Adverse perinatal outcomes in singleton IVF pregnancies have been most often explained by parental underlying diseases and so far laboratory conditions during embryo culture are still not explored well. The following review discusses the current state of knowledge on the influence of IVF laboratory procedures on the possible perinatal outcome. The role of improved media for human embryo culture is unquestionable. Addition of certain components to culture media and their effect on embryo survival and implantation rates have been taken into consideration recently and studied on animal model. Impact of media on perinatal outcome in IVF offspring has also been studied. It has been discovered that epigenetic changes and neonatal birth weight are probably associated with the use of specific culture media, as is the relation between placental size and its influence on perinatal outcome. There are still questions in the discussion about duration of embryo culture (cleavage stage versus blastocyst transfer). Some of the IVF methods, such as in vitro maturation of oocytes and freezing/thawing procedures, also require well-powered randomized controlled trials in order to define their exact impact on perinatal outcome. Constant further research is needed to assess the impact of laboratory environment on fetal and postnatal development.  After over 30 years of existence, in vitro fertilization (IVF) is now considered a routine medical practice in the management of infertility. Deliveries following IVF account for constantly increasing percentage of all births. According to 2012 estimates the number of children born after assisted reproduction techniques exceeded 4 million. Available national birth cohorts show that the proportion of IVF infants range from 0.8 to 4.1% of all deliveries [Potential adverse perinatal outcomes in singleton IVF pregnancies such as early loss, spontaneous abortion, ectopic pregnancy, preterm birth, low birth weight, and being small for gestational age (SGA) have been studied in depth and are well known. In vitro fertilization has been associated with an increased risk of pregnancy complications such as placenta praevia and placental abruption, gestational hypertension, preeclampsia, and cesarean delivery . HoweverMost of the abovementioned complications may be explained by maternal and paternal underlying medical conditions associated with subfertility and infertility such as sperm factors, the use of fertility medications, laboratory conditions during embryo culture, culture media, cryopreservation and thawing, prenatal genetic diagnosis (if performed), differences in obstetrical management, increased proportion of multiple gestations and vanishing twins, or a combination of these factors , 5.Until now clinical practice has shown that the in vitro culture of human embryos does not confer major adverse effects on the offspring, but possible consequences in late childhood or adulthood are still to be explored, as even the eldest children conceived by ART are still quite young.The presented literature review is focused on the impact of embryo culture conditions on perinatal outcome after in vitro fertilization procedures. Alongside infertility causes, the factors related to culture conditions seem to be the most difficult to explore.It is obvious that the quality of an embryo is one of the main factors, if not the most important, affecting the cumulative pregnancy rates after in vitro fertilization treatment, especially when elective single embryo transfer (eSET) policy is applied. In order to be a successful IVF center with eSET policy, having a highly efficient embryo freezing program is a must. Nordic countries are European leaders of the abovementioned approach, reaching 51.3% cumulative live birth rates per oocyte pickup . It is vThere would never be any success of human IVF if it was not for the efficient laboratory techniques. The technical and scientific progress, together with the proper quality control in the IVF laboratory, has an undisputed role in maintaining satisfactory clinical results. Alongside technical aspects, the role of improved media for human embryo culture is unquestionable. Extensive knowledge regarding the composition of culture media used in human IVF comes from animal studies. It has been well documented that, for example, certain amino acids are integral components of embryo culture media, as they are important at all stages of development and serve as energy source needed to maintain homeostasis . NeverthSome authors also studied the addition of certain components to culture media and their effect on embryo survival and implantation rates. Ziebe et al. analyzed the effect of addition of granulocyte-macrophage-colony stimulating factor (GM-CSF) to the culture media. The authors proved that it had some protective effect on culture-induced embryo stress, therefore decreasing the miscarriage rates. There was, however, no effect on birth weight and abnormalities in comparison to the control group .The research groups of Dumoulin et al. and Nelissen et al. were the first to have documented the effect of culture media on the birth weight of IVF singletons in humans. The authors agree that culture media influence perinatal outcome of IVF singletons and twins, with the exception of gestational age. Nelissen et al. compared two commercially available culture media and concluded that in vitro culture of embryos in media made by Cook Corporation resulted in singletons with a lower mean birth weight (adjusted mean difference 112 g) and more singletons with low birth weight  when compared to singletons born after culture media produced by Vitrolife . These rNevertheless, a few studies with somewhat conflicting results regarding growth patterns have been published since 2010. Several studies comparing cultures in G1.3/Global/G1.5, HTF/Sage, G5tm/Global/Quinn's advantage, and Medicult/Cook/Vitrolife media found no significant differences in mean birth weight between singletons cultured in those media –21. One In contrast to Dutch studies, Belgian group of De Vos et al. published a large retrospective study which does not support earlier concerns that both the type of culture medium and the duration of embryo culture affect birth weight of IVF singletons. The authors analyzed 2098 singleton live births derived from culture in sequential media by Medicult and Vitrolife and they showed no significant differences in neonatal birth weights between two culture media. They also claim that continuous surveillance of human embryo culture procedures should remain a priority of IVF centers .There are also papers examining if the source of proteins in embryo culture media can influence neonatal birth weights of IVF singletons. Zhu et al. proved that such a source might be an independent factor affecting birth weight based on multiple regression analysis . AnsweriSeveral animal studies have been performed to address the same issue. Different culture conditions, mainly achieved by the addition of serum, have led to an increased birth weight in sheep and cattle and a decreased birth weight in mice. An explanation for these culture medium-induced effects could be that in vitro culture leads to epigenetic disturbances, possibly in more fragile mitochondrial DNA particles, which in turn might affect developmental programming of fetal and placental tissues. Preimplantation embryo culture has been shown to affect methylation and expression of imprinted genes in several animal models, as mentioned above –28.It seems that there is still lack of knowledge in this aspect of assisted reproductive technology. Unfortunately, the complete composition of commercially available media is unknown, so it makes it harder to conclude and speculate as to which components might be more beneficial or deleterious for development and competence. It is obvious that further studies are definitely required to make in vitro fertilization procedure a safe and responsible choice for patients.Several studies have evaluated the outcome of singleton pregnancies after blastocyst versus cleavage stage embryo transfer. Higher incidences of preterm birth (PTB), very preterm birth, low birth weight (LBW), and congenital malformations were identified in a few of them. The systematic literature review of Dar et al. compared 9506 births after day 3 transfer with 3206 babies born after blastocyst transfer. The authors concluded that embryo transfer after prolonged culture to the blastocyst stage was associated with higher odds of preterm birth compared to cleavage stage transfer. In order to explain the above they suggested that longer in vitro culture might have a deleterious effect on subsequent placentation .A large Swedish study by Källén et al. examining neonatal outcomes after cleavage versus blastocyst transfer also indicated a significantly higher likelihood of preterm birth after blastocyst transfer. The authors compared 1311 infants born after blastocyst transfer versus 12562 babies after cleavage stage transfer and attributed PTB to the possibly longer period of culture .In contrary to the latter study, Fernando et al. in a large cohort of 4202 women who conceived after IVF and IVF-ICSI found no differences in any of the perinatal outcomes when births resulting from transfer on days 5 to 6 were compared to transfers on days 2 to 4 .The systematic review and meta-analysis by Maheshwari et al., based on the data from observational studies, found an increased risk of preterm delivery, but not small-for-gestational-age babies, in pregnancies after blastocyst transfer compared to those after cleavage stage transfer. The authors are very critical to the obtained results and suggest combining data from all existing randomized trials in an individual patient data meta-analysis to provide enough power to determine any differences when designing further studies. Although individual patient data from various registries will have higher power and can be adjusted for important confounders, one has to remember that the patients who are eligible for extended culture have very different prognosis compared with those who do not fulfill the criteria for blastocyst transfer .Future research should be aimed at improving both the safety of culture media and incubation systems used for prolonged culture to the blastocyst stage.Only few studies have examined placental size as a perinatal outcome in pregnancies achieved by in vitro fertilization. An extensive analysis in the Norwegian population compared placental and newborn weights in 536 567 singleton pregnancies, including 4557 conceived by IVF, 3192 through ICSI, and 88 pregnancies by the combined use of IVF and ICSI or other unspecified ART (assisted reproductive technologies) procedures. As previously reported, ART singletons had significantly lower birth weights than spontaneously conceived controls but were additionally characterized by significantly bigger placentas. The changes in birth weight, placental size, and fetal: placental weight ratio remained significant regardless of the form of fertilization even when adjusted for length of gestation, sex, parity, maternal age, and pregnancy complications . It all In vitro maturation (IVM) is an emerging type of ART, in which immature as opposed to mature oocytes are harvested from the ovaries and allowed to mature in vitro prior to fertilization by either standard IVF or intracytoplasmic sperm injection (ICSI). Since the first report of a human birth resulting from IVM in 1991 it is estimated that over 1,300 babies have been born following this technique, in contrast to estimated 4 million babies conceived by standard IVF and ICSI over the last 30 years. In vitro maturation, or the maturation of prophase I oocytes to metaphase II in culture dish prior to fertilization, is a newer technology with limited follow-up on childhood outcomes –36.The largest and best study designed to investigate the outcomes of IVM children was published by Buckett et al. from McGill University in Montréal. The authors examined obstetric outcomes and congenital abnormalities in pregnancies conceived after IVM (55 cases), IVF, and ICSI and compared with those of spontaneously conceived controls. They concluded that while all ART pregnancies were associated with an increased risk of multiple pregnancy, cesarean delivery, and congenital abnormalities, IVM was not associated with any additional risk compared with IVF or ICSI. Interestingly, the mean birth weight of IVM infants was higher than in controls, IVF, and ICSI infants, which may be suggestive of epigenetic differences in IVM children. Different culture media used in this particular procedure should also be taken into account .Further analyses of perinatal outcome of babies born after IVM are required, as this method has emerging promises for patients at risk of fertility loss due to emergency gynecological surgery or oncological states requiring fertility preservation.Constantly improving cryopreservation techniques are a guarantee for future success of ART. Cryopreservation and thawing processes can uniquely affect pregnancy outcomes. The available literature regarding that subject is generally in favor of frozen/thawed embryo transfers. Singleton pregnancies after transfer of frozen/thawed embryos are associated with a lower risk of perinatal mortality, small-for-gestational-age babies, preterm birth (<37 weeks), low birth weight (<2500 g), and antepartum hemorrhage when compared with those after fresh embryo transfers. They are also at increased risk of macrosomia or being large for gestational age , 39. A sCulture media are one of the puzzles pieces in the success of IVF. Other factors such as incubator gases, temperature, culture oils, and supplements are equally important and can change the dynamics of how the embryo interacts with components of the medium. Characteristics of other parameters, such as pH and oxygen concentration, can also affect the embryos and should be considered together when analyzing the overall result of IVF. The implementation of new technologies brings us closer to robotic IVF, but we still need to provide the early human being with the conditions mimicking the female reproductive tract. Therefore, culture media will always play a major role in the IVF laboratory. The variety of factors affecting the results such as adverse perinatal outcome, neonatal birth weight, placental weight, and epigenetic changes makes it very hard—almost impossible—to draw conclusions. This results in many papers showing conflicting data. Hopefully, unification of products and clear indications for the procedure will allow the performance of prospective, randomized studies with satisfactory properly powered results and conclusions.Regarding the obstetric and perinatal outcome, it seems that “freeze-all” strategy may one day dominate IVF procedures, as there is increasing evidence of favorable outcome in such cycles.


Preterm birth is a serious public health problem, as it is linked to high rates of neonatal and child morbidity and mortality, with Brazil listed among the countries with the ten highest numbers of premature births. Nonetheless, knowledge is scarce regarding prematurity and associated factors in mid-sized cities. The objective of this study was to analyze the trend of preterm births and associated factors in a municipality located in the state of Paraná, Brazil.This was an ecological time series study of births recorded into the Live Birth Information System for residents of Maringá, Paraná, Brazil, between 2000 and 2013. The polynomial regression model was used for trend analysis of preterm birth, characteristics of the mother, gestation and delivery, and newborn. The association with preterm birth was analyzed using odds ratio (OR).2 = 0.93)–with a growing share of moderate preterm births (32 to <37 weeks), which rose from 7.0% in 2000 to 9.7% in 2013. Between 2011 and 2013, multiple pregnancy , inadequate number of prenatal visits , Apgar score below 7 at 1  and 5 minutes , low birth weight  and congenital malformations  were associated with preterm birth. A growing trend was observed for multiple pregnancies, with an average annual increase of 0.32% (r2 = 0.90), as well as for C-section birth (2.38% yearly increase). Of all newborn characteristics, Apgar score below 7 at 5 minutes (-0.19% per year) and low birth weight (-1.43%) decreased, whereas congenital malformations rose (0.20% per year).A total of 61,634 live births were analyzed, of which 5,632 were preterm births. Prematurity increased from 7.9% in 2000 to 11.2% in 2013 –an average increase of 0.54% per year (rEfforts are required to prevent premature delivery, particularly during the moderate period, as well as greater care during the prenatal period towards expectant mothers bearing multiple pregnancies, birth defects, in addition to reducing C-section birth as it may be linked to preterm birth. The prevalence of preterm births is high, with a tendency for further increase in several countries. In the United States, prematurity rose from 9.5% in 1981 to 12.3% in 2008, leveling off between 12% and 13% [In Brazil, a similar increase has been observed in preterm births, from 5% in 2005 to 11.8% in 2012; the state of Paraná has shown similar rates as the rest of the country, increasing from 6.2% to 11.9% in the same period . For theth week of gestation, is a serious public health problem, as it is linked to high rates of neonatal and child morbidity and mortality, as well as the possibility of disabilities during childhood and adult life [Preterm birth, defined as a birth that occurs prior to the 37ult life –5. It isult life . ChildreThe etiology of preterm birth is complex and involves socioeconomic factors, the mother’s reproductive history, quality of prenatal care, maternal incidents during gestation, fetal traits, among others . Possiblth gestational week may continue [Despite the consensus regarding the multifactor etiology of preterm birth, accrued knowledge is still insufficient to explain the rise in prematurity observed over the last few decades. Without deepening this knowledge, births prior to the 37continue ,9.Research studies are needed to identify the risk factors related to prematurity –11, in oThe objective of this study was to analyze the trends in preterm births and associated factors in the city of Maringá, Paraná, Brazil. The expectation is to contribute to planning more effective interventions in order to reduce preterm births as well as perinatal and child morbidity and mortality.This was an ecological, time series and cross-section study of births listed in the Live Birth Information System (Sinasc) for residents of the municipality of Maringá, Paraná, in the period between 2000 and 2013.th State Regional Health Office. It features a 98.6% rate of urbanization and Human Development Index (HDI) of 0.80, ranking sixth in the state and 67th among all Brazilian cities [Maringá, the third largest city in Paraná State with an estimated population of 391,753 in 2013, population density of 793 inhabitants per km², is the seat of the Maringá Metro Area (RMM) and the 15n cities .th State Regional Health Office, because they were not yet available online on Datasus. The period between 2000 and 2013 was defined after exploring the thoroughness of the variables listed on Sinasc: gestational age, sex, Apgar at 1 and 5 minutes, race/color, birth weight, birth defect, mother’s age, marital status, mother’s educational level, profession, number of children born alive and stillborn, pregnancy type, type of delivery, number of prenatal visits and delivery site.To analyze the evolution of preterm births, data from 2000 to 2012 were obtained from Sinasc, and 2013 data were provided by the 15excellent, a percentage of undeclared variables below 1%; good, between 1% and 2.99%; regular, from 3% to 6.99%; and poor, when the percentage of undeclared variables is equal or greater than 7% [To determine the quality of the database and its potential use, the study analyzed the rate of undeclared variables—variables that were ignored or left blank. To that end, a scale was adapted to fit the reality of the satisfactory quality of information included into Sinasc in the state of Paraná:  than 7% .Overall, the year with the worst rate of data completion was 1999, in contrast with 2013 when all variables were regarded as having excellent rates of completion. Accordingly, it was decided not to include the year 1999.Preterm birth rates were calculated according to gestational age (GA): under 28 weeks (extreme prematurity); from 28 to <32 weeks (very premature); from 32 to <37 weeks (moderate preterm birth); and <37 weeks . Also analyzed were the delivery site , the mother’s age , partner (yes or no), education level (<8 or ≥ 8 years of schooling), parity (primigravida or multigravida), pregnancy type (single or multiple), type of delivery , number of prenatal visits (<7 or ≥ 7 visits), sex of the newborn , Apgar at 1 and 5 minutes (<7 or ≥7), birth weight  and congenital malformations (yes or no).Rates were calculated year-to-year for trend analysis of preterm birth and trends in the characteristics of the mother, gestation and delivery, and newborn. To analyze the factors associated with preterm birth, the data were clustered into two three-year periods (2000 to 2002 and 2011 to 2013), using odds ratio (OR), with a confidence interval (CI) of 95%.0+β1x1), quadratic (y = β0+β1x1+β2x2), and cubic polynomial regression models (y = β0+β1x1+β2x2+β3x3) were tested. Any trend whose estimated model reached a p-value <0.05 was considered significant. In order to select the best model, the choice for best model also took into consideration the analysis of the scatter plot, the value of the coefficient of determination (r2) and residual analysis . When all criteria were significant for more than one model and the coefficient of determination was similar, the simpler model was chosen. All analyses were carried out using SPSS software, version 20.1.The polynomial regression model was used for trend analysis, in which preterm birth rates were regarded as dependent variables (y) and the years of the study were the dependent variable (x). The ‘year’ variable was transformed into the year-centered variable (x-2006) and the series were smoothed using a three-point moving average. The linear (y = βhttp://datasus.saude.gov.br/). All data were anonymized.The research project was approved by the Standing Committee for Ethics in Research of the Paraná State Health Secretariat/Workers Hospital . All data were obtained from public databases  were preterm births. In 2000, the rate of preterm births was 7.9%, rising to 11.2% in 2013, with a larger share of moderate preterm births (from 32 to <37 weeks), accounting for 84.8% of total preterm births analyzed, and which went from 7.0% in 2000 to 9.7% in 2013 , with a 2 = 0.93), when calculated in relation to all births in each year. Moderate preterm births—between 32 and <37 weeks—were the greatest contributors to the growth in prematurity, with an average increase of 0.49% per year (r2 = 0.91). Behavioral analysis of the different stages of prematurity—calculating the percentages in relation to all preterm births—showed a negative trend for extreme premature births (-0.43% per year) and very premature births (-0.15%), and a growing trend for moderately premature births (0.73% per year)  .p = 0.001) or 35 years or older , having less than eight years of formal education , and having a first pregnancy . With regard to characteristics of gestation and delivery: multiple pregnancy , and having had less than seven prenatal visits . With regard to newborn characteristics: having had Apgar score below 7 at 1 minute , and at 5 minutes , having birth weight lower than 2,500 gr , and the presence of congenital malformations . In that three-year period, having a C-section birth was a protective factor for preterm birth   .p<0.001), having had fewer than seven prenatal visits ; Apgar score below 7 at 1  and 5 minutes , as well as low birth weight  and congenital malformations , were factors associated with preterm birth (For the second three-year period (2011 to 2013), only the variables regarding gestation and delivery and the newborn showed a significant association with preterm birth. Multiple pregnancy . C-section birth showed a tendency towards increase, only among premature births, with an average annual increase of 2.38% , and an increase in preterm births among women without a partner (0.85% per year) and with eight or more years of formal schooling (mean percentage of 83.26% and a 1.5% increase per year). With regard to pregnancy type, a growing trend was observed in multiple pregnancy births, with an annual increase of 0.32%, and high explanatory power for the model , which are largely ignored and left without early detection of problems related to their development .With regard to the factors associated with preterm birth, it is possible to detect a transition in the epidemiological profile of maternal factors, signaled by the changes seen in the results found in the analysis of maternal sociodemographic variables, which showed no link to preterm birth during the three-year period from 2011 to 2013. Similar results were observed in a case-control study on preterm births carried out in the city of Londrina, state of Paraná, in which the mother’s age, education level and parity were not associated with prematurity, either .Both in Brazil and others middle income countries, the increase of preterm births has been recorded in a context of demographic and epidemiological transition, with modifications in some characteristics of the population of women including a diminution of fecundity rate, and decrease amount of marriage . During With regard to gestation and delivery variables, only multiple pregnancy and the insufficient number of prenatal visits remained associated with prematurity in both three-year periods.Research studies indicate that perinatal morbidity/mortality and the occurrence of prematurity are higher in multiple pregnancies, as a result of distention of uterine fibers, causing early maturing, as well as other common complications in pregnancies with more than one conceived fetus , and thath week of gestation. However, it is important to consider that 84.8% of premature births analyzed in this study were moderately premature births (32 to <37 weeks), and that some characteristics such as multiple pregnancy and gestations with fetal malformations—often linked to preterm births—indicate the need for a closer look by health services towards expectant mothers with these characteristics, by scheduling more prenatal visits, given that six is only the minimum recommended number of appointments [The results of this study showed that an insufficient number of prenatal visits was significantly associated with preterm birth . It should be taken into account that if a birth occurs prematurely, a lower number of visits may have occurred because there was not enough time for them to take place, particularly because appointments are scheduled with greater frequency starting in the 28C-section birth was performed in 79.7% of all deliveries in that period—much higher than the upper limit of 15% recommended by the World Health Organization . DespitePolynomial regression analysis showed that preterm birth has increased, and although it is possible to observe that factors related to prematurity are mostly decreasing, the sociodemographic improvement observed was not enough to reverse the scenario of prematurity in Maringá.In several developed countries, there are references to this same growing trend in prematurity, attributed in large part to obstetric interventions, such as elective C-section, assisted reproduction and increase in multiple births ,7. One sThe data presented herein show a growing trend in multiple pregnancies among women who had premature births, possibly due to earlier motherhood and the popularization of assisted fertilization procedures, particularly those featuring induced ovulation . ChildreAlso noteworthy is the increase in pregnancies among women 35 or older , a factor that may contribute to the increase in gestations with birth defects, considering that these pregnancies are often subjected to ovular stimulation, artificial insemination, in addition to frequently showing high blood pressure, diabetes and choice for elective C-section—all of which are risk factors for preterm birth.In that regard, pre-conception interventions, such as iron supplementation to prevent neural tube closing defects  and gestFrom the polynomial regression model, it was possible to detect that favorable vitality conditions at birth  have increased among premature births, which may be linked to improved preparation in delivery care, as well as the marked increase in moderately premature newborns.It is also necessary to consider the complexity that involves the occurrence of preterm births, in that prematurity may be associated with several other factors such as stress during gestation and maternal incidents, which were not analyzed in this study because they were not available in Sinasc; they require more profound population studies, including analysis of births according to prematurity stages at the local level.Knowing and understanding the complex process of preterm birth and its interfering factors is essential to provide quality assistance to the mother and child, as well as to perfect and rationalize the care given during all stages or the reproductive cycle, in order to prioritize actions of prevention, recovery and life maintenance, and furthermore, to direct and adopt preventive and curative measures in consonance with local reality .Some limitations of this study are noteworthy, such as those related to information obtained from secondary databases, subject to data completion and reliability. Another limitation concerns the impossibility of working with borderline prematurity, from 34 to <37 weeks, given the manner of data collection regarding gestational age by Sinasc—until 2009, it was categorized directly into intervals; only starting in 2010 did it come to be recorded as number of weeks of gestation .The present study evidenced a growing trend for preterm birth in a mid-sized city in southern Brazil, which occurred mainly in the moderate period. It is important to maintain health actions and programs that are showing a positive effect on very premature and extremely premature births in Brazil, but health actions and programs geared towards moderate preterm birth are necessary as well, as they concentrate the highest number of premature births.To that end, according to the results of this study, closer attention must be paid during the prenatal period to expectant mothers with multiple pregnancy and fetuses with birth defects, which have shown to be a risk for this unfavorable event.Also important is the need for further and more detailed population studies, including an analysis of preterm births and associated factors according to gestational age.


Occupation is a significant factor affecting life, health and well-being. Long-term military service is a unique career path that may have an influence on life expectancy, even after excluding obvious risks such as battlefield mortality. However, it remains unclear what the effects of a military career are on the life trajectory of personnel after retiring from service. This study compared life expectancy among retired military personnel (RMP) to their sex and birth cohort-specific reference populations.For this historical cohort study, we collected data on the sex, year of birth, year of death, time in service, and rank at end of service for 4862 Israeli RMPs. Data on reference populations were provided by the Israel Central Bureau of Statistics by birth decade from 1900 to 1989. We calculated the difference between each individual RMP’s age at death and the “expected” age at death, based on sex and birth cohort-specific means in the reference populations.P < 0.002), whereas no significant trend was identified among females. Length of service and rank were not associated with relative longevity for RMPs.Overall, 67.9 % of RMPs lived longer than average relative to their sex-specific birth cohort. This difference in life expectancy was more pronounced among women than among men. There was a significant trend of increasing differences between RMP males and reference males over time (The mechanism of the protective effect of military service on life expectancy remains unknown, but our findings indicate that it affects men and women differently, with women being more likely to benefit from the potential protective effect of military service. The healthy worker effect is known to vary from one occupation to another, and to the best of our knowledge, this is the first attempt to quantify the magnitude of the healthy worker effect among career military servicemen and women. Occupation is a significant factor affecting life, health and well-being. Several factors likely contribute to this situation, including income, environmental conditions and personal satisfaction and fulfillment , 2. The While the association between occupation and life expectancy may be complicated by self-selection, demographic and socioeconomic characteristics may affect opportunities for entry into certain careers. There is also evidence that occupation may causally influence life expectancy. For example, for black and white athletes a career in professional sports was found to substantially narrow the typical racial gap in life expectancy, a phenomenon attributed to the unique characteristics of this occupation .Long-term military service is another unique career path that may influence basic health outcomes such as life expectancy. After excluding obvious reasons for shortened life expectancy, such as battlefield injury and death, it is unclear what the effects of a military career are on the life trajectory of personnel once they retire from service. On one hand, some researchers have suggested that military service may serve as a protective factor for life course events such as education, family, earnings and post-service employment –8. DespiOn the other hand, military personnel, on average, spend less time with their families, have spouses who are less likely to work, and experience higher divorce rates . Some reThe aim of this historical-cohort study was to compare life expectancy among retired military personnel (RMP) to their sex and birth cohort-specific reference populations.Data on military personnel were provided by the Israel Defense Forces (IDF) veterans’ organization and included sex, date of birth, date of death, time in service, and rank at end of service. The veterans’ organization maintains ongoing contact and communication with RMPs, up to and including notification of death, at which time insurance benefits are paid to surviving family members. Thus, these data were deemed complete and reliable. Data on medical screening procedures prior to recruitment were not available. Rank was categorized as junior commissioned officer , senior commissioned officer , and noncommissioned officers (sergeant [OR-5] through chief warrant officer [OR-9]).Data on mean age at death in the reference population, stratified by sex and birth cohort, were provided by the Israel Central Bureau of Statistics. To limit the reference populations to those most appropriate for comparison to the RMPs, we excluded non-Jewish persons, individuals who died prior to the age of 20 and immigrants who completed fewer than 10 years of life in Israel before their death.RMPs and their reference populations were categorized by birth decades from 1900 to 1989. On initial descriptive analysis of the data, we discovered no deceased RMPs in the 1980–1989 birth cohort and only one RMP in the 1970–1979 birth cohort who had died by the time of the study. Thus, we limited our final analysis to the seven birth cohorts between 1900 and 1969.P values <0.1 on univariable analysis were included in the multivariable model. The Mann Kendall test for trend was used to examine age differences over time. Statistical analysis was carried out using IBM SPSS version 21.We conducted univariable and multivariable analyses to evaluate the association between military service and age at death. To facilitate this comparison, we calculated the difference between each individual RMP’s age at death and the “expected” age at death, based on sex and birth cohort-specific means in the general population. For logistic regression analysis, we included the predictor variables sex, rank, length of military service and birth cohort. Rank and length of service were introduced into the model as dichotomous variables , as these categorizations were found to provide the best goodness of fit and prevented collinearity. The outcome variable was defined as older or younger than the expected reference value. Variables with n = 4862) are presented in Table The characteristics of the RMPs included in the study (P < 0.02). These observations were not apparent in the respective birth cohorts in the general population. Overall, the mean age at death among the reference population was also higher for females than for males, although in the birth cohort of 1900–1909, the difference was reversed (difference = −0.59 years) and for the birth cohort of 1940–1949, the difference between the sexes was not significant. The absolute values of the sex differences in each reference birth cohort were substantially lower than in the respective RMP cohorts; the trend in difference widths did not maintain a linear progression over time (P > 0.20).Table P < 0.002), whereas no significant trend was identified among females.Table P < 0.001). There was a clear increase in the odds of relative longevity for RMPs in progressive birth cohorts with a dramatic increase for the latest birth decades , while among the reference population, the sex differences were generally stable and of a smaller magnitude. The fact that this sex difference narrowed over time in RMPs but not in the general population seems to indicate that this is a phenomenon unique to military careers.The difference in longevity between RMPs and their civilian reference populations was pronounced in young males (later cohorts) but diminished among older men (earlier cohorts). In contrast, the difference in females was prolonged and remained stable into older age. The mechanism of the protective effect of military service on life expectancy is unknown, but this finding seems to indicate that it affects men and women differently, providing shorter-term protection for males than for females. Alternatively, military service may provide better protection against causes of death that are more frequent among females than among males. However, this latter point could not be verified with the available data as this study was not designed to investigate specific causes of death.Younger military retirees had the greatest odds of outliving their civilian counterparts. These increased odds of longevity extended into older age but were substantially reduced with increasing age. This observation remained unchanged after adjusting for any changes in the male–female composition of the military over time. Thus, both sex and birth cohort remained independent predictors of relative longevity in RMPs relative to the general population.Females were more likely than males to benefit from the protective effect of military service. Because the recruitment screening process is identical for males and females, differential health selection of females is not a plausible explanation for this observation. Given that women generally outlive men, the phenomenon of a substantially longer life expectancy observed among female RMPs in this study may possibly be due to the military selection process, which favors healthy individuals. This selection process may enhance and amplify the expression of any anticipated life expectancy differences between sexes in this unique occupational group .Interestingly, neither rank nor long-term military service was associated with outliving civilian counterparts. This finding might indicate either that the protective effect of military service occurs in the earlier stages of a military career or that it is the result of a selective process, such as the medical, mental and cognitive screening inherent in recruitment procedures.The main results of this study are likely attributable to the healthy worker effect, first described some 150 years ago , in whicWe compared the mean age at death among military personnel to that of the Jewish population in Israel, excluding non-Jewish minorities such as Arabs and Druze. For females, there is no question of validity in the comparison, as nearly no non-Jewish females serve in the IDF. As for males, the vast majority of RMPs are Jewish, with nearly no Arabs and only a small proportion of Druze in service. We initially used a weighted standard population average to adjust the mean age at death of the overall population for the differences in life expectancy between Jewish and Druze males. This difference, while evident among earlier birth cohorts, was negligible among birth cohorts born after 1940. Due to the very small proportion of Druze in the overall Israeli population, the weighted averages were identical to those of the Jewish population, varying by no more than ±0.01 years. Thus, we adopted the simpler, more intuitive and identical population data over a weighted, calculated and indirect adjusted population average for purposes of comparison. In sensitivity analysis, no differences were found in our results.While our study design  precluded potential confounding effects by sex, religion, untimely deaths, immigration status and the cohort effect, we were unable to adjust for income level. Additionally, this study was limited to all-cause mortality, while future studies might investigate specific causes of death and their effects on the population of RMPs. Furthermore, we did not study the sex-specific attributes of additional “exposure” variables, such as the utilization of health care services, periodic health screening, medical discharge and working patterns during and after military service. Future studies might examine the effects of these characteristics on differential life expectancy.In this large historical cohort study of nearly 5000 career military personnel and over 1 million civilians, retired soldiers, sailors, airmen and airwomen were shown to outlive their peers in the general population. The magnitude of this benefit varied by sex and by birth cohort. Future studies should examine the differential effects of military service on specific causes of death in order to better understand the mechanisms of this occupational observation.


Cytokines are cell signalling proteins that mediate a number of different physiological responses. They are also biomarkers for inflammatory conditions and potential diagnostic references for diseases. Until recently, simultaneous quantification of cytokine profiles had not been possible. Now however, fluorescent microsphere immunoassays (FMIA) are able to measure multiple cytokines in a single sample. The following pro-inflammatory and anti-inflammatory cytokines were quantified in equine plasma and serum samples: interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α.•® 200 system in equine EDTA-plasma and serum.The objective of this study was to quantify six equine cytokines simultaneously using the BioPlex•It demonstrates an increased number of detectable cytokines over published studies.•This technology has the advantage of reduced sample volume and assay time compared to traditional sandwich ELISAs. Recently the advance of fluorescent-bead-based technology, based on the principle of a sandwich ELISA allows the use of multiplex assays known as a fluorescent microsphere immunoassay (FMIA) Recombinants, polyclonal capture and detection antibodies for the FMIA were purchased from R&D systems  and Genorise Scientific  . The mic6 microspheres) of fluorescently distinct microspheres were washed, then capture antibodies (Capture antibodies were coupled to magnetic microspheres using the Amine Bioplex Pro Magnetic Beads COOH coupling kit (BioRad cat #4110012C). For each cytokine, the relevant capture antibody was coupled following manufacturer’s instructions, using reagents from the Bio-Plex amine coupling kit (171-406001) except where specified. Briefly, 100 ml . Samples were centrifuged immediately at 4 °C at 800 g for 15 min and plasma was aliquoted  and stored at −80 °C. Frozen samples were sent on dry ice to SRUC Edinburgh for cytokine analysis.Blood was collected from 40 healthy male horses of different breeds before undergoing routine castration. The plasma was collected by jugular venipuncture into EDTA Monovette® instrument and analysed with the Bio-Plex Manager software version 6.1. During a run, the BioPlex Manager software produces a histogram and a bead map  at room temperature (RT) for 110 min. The plate was washed three times as before, then 100 μl of the detection antibody (in assay buffer) was added to the wells and the plate was incubated as before for 50 min. The plate was washed three times as previously, then 50 μl of 1× Streptavidin-phycoerythrin SAPE (BioRad) was added and the plate was incubated for 30 min as before. The plate was washed for a final three times, and then 125 μl of assay buffer was added. The plate was incubated with shaking for 5 min then the reaction was measured using a Bio-Plex 200bead map , which sThe equine multiplex was validated using a number of measures.For each cytokine the recovery of known amounts of cytokines  had to be within the 70–130% range to be included as successful detection n = 3) of seven recombinant cytokine standards with known concentrations during a single assay run and expressed as the coefficient of variation (CV (%)) of repeated measurements. Inter-assay variability was studied using seven different concentrations of standards, analysed in triplicate over three different days and expressed as the CV (%) of repeated measurements To verify the repeatability of the assay, dilutions of recombinant cytokine standards were prepared for each cytokine and used in triplicate in the FMIA over a 3-day period of time A multiplex FMIA was set up with standards containing all capture and detection antibodies and all recombinant cytokines. Unknown wells contained all capture antibodies and cytokine recombinants but only one detection antibody to test specificity for each matched pair of antibodies. Cross reactivity was defined as the percentage of non-specific, cross reacting signal detected relative to the specific signal for that analyte.This is defined as the lowest value at which a cytokine can be found below the predetermined standard value.This was the range over which cytokines could be detected and measured within the 70–130% recovery.The analytical sensitivities and the assay range for the detection of the equine cytokines were determined in the multiplex assay using serial dilutions of the standards of all cytokines for quantification. The range found for all the six were between 40.96 and 10,000 pg/ml, which is comparable to many ELISA cytokine kits. The sensitivity of detection varied for each cytokine . The repThe six-plex had very low cross-reactivity scores, showing that the antibodies used were specific for individual cytokines . AverageStandard curves were similar for single-plex and multiplex assays, with multiplex assays showing increased fluorescence for IL-4 and IL-6, however the reason for this is unclear .Cytokines are polypeptides or glycoproteins produced by many types of cells present at the sites of immune response. They mediate cellular interactions and regulate differentiation, proliferation and survival of immune cells by triggering the production of other cytokines, which can increase (pro-inflammatory) or attenuate (anti-inflammatory) immune response. Cytokines act mainly by paracrine and autocrine mechanisms. Thereby, cytokines are biomarkers for inflammatory conditions and potential diagnostic references for diseases. Hence, cytokines are widely studied and the ability to detect them has become increasingly important for researchers and clinicians.Cytokines can be found in a variety of body fluids. Cytokines have been quantified in serum as well as in plasma ® system to develop a multiplex assay to measure six cytokines  simultaneously in horse samples. The choices of the cytokines were based on previous studies regarding their role in pain and diseases Recently the use of FMIA compared to gene expression measurements is preferred in cytokine analysis. To get a better understanding of the functional status of the immune system the actual protein level should be measured, because post transcriptional and post translational modifications cause an imperfect correlation in mRNA and real protein synthesis The six-plex assay developed can also detect cytokines in serum however, the use of plasma is preferred in horses as in humans During development of the plasma assay there were some sampling errors reported by the Bioplex machine when neat plasma was used, due to the complexity of the plasma matrix, whether physical  or presence of interfering factors (heterophilic or autoantibodies) The production of new antibodies is time consuming and impractical for all laboratories to perform so studies are testing the cross-reactivity of currently available antibodies with equine cytokines of interest A concern about multiplexing is the potential of cross-reactivity between antibodies present, which could result in false or elevated detection results. This six-plex however gave very low cross-reactivity results  assay, however this has currently not be validated or optimised to detect equine cytokines.We have demonstrated a six-plex assay to simultaneously measure cytokines in plasma of horses. This assay could be used to examine the cytokine profile associated with a number of different disease states.


Faster growth after preterm birth benefits long-term cognitive functioning. Whether these benefits extend to mental health remains largely unknown. We examined if faster growth in infancy is associated with better self-reported mental health in young adults born preterm at very low birth weight (VLBW) (<1500g).As young adults, participants of the Helsinki Study of Very Low Birth Weight Adults self-reported symptoms of depression and attention deficit/hyperactivity disorder (ADHD) (n = 157) and other psychiatric problems (n = 104). As main predictors of mental health outcomes in linear regression models, we used infant weight, length, and head circumference at birth, term, and 12 months of corrected age, and growth between these time points. Growth data were collected from records and measures at term and at 12 months of corrected age were interpolated. Additionally, we examined the moderating effects of intrauterine growth restriction.Size at birth, term, or 12 months of corrected age, or growth between these time points were not associated with mental health outcomes . Intrauterine growth restriction did not systematically moderate any associations.Despite the high variability in early growth of VLBW infants, the previously described association between slow growth in infancy and poorer cognitive functioning in later life is not reflected in symptoms of depression, ADHD, and other psychiatric problems. This suggests that the development of cognitive and psychiatric problems may have dissimilar critical periods in VLBW infants. The cohort has been described elsewhere.[The original lsewhere. In 2004–lsewhere.,18: 166 lsewhere.,27,28 inAll participants gave their written informed consent as adults, and the Ethics Committee for Children and Adolescents’ Diseases and Psychiatry at the Helsinki University Central Hospital approved the study protocol. In order to protect the privacy of the cohort members, the data were anonymized during collection and provided to the researchers of the current study in a format where official personal identification numbers and other personal information such as names and addresses had been removed, and participants could only be identified using study identification codes.term , we interpolated between true measurements, provided a measurement had been made within 28 days. Median time period between term and closest true measurement point was 1 day for weight, 5 days for length, and 4 days for head circumference. We interpolated size at 12 months (52 weeks) CA if a measurement had been made within 42 days, allowing a wider range to increase sample size. Median time period between 12 months CA and closest true measurement point was 14 days for weight, and 15 days for length and head circumference.Weight, length, and head circumference measurements came from hospital and child welfare clinic records. To obtain measures at z scores by sex and age according to Finnish charts[z scores for length and head circumference, and a percentage score of current weight in relation to expected weight for sex and CA. Therefore, we converted length and head circumference at 12 months CA into standardized z scores by sex and age, whereas weight at 12 months was first converted into percentage scores for sex and age and thereafter, to facilitate comparison of effect sizes, into z scores within the VLBW cohort.We converted size at birth and at term into sh charts. Finnishsh charts,31 proviDuring the first clinical visit, the participants completed the Beck Depression Inventory (BDI) and the From medical records, we extracted sex , gestational age (weeks), date of birth for calculating age during the visit (years), time period between closest true measurement point and term /12 months CA (days), self-reported maternal smoking during pregnancy (yes/no), and neonatal complications/illnesses . 47 participants lacked data on intraventricular hemorrhage, and 10 participants lacked data on maternal smoking during pregnancy, and were considered separates groups when dummy coding the variables. None of the participants were diagnosed with necrotizing enterocolitis. During the first clinical visit, participants reported diagnosed neurosensory impairments  and highest education of either parent (basic /secondary /lower tertiary /upper tertiary).As main outcomes in linear regression models, we used BDI and CES-D , and APQ sumscores, and ASR Total Problems score and Internalizing and Externalizing Problems subscores . We standardized these outcomes by sex within the sample  to facilitate interpretation of effect sizes.As main predictors of mental health outcomes, we used infant growth in weight, length, and head circumference from a) birth to term and b) from term to 12 months CA. We used standardized residual change scores from linear regression models where a) body size z scores at term were regressed on the corresponding measure at birth and b) body size z scores at 12 months CA were regressed on the corresponding measure at term, creating uncorrelated residuals that reflect growth conditional on previous history.,38 In suTo control for covariates and confounders, we included gestational age at birth, sex, age during the visit in question, time period between closest true measurement point and term , and parental education (as a proxy of socio-economic background) in all analyses. In additional models, we further adjusted for neonatal complications/illnesses and maternal smoking during pregnancy, and excluded those with cerebral palsy (n = 13) and blindness (n = 2).We first performed the analyses in the whole VLBW group. We then tested if the effects of growth on mental health varied by SGA/AGA status. We did this by including the interaction term ‘SGA vs. AGA status for weight /length /head circumference x corresponding growth measure’ into the regression equation followed by main effects. All participants were <+2SD in birth weight and head circumference. The two participants scoring >+2SD in birth length were classified as AGA for length.Data on participants’ growth in infancy and on neonatal and adult characteristics and mental health outcomes are presented in Sample size varied according to growth data availability . We foungrowth from birth to term and from term to 12 months CA were associated with adult mental health outcomes. Firstly, we tested if body size at birth, at term or at 12 months CA  . Faster We show that self-reported symptoms of depression, ADHD, and other psychiatric problems in young adulthood were not associated with growth from birth to term or from term to 12 months CA after preterm birth at VLBW. We conducted 36 analyses examining associations between growth in weight, length, and head circumference during two time periods, and mental health questionnaire scores, and 54 analyses examining associations between body size at birth, at term, and at 12 months CA, and mental health questionnaire scores, and found no statistically significant effects. Further, we examined 36 interactions with intrauterine growth restriction, and found only two statistically significant interactions. In sub-analyses following one of them, we found statistically significant main effects: faster growth in head circumference from birth to term in those born SGA was related to higher depressive symptoms in young adulthood.Our findings are contrary to what we expected based on previous literature, which has however concentrated on cognitive functioning as the long-term outcome. These studies have shown that faster growth after preterm birth may provide long-term benefits for intelligence and executive functioning–22. Our Our findings are in agreement with the two previous studies that we are aware of, which reported that growth in head circumference during the first two years of life after preterm birth at extremely low birth weight was not associated with parent- or teacher-reported ADHD symptoms in childhood, and weiWe did not find that the effects of growth on mental health among the preterm VLBW group would vary systematically by intrauterine growth restriction, as reflected by AGA/SGA status, either, and cannot rule out that the few significant associations reflect type 1 error. This finding is in agreement with our own previous reports on cognitive functioning and autiIn addition to neurocognitive outcomes, earlier studies have shown increased cardiometabolic risk factors such as higher blood pressure and impaired glucose regulation in adults born preterm. In relain utero: associations between growth and mental health in term-born populations, and conclusions about potential differences between preterm and term-born individuals were beyond the scope of our study. Finally, the participants in this study were born between 1978 and 1985, and thus the findings may not be representative of preterm infants born today in high-income settings, whose postnatal care and nutrition have much improved, and who grow on average substantially faster than the infants we have studied. This is an unavoidable limitation, when examining adult outcomes. This can also be considered a study strength, as the group showed high variability in early growth, and thus provided a unique opportunity for studying variation in early growth.Strengths of this study include the longitudinal study design, extensive early life and adult mental health data, and the use of well-validated mental health questionnaires. Limitations include sample size: while these 157 and 104 adults provide insight into the associations between early growth and mental health in the VLBW population as a whole, postnatal growth may be associated with adult mental health in some VLBW individuals  and notIn conclusion, we show that growth in infancy after preterm birth at VLBW seems largely unrelated to self-reported symptoms of depression, ADHD, and other psychiatric problems in young adulthood. We also show that intrauterine growth restriction, as reflected by SGA and AGA status, did not systematically moderate effects of growth. Further studies are clearly warranted that unravel factors explaining why some individuals born preterm at VLBW are rendered vulnerable to mental disorders, while others remain resilient.S1 TableChange in ASR Internalizing and Externalizing subscale scores (in SD units) per one SD faster growth in weight (Panel A), length (Panel B), and head circumference (Panel C) during two time periods in infancy: birth to term, and term to 12 months CA, in individuals born at very low birth weight (<1500g).(PDF)Click here for additional data file.S2 TableChange in mental health questionnaire scores (in SD units) in young adulthood per one unit difference in relative weight (Panel A), length (Panel B), and head circumference (Panel C) at birth, at term equivalent age, and at 12 months of corrected age in individuals with very low birth weight (<1500g).(PDF)Click here for additional data file.S3 TableChange in adult mental health questionnaire scores (in SD units) per one SD faster growth in weight (Panel A), length (Panel B), and head circumference (Panel C) during two time periods in infancy: birth to term, and term to 12 months CA. Results are shown separately for small-for-gestational-age (SGA) and appropriate-for-gestational-age (AGA) individuals, who were all born preterm at very low birth weight (<1500g).(PDF)Click here for additional data file.


The study assessed birth trends per decade in offspring of females with inflammatory joint diseases (IJD) compared with women without IJD.n = 7502) and compared with births from the general population (n = 2 437 110). Four periods were examined: 1967–79, 1980–89, 1990–99 and 2000–09. In the logistic regression analysis adjustments were made for maternal age at delivery and birth order. Odds ratios were obtained for the associations between IJD and birth outcome for each period.This retrospective cohort study is based on data from the Medical Birth Registry of Norway from 1967 to 2009. We investigated singleton births in females with IJD  in the 1970ies and 274 deliveries / year  from 2000 to 2009. Adjusted Odds ratios (aOR) for newborns small for gestational age were 1.5  in the earliest and 1.1  in the last period. Correspondingly, for birth weight < 2500 grams aOR decreased from 1.4  to 1.1 . For preterm birth aOR was 1.1  in the first and 1.3  in the last period.An increasing number of births among females with IJD were observed in the study period. Birth weights of newborns of IJD women approached to birth weights in the general population, but preterm birth remained a problem.The online version of this article (doi:10.1186/s12884-016-0919-7) contains supplementary material, which is available to authorized users. Inflammatory joint diseases (IJD), here including rheumatoid arthritis (RA), spondylarthritis (SA), psoriatic arthritis (PsA) and juvenile idiopathic arthritis (JIA) are characterised by chronic, systemic inflammation and may also affect internal organs. Previous studies have shown various effects of IJD on birth outcome –4. EspecIn the present study we wanted to assess secular trends in the number of births and the effects of IJD on preterm birth and birth weight in the newborns. The data were provided by the Medical Birth Registry of Norway from 1967 to 2009.The Medical Birth Registry of Norway (MBRN) is a population-based registry containing information on all births in Norway since 1967 comprising more than 2.4 million births 1967–2009. MBRN is based on compulsory notification of every birth or late abortion from 12 completed weeks of gestation onwards. Since 2002 MBRN has been organised under the Norwegian Institute of Public Health. A standard antenatal notification form is filled in at visits to a general practitioner, midwife or obstetrician during pregnancy and is brought by the mother to the place of birth. The midwife enters additional data recorded at the time of birth. Follow-up data are added to the form until discharge of hospital births. The attending midwife is responsible for the completion of the notification form at the end of each birth. The form is co-signed by the attending physician and sent to the MBRN within one week after birth. In the period January 1st 1967 to November 30th 1998 a notification form was used for all live births and stillbirths after 16 weeks of gestation. From December 1st 1998 a new and more comprehensive form was used for all live births and stillbirths after 12 weeks of gestation [Until December 1st 1998 diagnoses were recorded as stated by the International Classification of Diseases (ICD)-8 system. Afterwards the ICD-10 system has been used. It provides differentiation of the various diagnoses of IJD. Information on diagnoses was obtained from the patient’s doctor and from medical records. We have included data from females with IJD who gave birth in the period 1967 – 2009. The diagnoses of IJD were based on free text information in the birth registration form, as coded by MBRN using the ICD-8 or ICD-10 system, respectively.The study was performed in compliance with the Helsinki Declaration and approved by the Regional Ethics Committee of Central Norway (diary number 2011/127-3).We included anonymised cases with coding 712 in ICD-8, comprising rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA) psoriatic arthritis (PsA) and ankylosing spondylitis (AS). When the ICD-10 system was introduced we used M05.0, M05.1, M05.8, M05.9, M06.0, M06.8, M06.9 (RA), M45 (AS), M46.1 (spondylarthritis), L40.5 and M07.0, M07.1, M07.2 or M07.3 (PsA) and M08.0, M08.1, M08.2, M08.3, M08.4, M08.8, M08.9 (JIA). The different diagnoses within the IJD complex therefore had to be analysed as a collected group to obtain a comparison of the same diseases. We excluded unspecified arthritis, inflammatory connective tissue diseases and vasculitis in the selection of IJD patients  for the newborn, the proportion of newborns with BW < 2500 gram and the proportion born small for gestational age (SGA). SGA was defined as BW < 10 percentile for expected BW for gestational age. Gestational age at birth was based on ultrasound before 20 weeks or computed by the last menstrual period. Birth before 37 + 0 gestational weeks was defined as preterm.Births for females with IJD and reference deliveries were stratified into four periods: 1967–79, 1980–89, 1990–99 and 2000–09. Comparison of groups was studied by Student’s t-test for continuous variables and chi-square tests for categorical variables. Logistic regression analysis was used to assess associations between IJD and perinatal outcomes. We adjusted for parity and maternal age at delivery. Multivariable linear regression analysis was used to analyse mean differences in BW of the newborns of mothers with IJD and reference women. The explanatory variables were maternal age at delivery, gestational age, gender of infant and parity , 11. Valn = 2736 births) when detailed IJD diagnoses were available through ICD-10, the distribution of diagnoses was: RA 51 %, SA 32 %, PsA 10 % and JIAFrom 1967 to 2009 a total of 7502 births were registered in females with IJD forming 0.3 % of totally 2.4 million births in Norway in the period. In the earliest period, 1967–1979, the births in females with IJD constituted 0.08 % of all births. This increased to 0.5 % in 2000–2009. Correspondingly, in average 65 deliveries annually were registered in females with IJD during 1967–1979, increasing to 274 each year after year 2000. In 2000–2009 (7 %. The mean age at delivery for females with IJD was higher than among the reference women through all periods, and parity was higher from 1990 onwards. In the period 2000–2009 more females with IJD were smoking at time of conception than among the references Table . The risSince 1967 births among females with IJD have significantly increased. The low number of births during the earliest period may have several explanations. In accordance with previous reports females with IJD may have been advised not to have children . After 1We observed that the mean BW in newborns of IJD women approached that of the reference women during the study period, and the mean difference was no longer statistically significant after year 2000. The proportion of newborns with BW <2500 grams declined from 6 % during 1967 – 1979 to 4 % after year 2000 in females with IJD. Correspondingly, the SGA proportion declined from 17 % to 8 %. Interestingly, these declines coincide with the introduction of better treatment options. Low BW has been associated with high disease activity at conception and during pregnancy , 16 and An advantage of the present study was the availability of population based data from a national registry. This limits selection bias of IJD cases and ensures representative references.Among potential limitations we cannot exclude the possibility of misclassification bias. However, a validation study of a selection of rheumatic diagnoses in MBRN including inflammatory arthritic diagnoses, reported that 97 % of the diagnoses were correct . We exclAn increasing number of births among females with inflammatory joint disease have been observed between 1967 and 1999. Birth weight was lower in newborns of mothers with inflammatory joint disease than in the general population until 1999, but not later. However, an increased preterm birth rate seems to persist.aOR, adjusted Odds ratio; BW, birthweight; CI, confidence interval; ICD, international classification of diseases; IJD, inflammatory joint disease; JIA, juvenile idiopathic arthritis; MBRN, medical birth registry of Norway; PSA, psoriatic arthritis; RA, rheumatoid arthritis; SA, spondylarthritis; SD, standard deviation; SGA, small for gestational age; TNF, tumor necrosis factor.


Cardiovascular magnetic resonance (CMR) can through the two methods 3D FLASH and diffusion tensor imaging (DTI) give complementary information on the local orientations of cardiomyocytes and their laminar arrays.Eight explanted rat hearts were perfused with Gd-DTPA contrast agent and fixative and imaged in a 9.4T magnet by two types of acquisition: 3D fast low angle shot (FLASH) imaging, voxels 50 × 50 × 50 μm, and 3D spin echo DTI with monopolar diffusion gradients of 3.6 ms duration at 11.5 ms separation, voxels 200 × 200 × 200 μm. The sensitivity of each approach to imaging parameters was explored.v1ST), intermediate (v2ST) and least (v3ST) extended orthogonal directions of signal continuity. The DTI data was analysed to determine the most (e1DTI), intermediate (e2DTI) and least (e3DTI) orthogonal eigenvectors of extent of diffusion. The correspondence between the FLASH and DTI methods was measured and appraised. The most extended direction of FLASH signal (v1ST) agreed well with that of diffusion (e1DTI) throughout the left ventricle (representative discrepancy in the septum of 13.3 ± 6.7°: median ± absolute deviation) and both were in keeping with the expected local orientations of the long-axis of cardiomyocytes. However, the orientation of the least directions of FLASH signal continuity (v3ST) and diffusion (e3ST) showed greater discrepancies of up to 27.9 ± 17.4°. Both FLASH (v3ST) and DTI (e3DTI) where compared to directly measured laminar arrays in the FLASH images. For FLASH the discrepancy between the structure-tensor calculated v3ST and the directly measured FLASH laminar array normal was of 9 ± 7° for the lateral wall and 7 ± 9° for the septum (median ± inter quartile range), and for DTI the discrepancy between the calculated v3DTI and the directly measured FLASH laminar array normal was 22 ± 14° and 61 ± 53.4°. DTI was relatively insensitive to the number of diffusion directions and to time up to 72 hours post fixation, but was moderately affected by b-value . Optimal DTI parameters were b = 1000 mm/s2 and 12 diffusion directions. FLASH acquisitions were relatively insensitive to the image processing parameters explored.The FLASH data showed laminar alignments of voxels with high signal, in keeping with the presumed predominance of contrast in the interstices between sheetlets. It was analysed, using structure-tensor (ST) analysis, to determine the most  contains supplementary material, which is available to authorized users. Myocyte orientation: mean orientation of aggregated myocytes within a local spatial regionSheetlet: localized sheet-like aggregations of myocytes ~ 6-cells thick extending as curved branching planesSheetlet-interstices: gaps between adjacent sheetlets which exist as potential spaces in vivo and open up on fixation. Collagen structure differs within sheetlets and adjacent to sheetlet interstices, and there is evidence that sheetlet-interstices function as shear layers in vivo nFI|, and defined as |cos−1 [v3ST/e3DTI].nFI)|. By definition 0° ≤ | [v3ST/e3DTI].nFI| ≤ 90°. In the lateral ROI, the distribution of the ST vector difference |∠v3STnFI| is narrow, unimodal and centered close to 0°, while the DTI vector difference |∠e3DTInFI| is broader, unimodal and centered on 22.5°. In the septal ROI, the ST vector difference |∠v3STnFI| is bimodal with a narrow dominant mode centered near 0° (90.7% of voxels) and a small second mode centered near 90° (9.3% of voxels). The DTI vector difference |∠e3DTInFI| is also bimodal but with a broad dominant mode centered near 65° (65.1% of voxels) and a small second narrow mode centered on 15° (29.1% of voxels). This data is summarized in Additional file In Figure 2 and λ3; a further 21% of voxels have less than 10% difference between λ2 and λ3; i.e. in 30% of voxels λ3 is at least 85% of λ2). The DTI median (±IQR) and mean (±SD) difference between laminar eigenvalues  are 14.1% ± 9.8% and 13.8 ± 6.0% respectively. This corresponds to a median difference of 77.8% ± 23.9% and a mean difference of 72.8% ± 18.7% for ST. The sets of eigenvalues which correspond to the myolaminae are not the same for DTI and ST . An implication of this much greater separation of ST laminar eigenvalues than DTI laminar eigenvalues is that misclassifications of e3DTI and e2DTI are more likely than misclassifications of v3ST and v2ST.Eigenvector misassignment (missorting) is the assignment of an eigenvector to the incorrect structural feature due to imaging noise and small differences in eigenvalue magnitudes. In order to explore whether ST or DTI eigenvector misassignment was a source of error in myolaminar measurement the distributions of eigenvalue ratios from the lateral ROI were examined Figure . Distribv3ST and e3DTI to nFI is limited as the FI method requires interactive segmentation and visualization and was carried out on four equatorial ROI of one rat heart. The analysis was extended to a series of 5 hearts from age matched rats in Figure The comparison of v3ST, β’v3ST, β”v3ST; and e3DTI, β’e3DTI, β”e3DTI) and the sheetlet in-plane vectors and angles are explored in rose diagrams for the septal ROI from one heart in Figure v3STe3DTI| has a spread out distribution skewed towards lower values, (median ± MAD: 27.9 ± 17.4°). The same pattern is observed in the derived sheetlet-normal elevation angles |∠β’v3STβ’e3DTI|, but the differences are less marked. It can be seen that the general properties of the angle difference of the vector are also observed in the distributions of the elevation and transverse angles. The patterns described for the septal ROI are also observed in the lateral ROI ; the putative sheetlet in-plane vector elevation angle |∠β’v2STβ’e2DTI| (ii) and transverse angle |∠β”v2STβ”e2DTI| (iii); angle between v3ST and e3DTI (iv); the putative sheetlet-normal vector elevation angle |∠β’v3STβ’e3DTI| (v) and, transverse angle |∠β”v3STβ”e3DTI| (vi). Corresponding data for the posterior and anterior ROI are shown in Additional file v1ST and e1DTI and the angles of local myocyte orientation, and this is discussed in the section ‘DTI and ST sensitivity analysis’). The general patterns described for the septal ROI in Figure e3DTI with respect to v3ST are also observed, but to a lesser degree, in the lateral, anterior and posterior ROI  is 15 ± 10°  and the maximum value of |∠v3STe3DTI| is 30 ± 15°. This analysis  includes all ST and DTI 200 μm voxels without filtering based on the simplicity/complexity of the contained laminar structure.In Figure e1DTI and v1ST can be compared. This is because it is not possible for FLASH to resolve individual cardiac myocytes at 50 × 50 × 50 μm3 resolution. Therefore the putative myocyte orientation vectors e1DTI and v1ST are compared to each other. The first part of this comparison is to evaluate the basis for eigenvector assignment (in the same manner as was carried out for laminar eigenvectors in section ‘2) in Figure e1DTI from the other DTI eigenvectors than in sorting v1ST from the other ST eigenvectors, as e1DTI is a primary eigenvector. This is not the case as there is greater difference between λ2ST and λ3ST than between λ2DTI and λ1DTI. The median difference between λ2ST and λ3ST is 61.1% ± 28.1%. This compares to a median difference of 23.9% ± 11.5%. As discussed above, the sets of eigenvalues which are relevant to assignment of the local myocyte orientation are not the same for DTI and ST . An implication of this greater separation of the eigenvalues relevant to v1ST assignment (λ2ST and λ3ST) than the eigenvalues relevant to e1DTI assignment (λ2DTI and λ1DTI), is that misclassification of e1DTI and e2DTI is more probable than misclassification of v1ST and v2ST. There is no absolute cut-off for the median difference between eigenvalues which is acceptable to allow confidence in the DTI or ST assignment of the putative local myocyte orientation (m). The order of the eigenvalue sets from greatest difference to least difference is: (i) λ2ST with λ1ST; (ii) λ3ST with λ2ST; (iii) λ2DTI with λ1DTI; and, (iv) λ2DTI with λ3DTI.Unlike the case of myolaminar orientation, there is no method to directly determine local myocyte orientation from the FLASH data against which e1DTI) follows the familiar smooth transmural change in orientation from large positive angles at the endocardium (+65° ± 5°) to negative angles at the epicardium (−65° ± 20°) [’ in the septal-apex, as identified with the black square in Figure e1DTI and α”e1DTI are visualized in the same equatorial short-axis slice for 5 hearts showing that: (i) α’e1DTI and α’v1ST are similar within individual rats; (ii) α”e1DTI and α”v1ST are similar within individual rats; (iii) α’ and α” are consistent between rat hearts, whether measured by ST (v1ST) or DTI (e1DTI); (iv) both α’e1DTI and α”e1DTI are very similar to rat α’e1DTI and α”e1DTI reported in the literature [v1ST to α’e1DTI in the equatorial slices  . The smocardium +5° ± 5° tcardium +5° ± 5° tterature . As obsecardium +5° ± 5° tcardium +5° ± 5° te1DTI and v1STI follows the classically described pattern [e1DTI and α’v1ST are remarkably similar, as are the profiles for α”e1DTI and α”v1ST. Greater angular standard deviations for ST v1 than for DTI e1 for both α’ and α” are a consequence of the greater noise apparent in the slice images in Figure v1ST – α’e1DTI| calculated for each voxel. This measure is particularly appropriate for this study where DTI and ST describe precisely the same voxel matrices. Perfectly matched measures will have a profile parallel to the x-axis at 0°. The pair-wise difference in α’ is small in the sub-endocardium and mid-myocardium: 5-10° ± 15°, increasing in the sub-epicardium: 15° ± 15° in the sub-epicardium. In Figure v1ST (5.3° ± 3.6°) and α’e1DTI (5.2° ± 2.7°) and of α”v1ST (3.1 ± 7.0) and α”e1DTI (5.7 ± 9.0). The distribution of α’ is spread across the −90° to +90° range, with a higher frequency of angles close to 0° for both ST and DTI. The distribution of α” is unimodal, centered close to 0° and predominantly in the range −22.5° to +22.5°.The qualitative comparison of local myocyte orientation in five hearts in Figure  pattern  and the  pattern  and the v1ST: e1DTI; α’v1ST: α’e1DTI and α”v1ST: α”e1DTI are explored in quadrant rose diagrams in Figure v1STe1DTI| are unimodal and narrow in the septal ROI (median ± MAD: 13.3° ± 6.7°) and in the lateral ROI (median ± MAD: 12.6° ± 5.9°). The distributions of |∠α’v1ST:α’e1DTI| have the same form, being unimodal and narrow in the septal ROI (median ± MAD: 8.5° ± 5.6°) and in the lateral ROI (median ± MAD: 9.1° ± 5.8°). Likewise, the distributions of |∠α”v1ST:α”e1DTI| also have the same form, being unimodal and narrow in the septal ROI (median ± MAD: 11.5° ± 7.8°) and in the lateral ROI (median ± MAD: 15.6° ± 11.1°). In the septal ROI the distributions of |∠v1STe1DTI|  are therefore in contrast to the distributions of |∠v2STe2DTI| and |∠v3STe3DTI| , the latter having greater bias and variation. This pattern is also seen in the lateral ROI in Additional file The comparative deviation angles of distributions of DTI and ST sensitivity analysis) and in Additional file 2 and 12 diffusion directions with post-fixation time (up to 72 hours) not being an important factor. ST was not sensitive to image processing parameters in the range explored.A series of imaging experiments carried out to explore sensitivity of ST and DTI to imaging parameters are presented in the Digital Supplement . Our approach has the benefit that: (i) direct comparison of DTI to FI method assumes no cardiac model of local myocyte orientation or of the relationship between local myocyte orientation and myolaminar orientation; and, (ii) no registration is required as FLASH imaging and DTI imaging are carried out sequentially without moving the heart, and using coincident CMR imaging matrices. Unlike all previous methods of validating DTI myolaminar measurements the method we use does not rely on first estimating n through prior knowledge about m. We directly measure the 3D orientation of the laminar normal from images (nFI) and compare this directly to the putative measure of sheetlet normal orientation e3DTI. The rationale for this approach is firstly that it is simple and secondly that it follows directly from the initial description of sheetlets (i.e. from examining images). This simple approach was possible as the 3D myolaminar structure is directly visible and well-defined in the contrast-enhanced FLASH [This approach of measuring DTI myolaminar orientation performance and sensitivity referenced to direct measurement in FLASH (the FI method) has several advantages over previously adopted methods. This is discussed in more detail in the Digital Supplement  [b-values, i.e. b-value < 1000s/mm2, where the b-value is defined below) the fast-component (vascular/IC) predominates, but this component still influences measurements at higher b-values. Blood vessels generally run parallel to local myocyte directions [Physical studies have shown theoretical and experimental evidence that the DTI model has shortcomings which may limit its application in the measurement of cardiac orthotropy ,23,41. Aartment) ,23. Thesartment) . In imagrections , and it rections . Howeverrections ,40.g is the strength of the diffusion sensitizing gradient pulses, δ is the duration of the diffusion gradient pulses, Δ is the time of separation between diffusion gradient RF pulses. b has the units of s/mm2 . The b-value (b) was introduced and defined by  [2, by variation of the strength of the diffusion gradient pulses (g) at fixed gradient pulse duration (δ = 3.6 ms) and fixed pulse separation (Δ = 11.5 ms), hence increasing the sensitivity to diffusion, but not changing the spatial scale over which the tissue anisotropy is probed by diffusion [2 and therefore TE and Δ must be shortened significantly to acquire sufficient signal [2 mapping of formaldehyde fixed rabbit myocardium a diffusion gradient separation of Δ = 12.6 ms was determined to be appropriate for balance between SNR and distinct DTI eigenvalues [2 and λ3 populations [−3 mm2/s [in vivo human clinical myocardial DTI [ex vivo unperfused unfixed calf myocardium and showed that the difference between λ1 and λ2 and the difference between λ1 and λ3 increase when Δ is increased from 34 ms to 800 ms. This results in greater overall myocardial anisotropy indices at higher Δ, as measured by the fractional anisotropy index, the kurtosis index and the lattice index. The change in the eigenvalue ratios was observed between Δ = 34 ms and Δ = 100 ms, with minimal change above Δ = 100 ms. The Kim et. al. study, however, did not report any change in the ratio  of λ2 to λ3 and did not report any change in the orientation of e2DTI or e3DTI . Although it has not been demonstrated in an experimental study it remains likely from theoretical considerations that the sensitivity of DTI to measuring sheetlet orientations (and hence the orientations of e2DTI or e3DTI) will change with changing Δ. Similarly, it is possible that the low Δ imposed by the process of fixing tissue may limit the DTI measurement of sheetlet orientation in fixed myocardium, and that a wider range of structural features may be resolvable in un-fixed myocardium by the wider possible range of Δ values. Detailed direct 3D evaluation of the performance of DTI in measuring the sheetlets requires a structural method that can directly resolve sheetlet structure (i.e. by a method other than diffusion). Current applicable methods all require tissue fixation, and therefore only a limited range of Δ can be explored. Given these considerations, this study evaluates the performance of DTI in measuring sheetlet orientation in fixed hearts, which is a commonly performed experimental method used for myocardial structural measurement. The results of this assessment of DTI cannot (or can only to limited extent) be extrapolated to DTI sheetlet orientation measurement in un-fixed or in vivo tissue where longer Δ times are commonly employed.The b-value is a factor of diffusion weighted sequences which summarizes the influence of the sensitivity of the image to the diffusion gradients. As such the b-factor characterizes the extrinsic (sequence-based) imaging contrast determining factors and higher b-value corresponds to greater strength and duration of the diffusion gradients. With higher the b-value the stronger the influence of diffusion weighting on the image, but also the longer the gradients are applied, and the longer the duration of diffusion over which the tissue is probed.., 1986) . The mos., 1986)  and on tiffusion . The grat signal ,47. Baseulations . No publ−3 mm2/s , so in ftion Δ = .5 ms, he−3 mm2/s  for unfidial DTI ,49. Kim dial DTI . imaged DTI and FLASH/ST both represent myocardial structure as a tensor and therefore both methods have inherent limitations in measuring myolaminar architecture in voxels which have 2 or more laminar orientations. The overall myolaminar orientation is simplified to a single laminar normal orientation vector. If a voxel contains two markedly different myolaminar orientations  this will be quantified as a sheetlet-normal vector in a non-physical intermediate orientation (0° for this example case). This limitation is not inherent in the FI method, as a distribution of orientations is obtained for each 200 μm isotropic voxel, however in practice it is necessary to simplify this measure to a single  of local myolaminar orientations. This limitation of DTI and ST could be addressed by increasing resolution. There are literature reports on DTI in cardiac tissue with up to 100 μm isotropic resolution , but as There has been ongoing controversy around the presence and nature of cardiac laminar architectural features . Most suin vivo contractile state [It is inevitable that fixation of hearts before imaging will result in some conformational change, such that the fixed structural state of the heart will not truly match an le state . The fix3 spatial resolution of the FLASH. The reliability of DTI sheetlet orientation measurement  compared to directly measured sheetlets varied depending on cardiac location: in the lateral ventricular wall the deviation angle (|∠e3DTInFI|) was 23 ± 14° (median ± IQR) and in the septal wall deviation angle was 61 ± 53.4°. ST/FLASH is more reliable as in the lateral wall deviation angle was 9 ± 7° and in the septal wall 7 ± 9°. Furthermore, we show erroneous assignment of e3DTI and e2DTI, in other words where the orientation assigned by DTI to the laminae sheetlet in-plane direction is actually much closer to the true laminar normal orientation. This was explored through assessment of the relative laminar eigenvalue magnitudes . DTI showed considerably less accuracy than ST when compared to the FI sheetlet normal orientation in both the lateral and septal ROI. In the septal ROI there was strong evidence of eigenvector missorting as the median difference between the DTI and FI laminar normals was 54.4 ± 47.3° and the deviation angle distribution was bimodal with the larger mode centered on 65° and including 65.1% of the lateral ROI voxels . The consequences of eigenvector misassignment are substantially deleterious to the accuracy of any imaging method that measures a tensor to encode structure, as the orthogonal system is rotated so as to produce a measure of the laminar-normal as different as possible to the correct values.Using the FI method we showed that the quality of the FLASH is adequate to clearly define the sheetlet normal orientation through visualization, and we demonstrated that the ST method was appropriately applied. The raw FLASH images .We showed that under the imaging parameter ranges explored in fixed myocardium there are greater grounds for sorting between the ST laminar eigenvectors than the DTI laminar eigenvectors. This was anticipated as: (i) ST is, from first principles, a method highly appropriate to quantifying laminar architecture in images where that structure is clearly visible on simple inspection; (ii) underlying myocardial diffusion has previously been shown to be complex, and the DTI model has limitations for describing this diffusion. We do not show that eigenvalue differences are insufficient to allow laminar measurement, but we show that there is little difference between the magnitudes of sheetlet eigenvalues and sheetlet-normal eigenvalues, and that e3DTI and v3ST differed from region to region, and overall there was poor agreement. Likewise we showed qualitatively and quantitatively that the degree of agreement between e2DTI and v2ST differed from region to region, and overall there was poor agreement . In this way we showed that the fixed-myocardium DTI secondary and tertiary eigenvector orientations are influenced by the underlying laminar architecture, but the assignment of these vectors to either orientation is not robust, and varies depending on cardiac location.The ST sheetlet angle and sheetlet normal angle maps largely recapitulate features described in the literature , v2ST in the plane of local consistent contrast, and v1ST in the direction of minimum contrast change. The FI method is only influenced by the local normal. It was noted that there was some eigenvalue misassignment in ST compared to FI. The probable reason for this is that unlike FI and DTI, in which just a single 200 × 200 × 200 μm3 voxel influences the local measured myolaminar normal orientation, in ST neighboring voxels also influence the tensor calculation . For at DTW = 3 each structure tensor component in the 200 × 200 × 200 μm3 representation has worked with intensities gathered from an effective 9 × 9 × 9 template at the original 50 μm resolution, i.e. around 450 × 450 × 450 μm3. However, the principal data weighting is at the center of that template. This will have minimal consequences when myolaminar structure is spatially conserved, but in regions of high-spatial change in laminar orientation then the neighbor influence will result in a different measure of laminar structure from ST/FLASH and from FI/FLASH.There is good agreement between the ST quantified laminar normal orientation and the FI quantified normal orientation . The mechanisms responsible for the systematic deviation between ST and the FI method based on FLASH have not been determined but it is likely associated with differences in the methodology used in the FI method to ST. ST is a tensor method so is influenced by the optimal orthotropic description of local contrast change . ST of entation , then, ientation , and thiIt was observed that for both DTI and ST α” did not have a simple circumferential pattern, but rather had high negative values in the anterio-lateral myocardium  [Here the term high-resolution is applied to FLASH CMR in the context of the whole heart geometry and with respect to clinical CMR, not with respect to the myocyte/sheetlet dimensions. The current study investigates conventional DTI on 200 μm isotropic voxels and b-values of 1000 s/mm (HARDI) ) may perex vivo cardiac tissue [in vivo or when applied to ex vivo physiologically perfused tissue, and the absolute values of sheetlet orientations may not reflect in vivo values. The high spatial resolution FLASH imaging requires ~18 hrs to image a rat heart and therefore requires cardiac fixation. As discussed in the section ‘b-value, diffusion gradient separation and DTI Imaging protocol used in this study’ above, fixation shortens myocardial T2 and therefore imposes short DTI TE and diffusion gradient pulse separation (Δ) compared to parameter ranges possible in unfixed myocardium. A consequence is that more accurate measurement of sheetlet orientation may be possible in unfixed myocardium than can be achieved in fixed myocardium. Therefore our conclusions on the accuracy of DTI sheetlet orientation measurement cannot be extrapolated to DTI in unfixed or in vivo myocardium.This study explores DTI and FLASH in fixed myocardium perfused with gadolinium (Gadopentetate Dimeglumine). Many DTI studies have explored structure in fixed c tissue ,60,64,65c tissue . It is n2 and 12 diffusion-directions with time post fixation up to 72 hours not being an important factor. ST was not sensitive to image processing parameters. FLASH and ST requires more time (~1 day) compared to DTI (~3 hours). However, FLASH directly resolves myolaminae and FLASH/ST more accurately quantifies the orientation of these structures. We conclude that the FLASH/ST framework is reliable, robust and the preferred option for myolaminar measurement in fixed myocardium. DTI has an important role for local myocyte orientation measurement, and a method could be developed for combining the optimal characteristics of ST and of DTI. The methods developed and assessed in this study will be useful in future development and refinement of diffusion CMR of cardiac structure, both in vivo and ex vivo. Future studies are required to quantify the accuracy of structural orientation measurement by DTI in unfixed myocardium (either in vivo or with ex vivo perfused hearts) against direct 3D imaging measurements.DTI and ST both produce tensors whose eigenvectors correspond to a greater-or-lesser degree with the cardiac orthotropic structural axes. DTI and ST predict globally similar myocyte orientations, and this evidence supports using DTI to measure local myocyte orientation. DTI produces smoother local myocyte orientation maps and is faster for imaging local myocyte orientation but with appropriate regularization, ST is likely to be useful for this purpose also. In fixed myocardium ST is a better measure of myolaminar orientation than DTI over the parameter ranges explored. The reliability of DTI sheetlet orientation measurement compared to directly measured sheetlets was low or markedly low depending on cardiac location and we showed that poor DTI performance over this parameter range could be a consequence of poor laminar eigenvector assignment. Sensitivity analysis showed that overall DTI sensitivity to time post fixation is low, to b-value is moderate (with fixed diffusion gradient separation time of Δ = 11.5 ms), and to number of diffusion directions is low. Optimal DTI imaging parameters were b = 1000 mm/s