes. While other studies have similarly found higher preconception blood pressure was associated with lower birthweight and higher preterm birth , 28, preWhen the life course pathway and conceptual model was considered as a whole (birth→childhood→pre-pregnancy→pregnancy→birth outcomes), associations could be seen for a path between birthweight, childhood BMI, adult BMI, weight gain, and child’s birthweight. Other pathways that started with own birth outcomes and led through childhood and pre-pregnancy cardiometabolic risk factors to offspring birth outcomes were minimal. Our conceptual model was therefore, again, supported to a degree but not fully. We are unaware of other studies that have been able to address a complete life course pathway. However, many developmental origins studies have found low and high birthweight or prematurity to be associated with adult hypertension, triglycerides, or diabetes –33. SomeLifetime cardiovascular risk could have effects on pregnancy in several ways. One, it could simply represent cardiovascular health during the pregnancy. Women with hypertension, dyslipidemia, and diabetes have a higher risk for poor pregnancy outcomes , 36. SubThis study examined relationships between cardiovascular risk and birth outcomes within a single generation, but the associations may go beyond this. Maternal cardiovascular health contributes to her child’s pre and postnatal environment, which subsequently affects the preconception and prenatal environment for the subsequent generation, and even beyond. Population-based three-generational effects on birth outcomes have been demonstrated. Grandparents’  higher incidence of cardiovascular mortality was associated with lower grandchild birthweight in a very large study in Norway ; U-shapeStrengths of the study include the standardized protocol for measuring cardiovascular risk factors, a prospective study design, and a community-based, unselected (not referred from clinic or high-risk) population. Most previous studies that have examined preconception health have been limited to Scandinavian populations , 29, 30 The implications of study results for public health should be considered. Interventions—improving cardiovascular health for both children and young women—would be likely be similar regardless of mechanism by which pre-pregnancy cardiovascular health has its effect. Determining the precise causal mechanism of these associations and thus the appropriate timing of interventions is an important area for future research. However, given the large proportion of unplanned pregnancies , generalS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Serum deprivation-response protein (SDPR), a phosphatidylserine-binding protein, which is known to have a promising role in caveolar biogenesis and morphology. However, its function in hepatocellular carcinoma (HCC) was still largely unknown. In this study, we discussed the characterization and identification of SDPR, and to present it as a novel apoptosis candidate in the incidence of HCC. We identified 81 HCC cases with lower SDPR expression in the tumor tissues with the help of qRT-PCR assay, and lower SDPR expression was potentially associated with poor prognostication. The phenotypic assays revealed that cell proliferation, invasion, and migration were profoundly connected with SDPR, both in vivo and in vitro. The data obtained from the gene set enrichment analysis (GSEA) carried out on the liver hepatocellular carcinoma (LIHC), and also The Cancer Genome Atlas (TCGA) findings indicated that SDPR was involved in apoptosis and flow cytometry experiments further confirmed this. Furthermore, we identified the interaction between SDPR and apoptosis signal-regulating kinase 1 (ASK1), which facilitated the ASK1 N-terminus-mediated dimerization and increased ASK1-mediated signaling, thereby activating the JNK/p38 mitogen-activated protein kinases (MAPKs) and finally enhanced cell apoptosis. Overall, this work identified SDPR as a tumor suppressor, because it promoted apoptosis by activating ASK1-JNK/p38 MAPK pathways in HCC. Despite a series of preventative and treatment measures that have been taken, HCC still remains the third leading cause of deaths that occurs from cancer in China, it also stands fourth among the common malignancy incidents in the same region2. Although several treatment procedures have been applied for HCC, the rate of recurrence is potentially high, and prognostication of HCC patients still remains very poor3. It is essential to identify pivotal antitumor molecules and establish potential therapeutic targets.Hepatocellular carcinoma (HCC) has been identified as the most common type of primary liver cancers, the latest epidemiological studies showed that HCC accounted for the third largest number of tumor-related deaths. During 2018, about 840,000 new cases of liver cancer have been reported worldwide, which caused 780,000 deaths, approximately4, apoptosis has been appeared to regulate cell proliferation and death balance, disruption of the balance may result in uncontrolled cell proliferation and eventually cancer or degenerative disorders5. The deregulation of cell death process is associated with aberrant cell proliferation, leading to growth of cancer and drug resistance6. As a major hallmark of tumorigenesis, apoptosis is considered to be one of the most promising antitumor mechanisms in cancer therapy7. However, treatment resistance still remains a major issue during the course of HCC incidence as effective interventions have not been fully discovered. Therefore, it is necessary to better understand the mechanisms of apoptosis and develop more effective molecular-targeted therapies in order to overcome treatment resistance of HCC.As a main type of programmed cell death8, but the potential function in cancer was rarely described previously. However, evidence from a recent study showed that SDPR was a metastasis suppressor through inhibiting epithelial–mesenchymal transition (EMT), migration and promoting apoptosis by relieving the inhibition of the extracellular-signal-regulated kinase (ERK) pathway in breast tumor9. A more recent study indicated that SDPR served as a key intermediary agent in miR-577-ERK-NF-κB pathway positive-feedback loop which regulated gastric cancer progression10. In addition, the study reported the lower expression of SDPR in the cancer of kidney, breast, and prostate11 and HCC12. Nevertheless, its specific functions and mechanisms in HCC still remains undiscovered.Here, we reported a novel tumor suppressor, serum deprivation-response protein (SDPR) , in inducing HCC cell apoptosis. SDPR was firstly identified as a phosphatidylserine-binding protein, as a member of cavin family, SDPR has been studied on the role of regulating caveolae formation and inducing membrane curvatureThe present study was conducted to identify the potential role of SDPR in the development and progression of HCC. We found that SDPR expression was markedly associated with the HCC cells’ malignant phenotypes and prognostication of HCC patients. Mechanically, SDPR induced cells apoptosis by promoting ASK1 N-terminus-mediated dimerization and activating ASK1-JNK/p38 MAPK pathways in HCC.2 at 37 °C. To avoid microbial contamination, culture medium was also added with 1% penicillin–streptomycin .The human HCC cell lines, HCCLM3, Huh-7, Hep3B, MHCC97L, HepG2, and SK-Hep1, and normal liver cell line L02 were obtained from the Cell Bank, Type Culture Collection, Chinese Academy of Sciences . Cell lines were authenticated by short tandem repeat profiling. Total cells were cultivated in the Dulbecco’s modified Eagle’s medium (DMEM)  added with 10% fetal bovine serum (FBS) , in a humidified atmosphere of 5% COIn this study, all those patients were included, who had been diagnosed with HCC from 2016 to 2020, who underwent hepatectomy in the Zhongnan hospital of Wuhan University of China, and those who did not receive any chemotherapy or radiation therapy before. Paired tissue specimens  were histologically confirmed by competent pathologists, collected within 30 min after the surgery, and were preserved in liquid nitrogen till further processing. All patients included were informed and approved the study.−ΔCt). All experiments were performed in triplicate, the sequences of the primers used were listed in Supplementary Table Using a Trizol reagent kit  with regard to the manufacturer’s instructions, total RNA was extracted from the tissues as well as cell lines. To determine the concentration and purity of RNA, the NanoDrop ND2000 instrument  was employed. Reverse transcription experiments were performed using HiScript II Q RT SuperMix  and then cDNA was obtained. The CFX96TM Real-Time System (Bio-Rad) was used to perform the QRT-PCR, and the level of the mRNA expression was assessed with the help of the comparative cycle threshold (Ct) method (2The BCA Protein Assay Kit (Thermo Scientific) was utilized to qualify the Cell Lysis solution. Proteins were transferred to polyvinylidene fluoride after being separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the next step, membranes were blocked with skim milk powder in TBST for 1 h and then incubated overnight at 4 °C with specific primary antibodies. Subsequently, membranes were added with the HRP-labeled antibody, then incubated at room temperature (RT) for a period of 2 h. Utilizing the ECL chemiluminescence reagent (Bio-Rad), antibody-bound proteins were identified. The information of antibodies utilized in this study were displayed in Supplementary Table Slides were prepared with 4 μm thick paraffin sections, then xylene and ethanol were used to deparaffinized rehydrate the slides, respectively. However, 0.3% hydrogen peroxide was utilized for the inactivation of endogenous peroxidase. While following the manufacturer’s protocol, all of the necessary steps were performed by employing an UltraSensitiveTM S-P kit .Full-length (FL) or truncated human SDPR and ASK1 gene sequences were amplified by PCR and cloned into phage-FLAG/HA/MYC vectors. The vector and lentivirus were transfected and SDPR was stably expressed in HCC cell lines. The small interfering RNA (siRNA) targeting SDPR were obtained from GENECREATE , then the transfection of HCC cells was accomplished with the help of SDPR-siRNA by utilizing the GenMute  in accordance with the manufacturer’s protocol. Moreover, total RNA and protein were extracted after 24 h of the transfection process. The sequences of siRNA have been displayed in Supplementary Table 3 cells/well and were kept for incubation in a complete culture medium. Every single well was added with 10 μl CCK-8 solution , and cells were incubated for a duration of 2 h. In the next step, absorbance was measured at 450 nm with the help of a microplate reader. Clonogenic assay was performed by seeding 1000–2000 cells per 6-well plate and were grown for a period of 2 weeks. The desired clones were stabilized with 4% paraformaldehyde, stained with 0.1% crystal violet solution, and counted under the light microscope.Cell proliferation was detected by using the colony formation assay and cell counting kit-8 (CCK-8). To perform CCK-8 assay, cells were first sown into 96-well plates at a density of 5 × 106 cells per well, and cultured without FBS for 24 h in DMEM. Wounds were created with the help of a 100-μl plastic pipette tip. Imaged of the scratches were captured at 0 and 24, 48, or 72 h. The digital pictures were used to calculate the areas of scratch without cells and the remaining areas.HCC cells were sown in a 6-well plate at a density of 1 × 105 cells were sown, however, the medium with 10% FBS was placed in the lower chamber. The cells holding the upper chamber were removed after a complete incubation of 24–72 h, and the invaded cells were stabilized and stained with 4% paraformaldehyde and 0.1% crystal violet solution, respectively.Transwell invasion was conducted in 24-well transwell plates, which possess 8-μl pore-size Matrigel-coated chambers. In the serum-free medium on the upper chamber, a sum of 1 × 10Cell apoptosis was detected by FC500 flow cytometer (Beckman-Coulter), with regard to the manufacture’s protocol of an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit .6) stably transfected with SDPR or negative control were suspended in PBS and then administered with direct injection into the armpit of 5 mice, respectively. Tumor size was measured with a vernier caliper. Mice were lost after 4 weeks, and tumors were dissected and imaged. Animals used in the experimental work of this study were treated humanely, with regard to the National Institutes of Health guide for the care and use of Laboratory animals . The study of animal was approved by the Ethics Committee of Zhongnan Hospital.The Central Laboratory of Animal Science located at Wuhan University , provided us with male athymic 5-week-old BALB/c nude mice, which were raised in a particular facility free from any pathogen. the mice were randomly assigned for the experiments using a table of random numbers. The sample size is estimated to effectively detect a significant difference among the groups. Investigators were blinded to the group allocation during the experiment. To evaluate the tumor proliferation in vivo, HCCLM3 cells . Then 1 μl FLAG antibody or IgG and 10 μl of Protein A/G magnetic beads (pre-washed with lysis buffer) were introduced into the cell lysate, and were incubated for 3 h at 4 °C. In the following step, the beads were sequentially rinsed two times with high salt and low salt wash buffer. Finally, proteins were eluted from the beads by 2× SDS loading buffer and boiled at 95 °C for 10 min prior to SDS-PAGE and immunoblotting with FLAG and ASK1 antibodies. For Exogenous co-IP, HEK293T cells were co-transfected with FLAG/HA-tagged SDPR and ASK1, the next steps are the same as for endogenous co-IP protocol.Huh-7 cells transfected with FLAG-SDPR were cultured in 10-cm dishes, following Co-IP as described previously. Proteins were analyzed using SDS-PAGE, and silver staining was carried out by utilizing Pierce Silver Stain Kit  with regard to the manufacturer’s recommendations. Then gels were subjected to mass spectrometry-based analysis.Cells grown on coverslips were washed three times with phosphate-buffered saline (PBS), and then paraformaldehyde was utilized to fix them for 15 min. Cells were blocked with 5% bovine serum albumin for a duration of 30 min after being permeabilized with 0.5% NP-40 for 20 min. In the following steps cells along with primary antibody were incubated for 2 h at RT, and subsequently incubated with fluorescein conjugated secondary antibody for 1 h. Finally, the DAPI and confocal microscopy were used to stain and image the slides, respectively.t test and χ2 as appropriate, parametric statistical analysis was conducted. For grouping, the median SDPR expression was considered as a cut-off value. A low SDPR group was defined as a value lower than the 50th percentile, in each of the 81 patients. However, a high SDPR group was defined as a value greater than the 50th percentile, in each of 81 patients. Following the Kaplan-Meier methodology, the survival curves were plotted. The entire values have been shown as the mean ± SD. P values were specified as follows: *P < 0.05; **P < 0.01; ***P < 0.001.GraphPad Prism 8.0 software and SPSS 21.0 software was used to carry out the Statistical analyses. Using the Student’s To identify the function of SDPR, the SDPR expression profile was accessed by Western blot and qRT-PCR. SDPR mRNA expression was detected in 81 HCC patients between tumor and adjacent non-tumor tissues by employing quantitative RT-PCR. The results revealed a considerably lower expression of SDPR mRNA in HCC tissues compared to peritumor tissues Fig. . MoreoveTo elucidate the antitumor role of SDPR, we firstly generated HCCLM3 and Huh-7 cell lines stably integrating SDPR. SDPR-overexpressed stable cell lines were shown in Fig. To further clarify the function of SDPR in HCC cells, we designed three small interfering RNAs (siRNAs). It has been displayed in Fig. https://portal.gdc.cancer.gov/) using the TCGA software 4.0.1. The single gene GSEA analysis showed positive enrichment for the gene signature associated apoptosis between the low and high heterogeneity groups  was performed on the gene data from TCGA (The Cancer Genome Atlas) database  Kinase, HA-ASK1679–936, and (3) C-terminal, HA-ASK1937–1374. The three ASK1 mutants and FL ASK1 were cotransfected with FLAG-tagged SDPR, respectively into HEK 293T cells, then coprecipitation using anti-HA antibody showed that SDPR were detected in FL-ASK1 and ASK11–678 groups, suggesting that NCC fragment may contain the SDPR binding site  and the activation of MAPKs including ERK1/2, JNK1/2, and P38 in HCCLM3 and Huh-7 cells transfected with SDPR. Consist with expectations, samples overexpressing SDPR displayed a considerable elevation in phosphorylated ASK1. However, only the phosphorylation of JNK1/2 and P38 were increased by SDPR overexpression, whereas phosphorylated ERK1/2 remained unaffected Fig. . SubsequBased on SDPR and ASK1 coordinately inducing HCC cells apoptosis and SDPR modulating the activation of ASK1, we hypothesized that the antitumor effects of SDPR might be mediated by ASK1. Accordingly, ASK1 inhibitor GS-4997 was utilized in HCC cells biological behavior experiments. We observed that the number of proliferating cells were much more in SDPR overexpressing cells after treatment with GS-4997 Fig. . Further10. Although previous study has identified decreased SDPR expression in HCC, how SDPR affected HCC progress and the underlying mechanisms were generally undiscovered. After discovering the differential expression of SDPR in tumor and non-tumor tissues, we speculated that SDPR might have an antitumor potential in HCC. Subsequently, we detected the relationship between SDPR expression with clinical characteristics and prognostication. The results demonstrated that SDPR was considerably linked with tumor size, histologic grade, PVTT, and TNM stage. Next, the phenotypic assays demonstrated that SDPR exerted significantly inhibiting effects on cell migration, invasion, and growth both in vivo and in vitro. The mechanisms of SDPR in lung cancer and breast cancer have been well studied, a study proved that SDPR might inhibit NSCLC cells malignant behaviors through p38 MAPK/ERK signals17, another research demonstrated that SDPR served as an apoptosis inducer thereby producing inhibitory effects on metastasis in breast tumor9. Then GSEA was conducted to further evaluate the mechanism of SDPR in HCC, the results identified positive enrichment of the apoptosis-related gene signature between the low and high heterogeneity groups. Accumulating with apoptosis assay, we reasoned that HCC progress inhibition might be leaded by activating cell apoptosis. What is more, SDPR significantly facilitated OXA-induced apoptosis. Previous works indicated that those agents which can directly trigger apoptosis pathways could also activate cell death pathways18. We hypothesized that SDPR might combine and interact with apoptosis pathway-related proteins. To confirm this, we detected SDPR-binding proteins by IP-MS assay and 259 proteins were discovered, among which Apoptosis signal-regulating kinase 1 (ASK1) aroused our attention.It is evident from the previous studies that SDPR served as an antitumor molecule in breast and gastric cancer21. The downstream cascade is subsequently initiated by activation of ASK1 which phosphorylates and activates two MAP2K pathways including MEE-4/7 and MKK-3/6, which thereby phosphorylates and activates c-Jun N-terminal kinase (JNK) and p38 MAPKs, respectively22. The basic roles of p38 and JNK MAPKs in cancer includes cell proliferation, differentiation, and apoptosis23. A study performed by Nakagawa indicated that the knockout of ASK1 inhibited HCC cells apoptosis apparently and promoted the development of HCC24. Another such study reported a key role of ASK1 in the event of HCC cell’s apoptosis, induced by sorafenib25. Therefore, it is extremely important to regulate the activity of ASK1 which in turn provides new insight into an anti-tumor strategy in HCC.ASK1, has also been identified as mitogen activated-protein kinase 5 (MAP3K5), which is stimulated by a series of stimuli such as calcium influx, endoplasmic reticulum (ER) stress, reactive oxygen species (ROS), and extracellular inflammatory signals including lipopolysaccharides (LPS) and tumor necrosis factor (ROS)20, proteins can affect ASK1 activity by combining and interacting with ASK1 N-terminal26. Thioredoxin (TRX) considerably inhibits ASK1 activity, particularly when it binds to ASK1 in its reduced form. However, in ROS condition TRX is converted to oxidized form and separated from ASK1. TNF receptor-associated factor 2 and 6 (TRAF2/6) have been appeared to promote ASK1 activation and homodimerization by subsequently binding to ASK128. In addition, there is a CIBI binding region at the N-terminal of ASK1 to detect Ca2+ stress signals, and more protein–protein interaction sites are being detected29. Among with our IP-MS results, we speculated that SDPR induced apoptosis through ASK1. Therefore, further PPIs experiments were performed to confirm SDPR-ASK1 interaction and the results revealed that SDPR bound to the NCC domain of ASK1 and facilitated ASK1 N-terminus-mediated dimerization and phosphorylation activation.As a crucial mediator of pro-apoptotic signals, the activation and inhibition of ASK1 are regulated by multiple mechanisms among which protein–protein interactions (PPIs) play an important role31. Even so, JNK and P38 MAPKs might be triggered by other MAP3Ks including TAK1, MLK, MEKK, and so on32. Thus, we utilized GS-4997, which binds to the ASK1 kinase domain and thereby inhibiting ASK1 selectively33, to assess the changes of p-JNK and p-P38. The results indicated that GS-4997 inhibited phosphorylation of JNK/P38 and cell apoptosis induced by SDPR, to a large extent, but not all. Consequently, we concluded that SDPR induced apoptosis primarily through ASK1-JNK/P38 pathways. Considering that GS-4997 did not totally block the pro-apoptosis effect of SDPR, the cross talk between SDPR and other apoptosis pathways was still needed further exploration. Mechanically, in HCC patients with high SDPR expression, SDPR combined with ASK1 NCC and facilitated N-terminus-mediated dimerization, subsequently ASK1 was phosphorylated and activated. Followed JNK/P38 pathways were activated and induced cells apoptosis. However, these pathways were largely blocked in patients with low SDPR expression (Fig. As the downstream kinases of MAP2Ks and MAP3Ks, JNK and P38 signaling pathways are selectively triggered by ASK1 and are recognized as major regulators in activating cell apoptosisIn conclusion, our findings supported the speculation that SDPR was a tumor suppressor, which inhibited cell invasion, proliferation, and migration by inducing apoptosis in HCC. What is more, SDPR overexpression could enhance the chemotherapeutic reactivity of HCC cells to OXA. Besides, we have identified a novel PPI that SDPR combined with and activated ASK1 and then leaded to activation of the JNK/P38 MAPK pathways. The finding provided a novel antitumor mechanism that may develop effective treatment strategies of HCC.Certificate of STR Analysis for huh7Certificate of STR Analysis for L02Certificate of STR Analysis for SK-Hep1supplementary table S1supplementary table S2supplementary table S3


PWM). Drying and warming piglets at birthhas been shown to reduce early postnatal temperature decline. This study evaluated theeffect of drying and warming piglets at birth on PWM and weaning weight (WW)under commercial conditions. A completely randomized design was used with 802 sows/litters; sows/litters were randomly allotted at start of farrowing to one of twoIntervention Treatments (applied at birth): Control (no drying or warming); Drying+Warming(dried with a cellulose-based desiccant and placed in a box under a heat lamp for 30 min).Piglets were weighed at birth and weaning; PWM was recorded. Rectal temperature wasmeasured at 0 and 30 min after birth on all piglets in a subsample of 10% of litters. Theeffect of farrowing pen temperature (FPT) on WW and PWM was evaluated bycomparing litters born under COOL (<25°C) to those born under WARM (≥25°C) FPT. Theeffect of birth weight on WW and PWM was evaluated by comparing three birth weightcategories . PROC GLIMMIX and MIXED of SAS were used to analyze mortality and other data,respectively. Litter was the experimental unit; piglet was a subsample of litter. Themodel included fixed effects of Intervention Treatment, and FPT or BWC as appropriate, theinteraction, and the random effects of litter. Piglet rectal temperature at 30 min afterbirth was greater (P ≤ 0.05) for the Drying+Warming than the Controltreatment (+2.33°C). Overall, there was no effect (P > 0.05) ofIntervention Treatment on PWM or WW, and there were no Intervention Treatment by BWCinteractions (P > 0.05) for these measurements. There was anIntervention Treatment by FPT interaction (P ≤ 0.05) for PWM. Drying andwarming piglets reduced (P ≤ 0.05) PWM under COOL (by 2.4 percentageunits) but not WARM FPT. In addition, WW were lower (P ≤ 0.05) under WARM(by 0.79 kg) than COOL FPT; however, there was no interaction (P >0.05) with Intervention Treatment. In conclusion, this study suggests that drying andwarming piglets at birth increases rectal temperature and may reduce PWM under coolerconditions, which are typically experienced in temperate climates during the majority ofthe year.Piglets are susceptible to hypothermia early after birth, which is a major predisposingfactor for preweaning mortality ( PWM) ofpiglets occurs within the first 3 d of birth . The objective of this study was to evaluate the effect ofdrying and warming newborn piglets on postnatal temperature changes and on piglet preweaninggrowth and mortality.One common method of limiting piglet heat loss without increasing farrowing roomtemperature is to include a localized area in the farrowing pen with a higher temperature. However, piglets are generally not confined to this heated area,and are often more attracted to the sow in the early postnatal period . WarmingThis study was conducted in the farrowing facilities of a commercial breed-to-wean farm ofthe Maschhoffs, LLC, located near Crawfordsville, IN, during the months of April–November2018. The experimental protocol was approved by the University of Illinois InstitutionalAnimal Care and Use Committee prior to the initiation of the research.A total of 402 sows and litters  were used in the study. Sows were fromcommercial dam lines of Yorkshire and Landrace origin that had been mated to commercialsire lines. The study used a completely randomized design, with litter as the experimentalunit and piglet as a subsample of the litter, to compare two Intervention Treatments(applied at birth): Control (no drying or warming); Drying+Warming . Sows/litters were randomly allotted to Intervention Treatment at the start offarrowing (after the birth of the first piglet), with the restriction that dam genotypeand parity were balanced across treatments.2; pen dimensions were 1.52 m wide by 2.07 m long, giving a total pen floorspace of 3.15 m2. Crates were equipped with a sow-operated feed dispenserattached to a feed trough, and a nipple-type water drinker for the sow. An infrared heatlamp was suspended in the center of the floor area on one side of the farrowing crate overan insulated rubber mat (average temperature under the heat lamp during the study periodwas 37.1 ± 3.22°C). For the Drying+Warming treatment, the heat lamp was suspended over aplastic box for the duration of farrowing. Room temperature was maintained using fans,heaters, and evaporative coolers as needed; the thermostat for each room was set at 22.5°Con the day of farrowing and was incrementally reduced to 18.0°C by weaning.Each sow was housed in an individual farrowing crate, located in the center of afarrowing pen, which had either woven metal or perforated plastic flooring. Cratedimensions were 0.55 m wide by 1.95 m long, giving a floor space within the crate of 1.07mManagement in the farrowing facility was according to unit protocols, which weregenerally in line with standard commercial practices. Sows that had not farrowed by day116 of gestation were induced to farrow on the following day using Lutalyse ; the identity of each sow that wasinduced and date of induction were recorded. The farrowing process was monitoredcontinuously by the investigators; if the interval between the births of piglets exceeded60 min, the investigator checked the birth canal for obstructions and assisted thefarrowing process as needed.At birth, piglets were given a uniquely numbered ear tag for identification, the allottedIntervention Treatment was applied, and they were returned to the farrowing pen(immediately for the Control and after 30 min in a warming box for the Drying+Warmingtreatment). Piglets were weighed within 12 h of birth using a Brecknell LPS-15 bench scale. Scales were calibrated daily prior to use with astandard test weight.Piglet rectal temperature was measured at 0 and 30 min after birth on a randomly selectedsubsample of 10% of the litters distributed throughout the study period . Rectal temperatures were measured on all sows at the startand end of the farrowing process. Piglet and sow rectal temperatures were measured at adepth of 2.5 and 10 cm, respectively, using a HSTC-TT-K-24S-36 thermocouple attached via aSMPW-K-M connector to a dual input K/J digital thermometer .Thermometers were calibrated each week during the study period by taking measurements in atemperature-controlled chamber that was set at temperatures that encompassed the expectedrange . A regression equation for the relationshipbetween measured and set temperatures was developed and was used to adjust rectaltemperature measurements taken during the following week of the study.The temperature in each farrowing pen at three locations [behind and at either side ofthe sow (one of these measurements being under the heat lamp)] was measured at thebeginning and end of the farrowing process using a digital infrared thermometer [TOOGOOGM320 LCD digital infrared thermometer gun ].The litter of piglets was the experimental unit for all measurements; piglet was asubsample of litter. The PROC UNIVARIATE procedure of SAS  wasused to verify normality and homogeneity of variances of the residuals. All variables thatconformed to the assumptions of normality and homogeneity were analyzed using PROC MIXEDof SAS . PreweanBWC). The maximum birth weight for the Light category represented that below which PWM increases substantially , Medium (1.0 to 1.5 kg), or Heavy (>1.5 kg) BirthWeight Categories . Thedivision at 25°C was chosen based on previous studies that suggested that piglet rectaltemperatures are higher above this point compared with lower, more typical farrowing roomtemperatures  or WARM (≥25°C) farrowing pen temperatures  between treatments for any of these except for temperatureunder the heat lamp before farrowing, which was greater (P ≤ 0.05) for theControl than the Drying+Warming treatment; however, this difference was relatively small(0.8°C). In general, the pigs used and temperature conditions in the farrowing facilitieswere typical of U.S. commercial production. The majority of sows on the study were betweenparities 1 and 8. Average sow rectal temperatures before and after farrowing were between38.28 and 38.70°C, which is typical for farrowing sows (Sow parameters and farrowing pen temperatures have been summarized by InterventionTreatment in ing sows . Averageing sows  were higP > 0.05) for the InterventionTreatments for both the entire dataset and the subsample. In addition, there were nodifferences between Intervention Treatments (P > 0.05) for either littersize or birth weight between BWC treatments or between FPT treatments for the entire datasetor the subsample  forthe two Intervention Treatments, which was expected as treatments were not applied untilafter birth temperature was measured. However, temperatures at 30 min after birth were2.33°C lower  of drying and warming of piglets onWW, PWM, or the age of piglets at death  under WARM thanCOOL FPT; however, this difference was relatively small (<0.2°C). The body temperatureof sows during farrowing has been shown to be higher under warmer than under coolerconditions (P ≤ 0.05) between FPT and InterventionTreatment for piglet temperature at 30 min after birth (P ≤0.05) under WARM than COOL FPT; in contrast, temperatures of piglets on the Drying+Warmingtreatment were similar (P > 0.05) for the two FPT. This resulted inthe Drying+Warming treatment producing a greater increase in piglet temperature under COOLthan WARM conditions  under COOL than WARM FPT; however,there was no interaction (P > 0.05) with Intervention Treatment (P > 0.05) between FPT and BWC (datanot reported), indicating that higher farrowing pen temperatures reduced WW to a similarextent for piglets of all BWC. Higher temperatures during lactation can reduce sow milkproduction  forPWM (P ≤ 0.05) PWM than the Control under COOL, but not WARM FPT. Inaddition, the average age of piglets at death tended (P = 0.08) to belower under WARM than COOL FPT; however, this difference was very small and there was nointeraction (P > 0.05) with Intervention Treatment. Hypothermia hasbeen shown to be an important predisposing factor for PWM, either directly or indirectly between BWC; however, differences were small (<0.2°C).There was an interaction (P ≤ 0.05) between Intervention Treatment andBWC for piglet temperature at 30 min (P ≤ 0.05) temperatures than the other two BWCfor both Intervention Treatments; however, this difference was greater for the Controlthan the Drying+Warming treatment. For example, the difference in temperature betweenLight and Heavy BWC was 2.49°C for the Control compared with 0.88°C for the Drying+Warmingtreatment. In addition, the Drying+Warming treatment resulted in greater(P ≤ 0.05) temperatures than the Control for all BWC, but thedifference between the two treatments was greater for Light than Medium or Heavy piglets. These resultshighlight that lighter birth weight piglets are more predisposed to hypothermia in theearly postnatal period than heavier littermates, which is in agreement with the findingsof a number of studies (P ≤ 0.05) between the two variables for both treatments;however, the relationships differed between treatments. Predicted temperatures were lowerfor the Control than for the Drying+Warming treatment for piglets of all birth weights,and the linear and quadratic coefficients were greater (P ≤ 0.05) for theControl than the Drying+Warming treatment. These relationships further illustrate thatdrying and warming generally reduced the variation in piglet temperature due to birthweight, and resulted in lighter birth weight piglets having temperatures at 30 min afterbirth that were relatively similar to their heavier littermates.The regression relationships between piglet birth weight and rectal temperature at 30 minafter birth for other studies that were carried out in the same facilities using the sameIntervention Treatments as in the current study are presP ≤ 0.05) WW and higher (P ≤0.05) PWM than Heavy piglets; Medium piglets were intermediate and different(P ≤ 0.05) to the other BWC for both measurements (r = 0.57). The average age of piglets at death was lower(P ≤ 0.05) for Light piglets compared with Medium or Heavy piglets,which were similar (P > 0.05); however, there was no interaction(P > 0.05) with Intervention Treatment for this measurement (Light piglets had lower (urements . A numbeurements . In addisurement . LeDiviP> 0.05) between Intervention Treatment and BWC for this measurement. This suggests thatDrying+Warming was ineffective at reducing PWM compared with the Control in piglets of allbirth weights . Thisresult was unexpected given that this intervention reduced postnatal temperature declineto a greater extent for lower birth weight piglets than their heavier littermates  for both piglet birth weight and rectal temperature;therefore, linear and quadratic terms for these factors were included in the three models.Piglet birth weight (Model 1) and rectal temperature at 30 min after birth (Model 2)independently accounted for 74.3% and 62.5% of variation in PWM, respectively. However,including piglet rectal temperature and birth weight in the model (Model 3) only increasedthe variation in PWM explained to 74.6% , whereas A number of studies have carried out retrospective multivariate analyses of commercialdata sets and have found that low birth weight is a major predisposing factor for PWM. In addiIn conclusion, the results of this study found that drying and warming piglets at birthreduced piglet rectal temperature decline at 30 min after birth with differences relativeto an untreated control that were consistent with those of previous research. However,there were no effects of drying and warming on WW or PWM, either overall or within any ofthe BWC. As expected, piglets of lower birth weight had lower WW and greater PWM thanheavier littermates. Drying and warming piglets reduced PWM under cooler, but not warmerfarrowing room temperatures. Preweaning mortality is complex, and postnatal change inpiglet temperature is only one of a multitude of potential factors involved. There is aneed for further large-scale controlled research studies to understand the potential roleof piglet temperature changes and possible interactions with other factors in influencingpiglet survival.


Apolipoprotein C-III (Apo CIII) is a crucial regulator of triglyceride-rich lipoproteins (TRLs) and influences the risk of cardiovascular diseases. High levels of Apo CIII have been also associated with cerebrovascular events and earlier works showed procoagulant effects of Apo CIII. The main aim was to assess whether the plasma concentration of Apo CIII could confer an increased risk of cerebral ischemic events in anticoagulated patients at high-risk of cardioembolism.We systematically checked medical records and quantified cerebral ischemic events in a selected cohort of 118 subjects , taking anticoagulant therapy with warfarin because of atrial fibrillation (AF) and/or mechanical prosthetic heart valves. All the subjects, enrolled between May 1999 and December 2006, were prospectively followed until death or July 31, 2018. Assessments of complete plasma lipid and apolipoprotein profiles, including Apo A-I, B, CIII, and E, were available for all subjects at enrollment.n = 15) and TIA (n = 9). Subjects with plasma concentration of Apo CIII above the median value (10.3 mg/dL) had an about three-fold increased risk of stroke/TIA than those with lower levels of Apo C-III [hazard ratio 3.08 ]. This result was confirmed in multiple Cox regression models adjusted for gender, age, CAD, AF, diabetes, hypertension, plasma lipids, and CHA2DS2-VASc score. By stratifying the sample on the basis of Apo CIII level and CHA2DS2-VASc score, an additive effect was observed with the highest risk in subjects with both high Apo C-III concentration and CHA2DS2-VASc score.After a median follow-up of 109 months , 24 subjects (20.3%) had cerebral ischemic events: stroke (High Apo CIII plasma levels may be associated with an increased risk of ischemic stroke/TIA in high-risk cardiovascular patients anticoagulated with warfarin. NotaOn the basis of these premises, we hypothesized that high Apo CIII levels leading to a prothrombotic diathesis could be associated with an increased risk of ischemic stroke/TIA in the specific clinical setting of patients taking anticoagulant therapies. Therefore, in order to investigate the factors potentially contributing to cerebrovascular risk despite anticoagulation, we selected from the previous study cohort  the subgn = 62 and n = 56, respectively), i.e., clinical conditions at high-risk of cardioembolic events. All subjects had a complete routinary laboratory evaluation, including Apo A-I, Apo B, Apo CIII and Apo E plasma concentrations . CHA2DSidelines .The subjects were followed until death or July 31, 2018. Follow-up data were available for all the subjects in the sample of the present analysis. Study subjects' mortality status was determined by searching in the National Population Register. Survival times were calculated starting from the date of enrolment. The electronic medical records of all the Hospitals in the District of Verona, Northeast Italy, including data of Emergency Units admissions were obtained for all subjects; ambulatory or telephone survey was performed in case of clinical doubt. In order to be included in the statistical computation, non-fatal ischemic stroke/TIA events had to be confirmed and validated by two independent neurologists after careful evaluation of all the available information . Ischemi2 test or χ2 for linear trend analysis when indicated. Ischemic stroke/TIA event rates during the follow-up period were assessed by using the Kaplan–Meier method with Log-rank statistic and Cox regression. Kaplan–Meier curves were used for survival plots, which stratified the study population according to Apo CIII plasma concentration. Taking into account the well-recognized role of CHA2DS2-VASc score in predicting the risk of cardioembolic events . As expected, subjects with high Apo CIII levels had an unfavorable plasma lipid profile, with increased concentrations of both total and LDL cholesterol, triglycerides, Apo B and Apo E , 24 subjects (20.3%) had non-fatal ischemic stroke  had an increased rate of ischemic stroke/TIA , with anSc score , and eve2DS2-VASc score, which was associated with ischemic stroke/TIA in the study sample . By thiate risk .As a necessary preliminary consideration, we must recognize that the current results are the by-product of a larger analysis showing that Apo CIII levels may confer an increased risk of ischemic cerebrovascular events in cardiovascular patients . This stThe findings showed that was just the case. With the limitations due to the above-mentioned premise, the present results are the first to provide evidence suggesting such a role for Apo CIII, with its high levels being associated with an about three-fold increased risk of ischemic cerebrovascular events during an about 10-year follow-up. Notably, three-quarters of subjects 18/24) with ischemic stroke/TIA during the follow-up had basal Apo CIII plasma concentrations above the median value . Apo CII/24 with 2DS2-VASc score as risk factors for recurrence of cerebral ischemic events . The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.MC declares consulting fees from Boehringer-Ingelheim and Pfizer-BMS, and advisory board from Daiichi Sankyo. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.