Restorative reproductive medicine (RRM) seeks to identify and correct underlying causes and factors contributing to infertility and reproductive dysfunction. Many components of RRM are highly suitable for primary care practice. We studied the outcomes amongst couples who received restorative reproductive medicine treatment for infertility in a primary care setting.Two family physicians in Massachusetts trained in a systematic approach to RRM  treated couples with infertility. We retrospectively reviewed the characteristics, diagnoses, treatments, and outcomes for all couples treated during the years 1989 to 2014. We compared pregnancy and live birth by clinical characteristics using Kaplan-Meier analysis. We employed the Fleming-Harrington weighted Renyi test or the logrank test to compare the cumulative proportion with pregnancy or with live birth.Among 370 couples beginning treatment for infertility, the mean age was 34.8 years, the mean prior time trying to conceive was 2.7 years, and 27% had a prior live birth. The mean number of diagnoses per couple was 4.9. Treatment components included fertility tracking with the Creighton Model FertilityCare System (80%); medications to enhance cervical mucus production (81%), to stimulate ovulation (62%), or to support the luteal phase (75%); and referral to female laparoscopy by a surgeon specializing in endometriosis (46%). The cumulative live birth rate at 2 years was 29% overall; this was significantly higher for women under age 35 (34%), and for women with body mass index < 25 (40%). There were 2 sets of twins and no higher-order multiple gestations. Of the 63 births with data available, 58 (92%) occurred at term.Family physicians can provide a RRM approach for infertility to identify underlying causes and promote healthy term live births. Younger women and women with body mass index < 25 are more likely to have a live birth.The online version contains supplementary material available at 10.1186/s12884-021-03946-8. Infertility is a common concern in couples , 2. It iTreatment strategies for infertility include those that accomplish some parts of the reproductive process outside of the body , and those that seek exclusively to restore normal physiologic fertility . Assisted reproductive technology techniques include in vitro fertilization (IVF), with or without intracytoplasmic sperm injection (ICSI), and intrauterine insemination . RRM incA specific model of RRM is called natural procreative technology , developed at Creighton University School of Medicine and the Saint Paul VI Institute for the Study of Human Reproduction. It includes a standardized system for educating couples about the fertility cycle, called the Creighton Model Fertility Care System (Creighton Model), and medical and surgical treatments to support conception in vivo , 12, 13.This paper presents results of a retrospective cohort study of RRM of all infertile couples referred to two primary care practices for evaluation and treatment. The primary outcomes are the cumulative proportion of couples experiencing conception and live births. The secondary outcomes are preterm birth and low birth weight. We assessed the impact of demographic and clinical characteristics on the primary outcomes. We also characterized the processes of care by evaluating the diagnoses and the treatments administered.In this retrospective observational study, we analyzed all infertility patients evaluated and treated by 2 family physicians in separate independent practices in Massachusetts between 1989 and 2014. Both physicians are formally trained and certified in NPT. Patients were received predominantly by referral from other physicians and fertility educators or lay referral, and were usually seeking fertility treatment not involving ART, for various reasons, including personal and religious values, or cost. Criteria for patient inclusion were at least 1 office visit during the study period; at least one lab evaluation related to fertility; the absence of clinical pregnancy despite at least 1 year  trying to conceive . Time trThe procedures of medical NPT used were similar to those reported previously , 16, 18.Treatments were prescribed to restore or optimize normal reproductive physiology to the extent possible, i.e., to assure regular ovulation, appropriate cervical mucus production, optimal timing of intercourse, and appropriate luteal phase hormonal function. Patients were encouraged to maximize preconception health, including appropriate weight loss, and treated any underlying condition that might contribute to impaired fertility, implantation, or successful pregnancy.Data were collected via review of medical records. These included patient characteristics, diagnoses, treatments employed, pregnancy, live births, number of fetuses, birth weight and duration of pregnancy. To ascertain pregnancy outcomes, patients were contacted, when possible, via mail and telephone. We used partially de-identified data for this analysis. Each physician obtained local Institutional Review Board approval, and the study was also approved by the Institutional Review Board at the University of Utah.We calculated descriptive statistics for all eligible patients. We compared specific fertility diagnoses before and after NPT evaluation using McNemar’s test statistic. We calculated frequencies of treatments received, crude proportions of couples conceiving or having a live birth over 2 years, and Kaplan-Meier survival curves to adjust for dropout from treatment. We conducted stratified analyses by clinical factors that we expected to impact the likelihood of pregnancy and birth, with the following factors chosen a priori, based on existing literature: woman’s age, time trying to conceive, prior pregnancy, prior live birth, prior IVF, prior intrauterine insemination (IUI). We also subsequently evaluated the impact of body mass index (BMI) and the treatment start date on the primary outcomes , 19, 20.Between 1989 and 2014, 559 patients were evaluated for fertility concerns. After excluding couples who did not meet criteria or who had missing data, there were 370 eligible couples. Half of eligible women were age 35 or older, 46% had experienced a prior pregnancy, and 27% had a previous live birth. The mean time trying to conceive prior to entry was 2.7 years. Additional characteristics of the couples are given in Table The mean number of fertility-related diagnoses per couple after evaluation was 4.9 . The most common diagnoses were endometriosis (74%), limited cervical mucus (65%), and ovarian dysfunction identified based on hormonal profiles (66%), the majority of which had a component of low luteal progesterone (56%). Male factor was diagnosed in 30% of couples. A female mental health diagnosis (primarily depression) was identified in 25% of couples. Details of diagnoses are given in Table The median number of office visits per couple was 4 . The large majority (80%) tracked ovulation and the fertile days with the Creighton Model , while 1The unadjusted proportion with a pregnancy and live birth were 31 and 18%. Adjusting for dropout with Kaplan-Meier analyses, the proportions were 39 and 29%, respectively Table . Dropoutn = 68 from 66 pregnancies), 58 (92%) were born at term; 5 (8%) at 32 to 37 weeks gestation; 3 had missing data. There were only 2 sets of twins, and no higher-order multiple births , and for those with BMI < 25. There were 2 sets of twins and no higher-order multiple births.Our study adds to the growing literature supporting an integrated RRM approach to infertility. It suggests that the RRM approach in a primary care setting can identify underlying causes or contributing factors for infertility and that treatment results in healthy births for a significant proportion of couples.This is the first study that has examined the impact of woman’s BMI within RRM, which we investigated as a secondary analysis. Women who had BMI < 25 had 40% probability of live birth, compared to 16% probability for women with BMI ≥25. This is concordant with studies showing lower live birth rates among infertile women with a high BMI who undergo other types of fertility treatment, including intrauterine insemination, IVF, or simply ovulation induction –26. FutuWomen > 35 years of age who had been trying from 6 months to 1 year based on standard clinical definitions and recommendations , had subWithout a control group we cannot identify the untreated spontaneous birth rate, which limits the ability to infer the impact of treatment. Because the patients were received predominantly by referral rather than being population-based, we believe a spontaneous birth rate for a referral population is a more relevant comparison than one for a population-based primary care practice; the former is about 50% lower than the latter . The proThe diagnoses prior to RRM evaluation Table  were repAlthough all patients received RRM evaluation, not all couples availed themselves of fertility tracking. Creighton Model Fertility Care System tracking is the foundation of NPT , 28, 29.Encouraging infertile couples to continue for a full trial of treatment represents a challenge. Over half of our study couples discontinued treatment before 2 years. This rate of discontinuation is similar for that of other infertility treatment cohorts, both RRM and ART , 32, 33.These are the first published data on RRM for infertility from family physicians in the United States, and complements previously published data from family physicians in Ireland and Canada. The cumulative adjusted proportion of live births in those studies was 53% in Ireland (32% for a separate study of only couples who had previous IVF), and 66% in Canada , 15, 16.Another difference relates to inclusion criteria. The criteria for this study were intentionally broad (only 1 visit and 1 lab test). In both the Irish and Canadian studies, at least 2 clinic visits were required for inclusion. The survival analysis should adjust for early drop out, but if those who dropped out after one visit from the present study had a lower potential for pregnancy than those who continued for two visits or more, this could contribute to a lower cumulative pregnancy probability in the present study, compared to studies that required two visits to be entered in the study, and thus excluded the couples who had only one visit. Finally, differences in the duration of follow-up may contribute to differences in survival analysis probabilities.A broad range of interventions was used in these patients (see Table The proportions of births with prematurity (8%) and low birth weight (2%) were very low, and compared very favorably to the recent Massachusetts state average prematurity rate of 8.6% in 2014 . This suFamily physicians can provide a RRM approach for infertility to identify underlying causes and promote healthy term live births. Younger women, women age > 34 trying less than 1 year, and women with body mass index < 25 are more likely to have a live birth.Additional file 1.


Lithium is prescribed during pregnancy, but there is limited information about pregnancy and neonatal outcomes following in utero exposure. Thus, this study aimed to investigate the associations between lithium use and adverse pregnancy and neonatal outcomes.This population-based cohort study examined associations between maternal lithium use and major adverse pregnancy and neonatal outcomes via inverse probability weighted propensity score regression models.n=9552), associations remained between lithium and spontaneous preterm birth, birth of a large for gestational age infant, and cardiovascular malformations; and a positive association with neonatal hypoglycaemia was also found. These associations were also apparent in a further analysis comparing women who continued lithium treatment during pregnancy to those who discontinued prior to pregnancy.Of 854,017 women included in this study, 434 (0.05%) used lithium during pregnancy. Among pre-specified primary outcomes, lithium use during pregnancy was associated with an increased risk of spontaneous preterm birth  and birth of a large for gestational age infant , but not preeclampsia nor birth of a small for gestational age infant. Among secondary outcomes, lithium use was associated with an increased risk of cardiac malformations . In an analysis restricted to pregnant women with a diagnosed psychiatric illness (Lithium use during pregnancy is associated with an increased risk of spontaneous preterm birth and other adverse neonatal outcomes. These potential risks must be balanced against the important benefit of treatment and should be used to guide shared decision-making.The online version contains supplementary material available at 10.1186/s12916-021-02170-7. Lithium is well-established as an effective first-line treatment for bipolar disorder, a major psychiatric condition affecting 2% of the global population . With anIn contrast, associations between lithium exposure in utero and the risk of adverse pregnancy or neonatal outcomes are unclear. Associations between lithium use and congenital malformations have been reported, including cardiac malformations, which suggest an increased likelihood of malformation –6. StudiBetter information on the possible impact of lithium administration and adverse pregnancy outcomes could aid clinical decision-making and surveillance in the management of psychiatric illness during and after pregnancy. To address this gap in knowledge, we performed a population-based study examining whether maternal lithium use was associated with adverse pregnancy outcomes.This retrospective population-based cohort study obtained data through linkage of 5 Swedish national registers using unique personal identity numbers that are assigned to all Swedish residents. Linked registers included the Swedish Medical Birth Register, the Patient Register, the Prescribed Drug Register, Education Register and Total Population Register. The Medical Birth Register prospectively collects data of over 98% of all births in Sweden and includes demographic data, reproductive history, complications during pregnancy and delivery and neonatal outcomes. The Patient Register includes information on all inpatient care and outpatient clinical services, including psychiatry. All procedures and diagnoses are documented using International Classification of Disease (ICD) diagnostic codes. The Prescribing Drug Register contains data on all prescriptions dispensed in Sweden and includes information on dispensed item, date of dispensing, substance, formulation, package size, dispensed amount and dosage. Linkage between the registers is possible due to the individual identification number given to all citizens in Sweden.All women giving birth to a liveborn or stillborn child at 22 gestational weeks or later in Sweden from January 2007 to December 2014 were included. Multiple pregnancies were excluded.Maternal lithium treatment prior and during pregnancy was obtained from the Prescribing Drug Register (ATC code N05AN). Lithium use during pregnancy was defined as a prescription dispensed during pregnancy or the 3 months prior to conception. To identify lithium exposure during this period, date of conception was calculated as date of birth − gestational age in days, with gestational age at birth obtained from the Medical Birth Register and estimated via ultrasound dating during the second trimester of pregnancy.Primary and secondary outcomes of interest were prespecified and selected based on potential impacts of medication exposure during pregnancy and findings from previous studies. The primary outcomes were preeclampsia , spontaneous preterm birth before 37 weeks’ gestation and birth of a small for gestational age or large for gestational age infant. Information on onset of birth is routinely recorded in a standardized manner by the delivery ward midwife and is categorized as spontaneous, induced vaginal or caesarean section before onset of labour. Spontaneous onset of birth was defined as a registered spontaneous start or if a diagnosis of preterm premature rupture of the membranes (ICD code O42) was present. Small for gestational age and large for gestational age were defined as a birthweight of more than two standard deviations below or above the mean weight for gestational age according to the Swedish national reference curve [2) and categorized as , schizophrenia (F20–F29), psychosis (F29), pre-gestational hypertension  and diabetes .Information on maternal age at delivery (</≥35 years), height, weight, smoking at first antenatal visit (yes/no), parity (nulliparous or multiparous), use of assisted reproduction (yes/no) and gestational disorders was obtained from the medical birth register. Body mass index (BMI) was calculated as weight (kg)/height , smoking status, country of birth, education, parity, maternal psychiatric disorders , other medical conditions  and antipsychotics (ATC code N05A), central stimulants (ATC-code N06BA), or lamotrigine (ATC code N03AX09) dispensed during or 3 months prior to pregnancy. Second, with the aim of achieving balance between exposed and unexposed groups, the estimated propensity scores were used to weight each subject, with a score of 1/propensity score assigned to the exposed individuals and 1/(1 − propensity score) to the unexposed. Propensity scores were also generated for subgroup analyses, with the following covariates included: maternal age (</> 35 years), body mass index (</> 30 kg/m2), smoking status, country of birth, education, parity, medical conditions (diabetes and hypertension) and antipsychotics, neuroleptics or lamotrigine dispensed during or 3 months prior to pregnancy. Propensity scores were checked for extreme values and overlap between exposure groups. The balance of individual covariates before and after inverse probability weighting was also assessed. Weighted standardized differences of less than 0.1 were indicative of covariate balance  on adverse pregnancy and neonatal outcomes was estimated using Stata’s ‘teffects’ command and presented as relative risk with 95% confidence intervals; within-mother clustering was accounted for in standard errors. Regression models were inverse probability weighted to achieve covariate balance between exposure groups. A propensity score for exposure was calculated for each individual using a logistic regression that included variables considered likely confounders based upon directed acyclic graphs and selected a priori for inclusion in the propensity score models. Included covariates were: maternal age (</> 35 years), body mass index .Of the 854,017 women studied, 434 (0.05%) used lithium during pregnancy. Among the whole cohort, the mean maternal age was 30.3 years (standard deviation (SD) 5.3), body mass index was 24.7 (SD 4.6) and most were born in Sweden  of 2.80 ). After adjusting via inverse probability weighting, lithium use remained associated with a two-fold increased risk of spontaneous preterm birth  and an adjusted relative risk of 2.64  , neonatal hypoglycaemia , a 5-min Apgar score < 6 , total malformations  or perinatal death . However, a higher incidence of neonates with cardiac malformations was observed among women who used lithium (2.1% vs 0.8%), with an aRR of 3.17  and of these, the majority (66.6%) were reported as malformations of the cardiac septa (ICD-10 Q21).n=434) to those who were prescribed lithium prior to pregnancy, but not during (n=871). Women using lithium during pregnancy, compared with those who discontinued lithium prior to pregnancy, had an increased risk of spontaneous preterm birth , birth of a large for gestational age infant , neonatal hypoglycaemia  and cardiac malformations  or not (n=9140). Associations observed were similar to the primary analysis: spontaneous preterm birth remained associated with maternal lithium use , as was the risk of birthing a large for gestational age infant , but not preeclampsia nor birthing a small for gestational age infant  and either prescribed lithium n=12 or notIn this nationwide population-based study lithium taken during or the 3 months prior to pregnancy was associated with an increased risk of spontaneous preterm birth, birth of a large for gestational age infant and cardiac malformations. Importantly, these associations remained in a pre-specified sub-analysis restricted to women with a diagnosis of bipolar disorder, schizophrenia or psychosis; and a further pre-specified analysis examining mothers who used lithium during pregnancy compared to those who took lithium prior to pregnancy but not during. Among these two subgroup analyses, an increased risk of neonatal hypoglycaemia was also observed in addition to the complications observed in the primary analysis. Reassuringly, however, we did not uncover associations between lithium use and preeclampsia, nor birth of a small for gestational age infant. These two complications can reflect placental dysfunction and foetuses that are small for gestational age incur an increased stillbirth risk .In contrast to our results, two meta-analyses’ have reported no association between maternal lithium use and preterm birth , 5. ImpoWe do note that like our study, the meta-analysis published during 2018 also included Swedish national data. Within the Swedish arm of the previous study, 238 lithium-exposed women were compared to a reference group of 13,407 women diagnosed with a mood disorder. This study investigated several pregnancy and delivery complications as well as neonatal readmission for congenital malformations. In contrast, the present study included 434 lithium-exposed women, who were compared to three reference groups. Additionally, using an a priori statistical analysis plan we focussed on four major pregnancy complications as our primary outcomes. Two of the four, spontaneous preterm birth and large for gestational age infants, were not reported in the prior study. Furthermore, we used a propensity score-based approach to control for important baseline differences between women using lithium and those not.Previous studies reporting congenital malformations have been conflicting. A meta-analysis published in 2020 concluded lithium was associated with an increased likelihood of congenital malformations overall and specifically, association with cardiac malformations . In the Given large for gestational age infants are at increased risk of neonatal hypoglycaemia , identifTo our knowledge, our study is the largest cohort from one country to examine perinatal outcomes among women using lithium during pregnancy. Importantly, we were able to perform two planned sub-analyses that corrected for underlying psychiatric illness . We also compared those who took lithium prior to, but not during pregnancy versus those who took the drug during pregnancy. The same associations held between lithium and major adverse outcomes remained in all these analyses . Another strength is that we found a likely association with cardiac malformation which has been previously shown . FinallyThere are some limitations to our study. Whilst we pre-specified our analysis and limited our choices for the primary outcomes to major obstetric conditions, four outcomes were investigated. This raises the potential for a type 1 error. Also, whilst the use of inverse probability weighting allowed us to adjust for a number of confounders, it remains plausible that residual confounding through unmeasured covariates may have occurred. Additionally, women with missing covariate data were not included in adjusted analyses. Lastly, medication use was obtained via the Swedish Prescribed Drug Register, which contains data on all dispensed prescriptions but does not provide data confirming that the lithium was consumed. However, this would have been expected to attenuate the strength of associations. Furthermore, future studies investigating the associations between actual lithium use or maternal serum levels and adverse obstetric and long-term offspring health and developmental outcomes are needed.The use of lithium during pregnancy is associated with a 2–3-fold increased risk of spontaneous preterm birth, birth of a large for gestational age infant and cardiac malformations. It is important to note that the absolute risk of malformations remained low. Lithium use may also be associated with neonatal hypoglycaemia. Lithium was not associated with an increased risk of preeclampsia or birthing a small for gestational age infant. These findings should be weighed against the need to effectively manage maternal psychiatric illness during pregnancy and should be used to guide shared treatment decision making and appropriate surveillance for those on treatment during pregnancy.Additional file 1: Table S1. Raw and weighted standardized differences for included covariates and each outcome.


Prolonged or non-progressive labour is the greatest risk factor for loss of newborn lambs in Australia and poses significant welfare and economic concerns worldwide. In this study, we set out to investigate whether pen-side technology could be used to predict which ewes would be at risk of prolonged labour. In our pilot trial, we found potentially useful markers. We next developed a sampling protocol by looking at changes in candidate markers over time in normal lambing events. Finally, we searched for blood markers that could distinguish between normal and difficult lambing events, sampling pre-birth (estimated one week before birth), at birth (within 3 h) and post-birth (16–26 h). Possible predictors of lambing difficulty were chloride, haematocrit and haemoglobin, sampled one week before birth; creatinine, sampled at birth; and acid–base related parameters after birth. In conclusion, we found that pen-side analysis of blood markers showed promise in identifying dystocic lambing events. More information is required to decide whether pen-side diagnostics could be useful to identify and predict dystocic lambing in the future.2, pO2, base excess, HCO3, TCO2, sO2, lactate, sodium, potassium, chloride, calcium, urea nitrogen, creatinine, haematocrit, haemoglobin and anion gap. From the pilot data, we identified creatinine, TCO2, chloride and calcium as potentially useful markers. To develop a time course and to establish variability of the selected blood parameters, a time series of samples was collected from 12 ewes, from mid-gestation to 48 h after birth. For the main trial, blood samples were collected at mid- and late gestation for glucose determination and for the full set of blood parameters at three time points before, at and after birth. Possible predictors of lambing difficulty were chloride, haematocrit and haemoglobin, sampled one week before birth; creatinine, sampled at birth; and blood pH and base excess after birth. In conclusion, we found that pen-side analysis of blood markers showed promise in identifying dystocic lambing events.Dystocia is the greatest contributor to neonatal lamb mortality in Australia and poses significant welfare and economic concerns worldwide. In this study, we set out to investigate whether pen-side analysis technology could be employed to detect blood parameters predictive of dystocic labour events in sheep. In a pilot trial, we collected and analysed blood samples in pen-side assays for glucose, lactate, pH, pCO Dystocia is an important welfare and economic issue faced in animal production industries in Australia and across the world. It is the greatest contributor to neonatal lamb mortality  and lambBlood biochemistry analyses are able to provide insights into individual animals’ health and welfare status by identifying disease presence, metabolic disturbances and stress hormone levels ,9. TradiIn this study, we made use of a series of lambing trials designed to collect behavioural data during lambing  to colleAnimal experimentation was undertaken at the CSIRO FD McMaster Laboratory, Armidale, NSW, Australia between March 2018 and November 2019 and conducted in accordance with the Australian Code for the Use and Care of Animals in Research and Teaching. Experimental protocols were approved by the CSIRO Armidale Animal Ethics Committee under Animal Ethics Approval numbers 18/11 and 19/10.All animals were treated in the same manner and followed the same lambing protocols unless stated otherwise. All trials were made up of pure Merino ewes of varying ages (2–7 years old), mated to either Merino or Border Leicester rams, and carrying either singles or twins. Each trial was balanced as far as possible for even numbers of these combinations. Approximately one week before lambing was due to commence, ewes were brought into the yards. The 20 ewes that were due to lamb first were drafted and were assigned to group 1, and the rest of the ewes were divided evenly into groups based on due date. All ewes were side branded with a unique number with stock safe paint  to facilitate identification from video records. Group 1 was moved into the supervised lambing enclosure equipped with 10 day- and night-vision cameras  recording continuously for the entire trial period, and the remaining groups were held in nearby holding paddocks. Ewes had ad libitum access to water and pasture grass and were supplemented with a mix of Faba beans and pellets once daily  and lucerne hay twice daily. Ewes in the main trial were supplemented with thiamine powder mixed in with their daily feed when thiamine deficiency symptoms appeared in this cohort , or there was no progress achieved 1 h after foetal membranes or foetal parts appeared. If assistance was given, the ewe was restrained in a lateral lying position by one handler, and progression of the lamb through the birth canal was assisted by another handler. The ewe and lamb were monitored closely until the ewe was observed to be grooming the lamb and allowing it to suckle.Once a ewe had lambed in the lambing enclosure, time of birth and birth intensity  were documented from the video recordings. Birth intensity was determined by a single observer familiar with lambing ewes. Ewe and lamb pairs that appeared fit, healthy and well bonded were moved into an adjacent paddock, or alternatively to hospital pens to allow treatment of ewes or to facilitate bonding. The ewe was replaced by a pregnant ewe from the holding paddock, keeping the number of ewes in the lambing enclosure at 20. This was continued until all ewes had lambed.Blood samples were collected by jugular venepuncture into 10 mL lithium heparin vaccutainer tubes. Samples were analysed with the iStat-1 handheld analyser  using a combination of cartridges . CartridPeripheral blood glucose was measured in the Time Course and Main Trial phases, using an AccuCheck handheld glucometer  via capillary puncture in the ear.n = 15; 13 single/2 twin births), the second in August–September , and the third in October–November . For the first cohort, if a ewe was found less than 2 h post-lambing, she was eligible for a blood sample. The ewe and her lamb(s) were brought into the lambing shed, a blood sample was collected, and the ewe and her lamb(s) were then moved to the adjacent paddock.The pilot trial was conducted over three lambing cohorts in 2018. A total of 37 ewes were sampled for the pilot trial from a total of 420 lambing ewes. The first cohort was lambed in April–May  were brought into the yards for blood glucose determination. Additionally, the ewes selected for indoor lambing had a blood sample collected for analysis with the iStat analyzer  with straw bedding and two day/night surveillance cameras. Ewes were provided with free access to water, ad libitum lucerne hay and a daily ration of sheep pellets and fava beans, as above.The ewes were checked every 2–3 h for signs of lambing, and when the first signs appeared, the ewe was monitored every 30 min until birth. Once a ewe had lambed, she was moved with her lamb into a single pen (1.5 m × 1.5 m) adjacent to the lambing pen. A blood sample was collected within 2 h of lambing and blood glucose was recorded. This protocol was repeated at 4 h, 8 h, 24 h and 48 h post-lambing. For ewes that lambed outside the observation period, no further blood samples were collected. After all blood was collected, the ewe and lamb were moved into a paddock with other ewes and lambs, and one of the other selected pregnant ewes was brought into the indoor lambing pen.Only complete sample sets for all time points were used for the time course analysis. A complete sample set was obtained from a total of six ewes for the time course of iStat analysis. n = 100) were brought into the yards for blood glucose determination. One week before lambing was due to commence, the 20 ewes that were expected to lamb first based on foetal size at pregnancy scanning were brought into the lambing shed, had their blood glucose measured and recorded and a blood sample was collected. The rest of the ewes were brought into the lambing shed in groups of 20 (based on due date) in two- or five-day intervals for the same blood sampling protocol. This method was employed to reduce stress on the ewes and to allow sampling of all ewes at around one week before lambing.At two time points during gestation, at 80 days post-joining for mid-gestation, and at 140 days post-joining for late-gestation, ewes  and on the day following birth within 16–26 h from 30 ewes .Blood samples were successfully collected at birth, within 3 h of birth, from 27 ewes . Excel was used to clean the data, organise it and perform basic descriptive statistics .p < 0.05.Statistical tests were performed using Genstat . Unpaired Student’s t-Tests were used to compare blood marker values between eutocic and dystocic births, and for comparing mid-gestation values and subsequent values in the time course. The significance level was All ewes in this study, including dystocic ewes, successfully delivered their lambs unassisted within the time limits allowed by the study protocol. n = 11; 9 single/2 twin births) and dystocic  lambing events. This cohort was analysed separately to cohorts 2 and 3 due to changes made to the sampling protocol. Potassium  and chloride  were significantly lower in dystocic ewes. Additionally, TCO2 , creatinine , base excess  and HCO3  were all significantly higher in dystocic ewes.In the first lambing cohort, 15 ewes were sampled 0–2 h after birth from eutocic  and dystocic  lambing events. In the third cohort, 12 ewes were sampled 2–4 h after birth, and again at 24 h after birth from eutocic  and dystocic  lambing events. When data from cohorts 2 and 3 were analysed together (17 eutocic ewes and 5 dystocic ewes), no differences were seen between eutocic and dystocic ewes at the first time point 2–4 h after birth for any blood parameters measured. At the second time point 24 h after birth, sO2  was significantly higher in dystocic ewes. In the second cohort, 10 ewes were sampled 2–4 h after birth, and again at 24 h after birth from eutocic (p = 0.005) was significantly lower in dystocic ewes; however, glucose  and potassium  were not significantly different. At 24 h after birth, TCO2  was significantly higher in dystocic ewes. Cohorts 2 and 3 lambed in late winter/early spring and late spring, respectively, of the Australian New England climate. To address the question of whether environmental conditions had an effect on the obtained results, data obtained for cohorts 2 and 3 were also analysed separately. When analysed separately, at the first time point for cohort 2, creatinine  was significantly higher in dystocic ewes. At 24 h after birth, chloride  and sO2  were significantly higher, and calcium , TCO2 , pCO2 , base excess  and HCO3  were significantly lower in dystocic ewes. At the first time point of cohort 3, calcium . There was no difference found between dystocic and eutocic ewes. During pregnancy, all 100 ewes of the larger cohort, to which the ewes involved in the time course experiment belonged, had blood glucose measured twice by the test strip method: once at 80 days gestation and again at 140 days gestation . Glucose was significantly higher at mid-gestation compared to late gestation  at 80 d and 140 d gestation for peripheral blood glucose determination. No differences were observed between singleton or twin-bearing ewes and ewes that would later experience eutocic or dystocic birth. For this trial, samples were collected from all ewes  and 9 dystocic (6 single/3 twin births) events); five ewes were affected by ‘stargazing’ disease/thiamine deficiency , one ewe died before sampling (Group 4) and one ewe was excluded as she was the only ewe delivering triplets (Group 2). At birth, blood samples were collected within 3 h of birth (n = 27), including 19 eutocic (11 single/8 twin births) and 8 dystocic (6 single/2 twin births) events. Samples from the day following birth were collected within 16–26 h from a total of 30 ewes, including 21 eutocic events (21 single/13 twin births) with samples collected at 16 h 25 min–25 h 42 min postpartum (1224 ± 163 minutes) and 9 dystocic events (6 single/3 twin births) with samples collected at 16 h 10 min–26 h 04 min postpartum (1253 ± 231 minutes). The full set of blood parameters was measured approximately one week before parturition for all ewes, except for seven individuals (Twin-bearing ewes were represented in 35% (33/93) of pre-birth samples ; 37% (10/27) of samples collected at birth (8/19 or 42% of eutocic and 2/8 or 25% dystocic ewes); and 37% (16/43) of samples collected at 16–26 h post birth (13/34 or 38% of eutocic and 3/9 or 33% of dystocic samples). Hence, twin-and singleton-bearing ewes were represented consistent with the overall average in pre-birth and post-birth samples. At birth, twin-bearing ewes were slightly underrepresented in the total sample set as one dystocic twin-bearing ewe lambed outside of the daily observation period. Overall, twin-bearing ewes were not overrepresented in the samples, and therefore, in the analysis of blood sample data, birth type (singleton or twin) was not further considered as a factor. p = 0.014). Additionally, dystocic ewes had a higher haematocrit  and a higher haemoglobin concentration  compared to eutocic ewes.Before lambing, dystocic ewes had a significantly lower chloride concentration compared to eutocic ewes . Blood creatinine concentration was significantly higher at birth for dystocic ewes than eutocic ewes .Blood glucose concentration was higher at birth for eutocic ewes compared to dystocic ewes, but the difference was not significant  than eutocic ewes (91 7.47 +/− 0.079), with a corresponding difference in base excess between eutocic (−1.38 +/− 2.80 mmol/L) and dystocic ewes (−3.44 +/− 2.55 mmol/L).In summary, before birth, dystocic ewes presented with significantly lower chloride and higher haematocrit and haemoglobin compared to eutocic ewes. Within 3 h of birth, blood glucose was significantly lower, and creatinine was significantly higher in dystocic ewes. In the 16–26 h after birth, pH and base excess parameters were significantly lower in dystocic ewes.During the pilot trial, we aimed to establish whether we could observe indicators promising to distinguish between dystocic or eutocic lambing events in blood samples collected from ewes in the peri-natal time period and analysed with pen-side diagnostics. In the first cohort, we found that potassium and chloride were lower in dystocic ewes. It is well documented that potassium and chloride are key ions involved in myocyte excitation and uterine contractility, allowing the proper progression of labour . Disturb2, base excess and HCO3 levels than eutocic ewes. This could be a compensatory effect, counteracting the acidosis that may have occurred during labour. Additionally, creatinine was higher in dystocic ewes. This was expected, as creatinine is a by-product of muscle and protein metabolism [Maternal acid–base function and dysfunction are well documented in humans  during gestation is to be expected; as maternal body weight increases, plasma volume increases . This phAt birth, blood glucose concentration was lower in dystocic ewes, but the difference was not significant. Larger group sizes and an earlier collection time will be required to address whether the observed difference has significance. Based on our time course results, the chosen at-birth collection time point may have missed the period of perinatal elevation, which may show differences between dystocic and eutocic ewes and epistatic mechanisms that tightly control blood glucose levels may have adjusted this difference already, if it had been there. Glucose appears to be the main energy source for myometrial cells ,33. DuriCreatinine concentration was significantly higher in dystocic ewes compared with eutocic ewes. Creatinine levels rise during and after exercise as a result of exercise-induced muscle breakdown , and parBy the time blood samples were collected at 16–26 h post-birth, most blood parameters had normalised, so there were no significant differences between eutocic and dystocic ewes. This further supports the idea expressed earlier that the acute adaptive stress response quickly regulates the body back to eustasis . An exceFor creatinine, which remains overall elevated for an extended time period after birth, we could not establish differences between eutocic and dystocic ewes. A more narrowly defined time window for collection of the second post-birth sample may have allowed such differentiation and more work will be required to address this possibility.In this study, we restricted the investigated parameters to those readily assessable through pen-side diagnostics. To get a more complete understanding of differences in the physiology of dystocic and eutocic ewes, analysis of additional metabolic markers, such as beta-hydroxy-butyrate and non-esterised fatty acids, may be of interest . FurtherIn this series of trials, we aimed to use pen-side analysis techniques to collect information on blood parameters in pregnant and periparturient Merino ewes to further understand the effect of dystocia on the physiology of the ewe. Pilot study results indicated that observable differences can be found between dystocic and eutocic ewes. The observed differences in the pilot study were dependent on cohorts, pointing to the strong influence of environmental conditions on individual parameters. The main trial confirmed observable differences between normal and difficult lambing events in the pre- and postpartum periods. Dystocic ewes presented in late pregnancy with significantly lower chloride and higher haematocrit and haemoglobin, compared to eutocic ewes. At birth, creatinine was significantly higher in dystocic ewes. On the day following birth, acid–base balance was affected in dystocic ewes. The observed differences may have potential as diagnostic markers. This study has demonstrated the potential benefits of pen-side diagnostics to predict and identify dystocic ewes. More research will be needed to confirm these results and to further our understanding of the underlying factors contributing to incidences of dystocia in sheep production systems.


Successful scale-up of antiretroviral therapy (ART) during pregnancy has minimized infant HIV acquisition, and over 1 million infants are born HIV-exposed but uninfected (HEU), with an increasing proportion also exposed in utero to maternal ART. While benefits of ART in pregnancy outweigh risks, some studies have reported associations between in utero ART exposure and impaired fetal growth, highlighting the need to identify the safest ART regimens for use in pregnancy.We compared birth anthropometrics of infants who were HEU with those HIV-unexposed (HU) in Cape Town, South Africa. Pregnant women had gestational age assessed by ultrasound at enrolment. Women living with HIV were on ART (predominately tenofovir-emtricitabine-efavirenz) either prior to conception or initiated during pregnancy. Birth weights and lengths were converted to weight-for-age (WAZ) and length-for-age (LAZ) z-scores using Intergrowth-21st software. Linear regression was used to compare mean z-scores adjusting for maternal and pregnancy characteristics.n = 431) were HEU and 51% (n = 457) HU. Of 431 HEU infants, 62% (n = 268) were exposed to HIV and antiretrovirals (ARVs) from conception and 38% (n = 163) were exposed to ARVs during gestation but after conception . In univariable analysis, infants who were HEU had lower mean WAZ compared with HU . After adjustment for maternal age, gravidity, alcohol use, marital and employment status the effect remained . Similar differences were noted for mean LAZ in univariable  but not multivariable analyses  after adjusting for the same variables. Mean WAZ and LAZ did not vary by in utero ARV exposure duration among infants who were HEU.Among 888 infants, 49% (In a cohort with high prevalence of ART exposure in pregnancy, infants who were HEU had lower birth WAZ compared with those HU. Studies designed to identify the mechanisms and clinical significance of these disparities, and to establish the safest ART for use in pregnancy are urgently needed.The online version contains supplementary material available at 10.1186/s12884-021-03836-z. Over 1 million women living with HIV (WLHIV) give birth annually . SeveralSouth Africa’s antenatal HIV prevalence is one of the highest globally, reported at approximately 30% in 2017 . In thisThe B Positive study was a prospective observational study conducted at a large primary healthcare facility in Gugulethu, a peri urban township in Cape Town, South Africa. The facility serves a population of about 350,000 with an estimated antenatal HIV prevalence of 30% .Consecutive pregnant women > 18 years of age were recruited into the study at their first antenatal care (ANC) visit, regardless of HIV status. Study enrolment occurred between January 2017 to July 2018. Women were eligible for this study if they planned to reside in Cape Town with their infants and had a confirmed maternal HIV status at time of study enrolment. For women not known to be living with HIV, a rapid antibody test was used to confirm their HIV status which is standard of care in routine ANC.All eligible pregnant women who were able to provide informed written consent were enrolled. Women were followed antenatally for one to three study visits depending on the gestational age (GA) at enrolment. GA was assessed by a dedicated study ultra-sonographer and repeated at all subsequent antenatal visits. Mother-infant pairs were evaluated postnatally at 7 days and birth anthropometrics were abstracted as recorded at birth in South Africa’s child Road to Health Booklet (RTHB). Birth weight of new-borns was measured within 24 h of birth by health facility nurses. To be included in this secondary analysis, a woman had to deliver liveborn, singleton infant and birth weight and/or length data had to be available on the child’s RTHB.Data collected included maternal demographics, pregnancy history and healthcare information. Questionnaires were administered to all women by trained study interviewers. Pregnant women with a negative HIV test at enrolment based on routine rapid antibody test, had repeat HIV testing up to once every 3 months during the antenatal period and immediately after delivery, as per South Africa’s ANC standard guidelines . For theFor this analysis, we used data collected during the antenatal study visits, at birth and 7 days after delivery/birth. We evaluated two primary exposures of interest. The first was exposure to HIV in utero (HEU versus HU), while the second focused on timing of ARV exposure only for infants who were HEU and consisted of a dichotomous variable of either ARV exposure from before conception and during gestation; or after conception but during gestation. Birth weight and length abstracted from the RTHB were used to derive anthropometric primary outcomes, birth weight-for-age (WAZ) and length-for-age (LAZ) z-scores, representing surrogate measures of fetal growth. WAZ and LAZ scores were generated using Intergrowth-21st software, which adjust for infant GA at birth and infant sex , and the2 test or Fisher exact test as appropriate. Proportions of secondary outcomes, preterm birth, low birthweight and small-for-gestational age were compared by in utero HIV exposure (HEU vs HU) and by timing of in utero ARV exposure in HEU infants (before conception vs after conception but during gestation). Univariable and multivariable linear regression models were used to compare the primary outcomes, mean WAZ and LAZ scores first by in utero HIV exposure status, then by timing of in utero ARV exposure in infants who were HEU. All covariates in univariable analyses with a p-value of 0.10 were included in multivariable analyses. Additionally, an a priori decision was made to include maternal age in the multivariable model, regardless of univariable p-value.Data were analysed using Stata 14.0  . MaternaThis study was approved by the University of Cape Town’s Faculty of Health Sciences Research Ethics Committee (UCT-HREC) and all women participating in the study provided informed written consent for their own participation and that of their infant’s.p < 0.001)]; more likely to be multigravid ]) and formally employed . (Table p < 0.001)]) and more likely to be multigravid  than women who initiated ART during pregnancy . There were no differences between WLHIV and women without HIV with respect to gestational age at delivery, parity, maternal education, marital status and alcohol use during pregnancy.Out of 989 women enrolled in the B Positive cohort, 888 mother-infant pairs with live singleton births with birth anthropometry data available were included in this analysis Fig. . Of the p = 18 and p = 0.004) respectively , p = 0.04]. Similar differences were noted for mean LAZ comparison with univariable linear regression analysis . However, in the multivariable analysis, there was no significant mean LAZ difference  after adjusting for the same variables.Infants who were HEU  had lower birth weight,  and  compared to infants who were HU,  and  (When we restricted the population to HEU infants only Table , WAZ andThere were no significant differences between WLHIV and women without HIV with respect to gestational age at delivery, parity, maternal education, marital status and alcohol use during pregnancy. Preterm birth, low birthweight and small-for-gestational age were similar among WLHIV compared to women living without HIV.In this prospective cohort of pregnant women seeking ANC at a public health care facility in a peri-urban township in South Africa, we found that infants who were HEU experienced lower mean WAZ and LAZ at birth compared to infants who were HU Tables  and 2. FOur finding of lower WAZ at birth among infants who were HEU is consistent with several studies in African populations , 3, 6, 8When we restricted our analysis to infants who were HEU, focusing on in utero fetal exposure to ART, there was no difference in anthropometric birth measures by timing of an infant’s exposure, either prior to conception or sometime after conception but during gestation. This is consistent with findings from a study of infants who were HEU in Brazil . UnfortuOur study had several strengths. The study had a comparator group of infants who were HU from the same community, with common socio-behavioural and economic characteristics between infants who were HEU and those who were HU. Another strength was use of a robust gestational age estimates by antenatal ultrasound. We used Intergrowth-21st New-born Standards to generate WAZ and LAZ which adjusts for gestational age and infant sex [Despite universal ART treatment during pregnancy and breastfeeding, which has improved maternal health and significantly reduced infant HIV acquisition, fetal growth remains impaired for infants with exposed to in utero HIV/ART compared to infants born to mothers without HIV. Studies to identify clinical significance of growth disparities between HEU infants and HU infants are urgently needed as well as establishing the safest ARV drug for use in pregnancy.Additional file 1.Additional file 2.Additional file 3.Additional file 4.


Introduction. Synovial cysts rarely develop in the atlantoaxial joint. We report a case of posterior C1-2 laminectomy for a synovial cyst of the atlantoaxial joint which passed through the dorsal dura and put pressure on the cervical spinal cord. Case Presentation. A 62-year-old man with rapid progression of pain and weakness in the left upper extremity presented to our hospital. A cervical spine X-ray showed left C5-6 and C6-7 stenoses. A cervical magnetic resonance imaging showed an intradural extramedullary cystic lesion on the right side of the ventral cervical spinal cord at the C1-2 level and severe compression of the cervical spinal cord. Because a cyst was partially enhancing, a tumor lesion was not identifiable. Due to severe spinal cord compression, we performed intradural cyst removal via a posterior intradural approach with C1-2 laminectomy and left-sided C5-6 and C6-7 foraminotomies. One year after surgery, the cyst did not recur, and atlantoaxial instability did not appear. Discussion. A compressive lesion on the cervical spinal cord was not identified preoperatively as a synovial cyst. However, intraoperative and pathological findings suggested that the compressive lesion can be a synovial cyst which passed through the dorsal dura. The surgical treatment strategy for a synovial cyst of the atlantoaxial joint is controversial due to factors, such as the presence of atlantoaxial instability, level of cyst causing compression of the cervical spinal cord, severity of myelopathy, and cyst location. In the present study, the cervical spinal cord was highly compressed and the cyst was located on the right side of the cervical spinal cord; we chose cyst removal through a posterior intradural approach with C1-2 laminectomy. A synovial cyst of the atlantoaxial joint is relatively rare. Even among reports on a synovial cyst of the atlantoaxial joint without complications of rheumatoid arthritis or other types of connective tissue disorders, a synovial cyst has seldom been found in the atlantoaxial region, and there are not so many reports on this subject. A synovial cyst of the atlantoaxial joint can be found accidentally or detected when cervical myelopathy develops as a result of compression on the cervical spinal cord –3. For tHerein, we report a case of posterior C1-2 laminectomy for a synovial cyst of the atlantoaxial joint which passed through the dorsal dura and put pressure on the cervical spinal cord.A 62-year-old man had chief complaints of pain in the area between the left axilla and upper extremity and weakness of the left upper extremity. The patient had a medical history of pneumonia and enlarged prostate. Two weeks before his first visit, he noticed pain in the area from the left axilla to the left upper extremity. Then, one week later, he developed left upper extremity weakness and presented to our hospital.In a physical examination, the patient complained of neuralgia in the area from the left axilla to the ulnar aspect of the left upper extremity. Results were as follows: grip strength , left brachioradialis ), and pronator teres muscle/supinator muscle and extensor digitorum muscle in the left forearm (2/5 MMT), showing a decrease in the left upper extremity. Impairment of skillful movements occurred in the left hand. In tendon reflex, reduced reflexes on left bicep and left brachioradialis were observed, and reflex on bilateral patellar tendon was enhanced.According to hematological and biochemical findings, a blood test did not show any abnormality, and tumor marker levels were not elevated.A cervical spine X-ray revealed the left C5-6 and C6-7 stenoses, but a kymograph did not show cervical spine instability .In addition to the left C5-6 and C6-7 stenoses, left C5-6 disc herniation was seen on a cervical magnetic resonance imaging (MRI) scan. Furthermore, an intradural extramedullary cystic lesion on the right side of ventral cervical spinal cord at the C1-2 level severely compressed the spinal cord .A cyst was partially enhancing in contrast-enhanced MRI, and a tumor lesion was, therefore, not identifiable . ContrasOn the basis of physical examination and imaging findings, left C5-6 disc herniation, left C6 and left C7 radiculopathy caused by the left C6-7 stenosis, and cervical myelopathy caused by an intradural extramedullary cystic lesion at the C1-2 level were identified.Because the cyst severely compressed the cervical spinal cord and was located on its right side, we performed a cyst removal through posterior C1-2 laminectomy and left-sided C5-6 and C6-7 foraminotomies. After dura mater incision at the C1-2 level, the denticulate ligaments situated on the right side of the spinal cord were incised. Then, an intradural extramedullary cystic lesion present in the right C2 nerve root  was observed. The cervical spinal cord was significantly compressed and displaced from the right ventral side by the cyst. When cyst puncture was performed, a jelly-like content with highly viscous fluid was drained, and the capsule was removed after aspiration. However, the capsule seemed to remain in the ventral side and extend to the posterior surface of the odontoid process. We observed the ventral side and found that the dura mater was partly missing. The dura mater was coagulated and closed . The surIn the postoperative course, upper extremity pain disappeared, and left-hand grip strength was increased. Pathological examination showed that slides contained both cyst contents and normal dura mater but tumor content . A synovSynovial cysts in the spine occur frequently in the lumbar facet joints, but synovial cysts in the atlantoaxial joint are rarely seen –3. SynovThe surgical treatment strategy for a synovial cyst of the atlantoaxial joint is controversial due to factors, such as the presence of atlantoaxial instability, level of cyst causing compression of the cervical spinal cord, severity of myelopathy, and cyst location. Previous reports include a case of cyst shrinkage after performing a posterior cervical fusion/an occipitocervical fusion on a patient associated with a synovial cyst of the atlantoaxial joint and atlantoaxial instability  and a caThe abovementioned surgeries have different characteristics. The transoral approach seems to be a rational surgical approach, which maximally decompresses the cyst causing anterior compression of the cervical spinal cord; however, this approach is also a maximally invasive surgery. According to the study by Van Gompel et al., a transoral approach requires nasogastric tube feeding for the heal of pharyngeal wound after surgery, and the length of feeding time was 14 days on average . In addiWith regard to a minimally invasive approach, there is a report of a patient with atlantoaxial instability and a relatively large synovial cyst in the atlantoaxial joint who underwent C1-2 posterior cervical fusion; as a result, the cyst regressed with immobilization, without direct removal [Although all the above surgeries require an occipitocervical fusion or a posterior cervical fusion, there is a more minimally invasive approach which consists of a cyst removal through a posterior approach and one of the following surgeries: a suboccipital craniotomy alone, a cervical laminectomy alone, and a combination of these two surgeries. When synovial cysts are located lateral to the transverse ligament, a posterior approach or a posterolateral approach is often used to remove the cysts –9, 11.There are further reports on a partial hemilaminectomy of the atlas to remove synovial cysts at the C1-2 level in a ventromedial region through an anterolateral approach , 13–15; The most minimally invasive approach is percutaneous aspiration . HoweverIn the present study, a compressive lesion on the cervical spinal cord was not identified preoperatively as a synovial cyst. According to intraoperative and pathological findings, it seems that the compressive lesion was indeed a synovial cyst which passed through the dorsal dura. The patient did not have atlantoaxial instability. Although it was not clear if myelopathy arose from a cystic lesion, tendon reflexes of both lower extremities were enhanced and the cervical spinal cord was highly compressed. From these points of view, we determined that the patient has an increased possibility of myelopathy in the future, and therefore, we performed a resection. A kymograph did not show atlantoaxial instability, and the patient did not have underlying conditions, such as rheumatoid arthritis. The cyst expanded to the ventral side of the cervical spinal cord, but we performed a cyst removal through posterior C1-2 laminectomy because the cyst was located on the right side of the spinal cord. One year after surgery, the patient remained without recurrence of the cyst. Careful attention must be given to the presence of atlantoaxial instability in the future.We experienced a case of compression of the cervical spinal cord caused by a synovial cyst of the atlantoaxial joint which passed through the dorsal dura without atlantoaxial instability. The cervical spinal cord was highly compressed, and the cyst was located on the right side of the cervical spinal cord. Therefore, we selected a relatively less invasive posterior intradural approach with C1-2 laminectomy, without posterior occipitocervical fusion. In the case of a synovial cyst without atlantoaxial instability, a resection via a posterior approach, which does not damage the structure of the atlantoaxial joint, must be one of the options, but surgical strategy should be determined after full consideration of patients' age and surgical risk.


Although the ratio of apolipoprotein B (apo B) to apolipoprotein A1 (apo A1) (apo B/apo A1) seems to be associated with mortality in hemodialysis (HD) patients, the association of apo B/apo A1 ratio with death remains not clear in peritoneal dialysis (PD) patients.The study targets to examine the relationship of apo B/apo A1 ratio with survival in patients receiving PD treatment.In this single-center prospective observational cohort study, we enrolled 1,616 patients receiving PD treatment with a median follow-up time of 47.6 months. We used a multivariable Cox proportional hazards model to examine the relationship between apo B/apo A1 ratio and cardiovascular (CV) and all-cause mortality. The association of apo B/apo A1 ratio with atherosclerotic and non-atherosclerotic CV mortality was further evaluated by competing risk regression models.P = 0.005] and 1.20 , respectively. The adjusted subdistribution hazard ratios for atherosclerotic and non-atherosclerotic CV mortality were 1.43  and 0.85 , respectively. For quartile analysis, patients in quartile 4 had higher CV, all-cause, and atherosclerotic CV mortality compared with those in quartile 1. Moreover, apo B/apo A1 ratio had a diabetes-related difference in CV, all-cause, and atherosclerotic CV mortality.During the follow-up, 508 (31.4%) patients died, 249 (49.0%) died from CV events, of which 149 (59.8%) were atherosclerotic CV mortality. In multivariable models, for 1-SD increase in apo B/apo A1 ratio level, the adjusted hazard ratios for CV and all-cause mortality were 1.26 [95% confidence interval (CI), 1.07–1.47; Elevated apo B/apo A1 ratio level was significantly associated with CV, all-cause, and atherosclerotic CV mortality in patients undergoing PD. Moreover, the association was especially statistically significant in patients with diabetes. Cardiovascular (CV) events have so far been the biggest obstacle to the survival of patients with end-stage renal disease (ESRD) . Even ifper se that determines atherosclerosis in hypertriglyceridemia  . A studyA prospective observational cohort study of 1,882 patients diagnosed with ESRD who began PD therapy between January 1, 2006 and December 31, 2013 in the First Affiliated Hospital of Sun Yat-sen University (SYSU) was performed. Individuals who met the inclusion criteria included those who were older than 18 years old, maintained PD therapy for at least 3 months, and signed a written informed consent. Patients who met the exclusion criteria included those who had maintained HD therapy for at least 3 months, who had suffered from malignant diseases, those with a history of failed kidney transplantation, and those with incomplete lipid data. In the end, only 1,616 patients entered the study, and the deadline for the follow-up was June 30, 2019. The entire study was carried out in line with the ethical principles of the Declaration of Helsinki and was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of SYSU. Written informed consent were obtained before all participants entered the study.When the patient was admitted to hospital, data on epidemiology and concomitant diseases would be collected. Individuals who met the diagnostic criteria for diabetes or using oral hypoglycemic drugs or insulin were believed to have diabetes. Patients who suffered from at least one of the following events were believed to have a history of CV events: angina, myocardial infarction, congestive heart failure, percutaneous coronary intervention, coronary artery bypass, or ischemic/hemorrhagic stroke . IndividLaboratory data were collected within 3 months of the start of PD treatment. Lipid parameters including TC, TG, LDL-C, HDL-C, apo A1, and apo B levels, as well as other biochemical indexes, were measured in the First Affiliated Hospital of SYSU . We adopThe primary and the secondary endpoint of this study was CV and all-cause mortality, respectively. Atherosclerotic and non-atherosclerotic mortality was further differentiated on the basis of the etiology of CV mortality. The definitions and the judgment methods of the endpoints have been described in our previous study exhaustively . Follow-The participants were categorized into quartiles according to apo B/apo A1 ratio levels: quartile 1, < 0.52; quartile 2, 0.52–0.66; quartile 3, 0.66–0.84; and quartile 4, > 0.84. Apo B, apo A1, and some conventional lipid parameters were also categorized into quartiles according to their baseline levels. Means ± standard deviations (SDs) are expressed for normally distributed variables, medians and 25th–75th percentiles are expressed for non-parametric variables. Frequencies and percentages are expressed for categorical parameters.Kaplan–Meier survival curves were performed to analyze CV and overall survival, and the method to assess the distributions of survival among apo B/apo A1 ratio quartiles was a log-rank test. We explored the shape of the relationship between apo B/apo A1 ratio and CV and all-cause mortality by using a restricted cubic spline model with four knots. A Cox proportional hazards model was used to analyze the independent association of apo B/apo A1 ratio with CV and all-cause mortality. The proportional hazards assumptions were tested by Schoenfeld residual plots. Hazard ratio (HR) with 95% confidence interval (CI) was used to present the results. Since atherosclerotic and non-atherosclerotic CV mortality are competing events, cumulative incidence function (CIF) curves were performed to analyze cumulative incidence of these two outcomes, and the method to assess the distributions of incidence among apo B/apo A1 ratio quartiles was a Fine-Gray test. The association between apo B/apo A1 ratio and atherosclerotic and non-atherosclerotic CV mortality was evaluated by competing risk regression models. Subdistribution hazard ratio (SHR) with 95% CI was used to present the results.P < 0.05. All analyses were carried through SPSS software, version 13.0  and Stata software, version 14.0 .Subgroup analyses were conducted to further examine the relationship between apo B/apo A1 ratio and mortality based on several groups of clinical parameters by forest plots. A formal interaction test was carried out to investigate the interaction between diabetes and apo B/apo A1 ratio. The standard of statistical significance is Among 1,882 patients treated with PD, 1,616 eligible patients entered the final study . BaselinThe median follow-up time was 47.6  months, and 508 (31.4%) patients died. The leading cause of death was CV mortality . The proportion of atherosclerotic and non-atherosclerotic CV mortality was 59.8% vs. 40.2%, respectively. Other causes of death were listed in P = 0.005 and P < 0.001, respectively).Kaplan–Meier analysis is used to assess CV and overall survival among quartiles of apo B/apo A1 ratio levels . PatientP = 0.005) and 1.20 , respectively. For quartile analysis, a statistically significant association existed between quartile 4 and CV  and all-cause  mortality relative to quartile 1.A trend toward linear relationship between apo B/apo A1 ratio and CV and all-cause mortality was observed when we examined apo B/apo A1 ratio as a continuous variable in restricted cubic spline models . A multiWith regard to apo B or apo A1 alone or some conventional lipid parameters, apo B was significantly associated with CV and all-cause mortality for continuous variable analysis, however, the quartile analysis did not show statistical significance for all-cause mortality. No association was found between apo A1 and worse prognosis. The association of LDL-C/HDL-C ratio with mortality exhibited similar pattern to apo B/apo A1 ratio. TC/HDL-C ratio level was significantly associated with CV and all-cause mortality for continuous variable analysis, but the quartile analysis did not show statistical significance for CV mortality .P < 0.001) but lower incidence risk of non-atherosclerotic CV mortality (P = 0.001) than those in other three lower quartiles.The CIF curves were shown in P < 0.001) and 0.85 , respectively. For quartile analysis, apo B/apo A1 ratio in quartile 4 was significantly associated with a higher risk of atherosclerotic CV  but not non-atherosclerotic CV  mortality relative to quartile 1.The relationship of apo B/apo A1 ratio with atherosclerotic and non-atherosclerotic CV mortality was further investigated by competing risk regression models in order to clarify the impact of different etiologies on the association of apo B/apo A1 ratio with CV mortality. As shown in With regard to apo B or apo A1 alone or some conventional lipid parameters, increased apo B, LDL-C/HDL-C ratio, and TC/HDL-C ratio levels were significantly associated with an increased risk of atherosclerotic CV mortality, but had no association with non-atherosclerotic CV mortality. Apo A1 levels were not associated with both the two different etiologies of CV mortality .Next, subgroup analyses were carried out and we found that the CV, all-cause, and atherosclerotic CV mortality significantly increased with the elevation of apo B/apo A1 ratio levels in all predesignated subgroups except in patients without diabetes .P = 0.035), all-cause (P = 0.012), and atherosclerotic CV (P = 0.011) mortality, but not on non-atherosclerotic CV (P = 0.498) mortality (data not shown). As shown in P < 0.001) and 1.49 , respectively, the adjusted SHRs for atherosclerotic and non-atherosclerotic CV mortality were 1.96  and 0.79 , respectively. However, we did not observe an association between apo B/apo A1 ratio and mortality in patients without diabetes.We then proceeded a formal interaction test to examine whether the association of apo B/apo A1 ratio with survival differed due to diabetes. The results indicated a significant interaction between diabetes and apo B/apo A1 ratio on CV (P < 0.001), all-cause , and atherosclerotic CV  mortality was closely associated with the elevation of apo B/apo A1 ratio levels in patients with diabetes.The results of this study indicated that elevated apo B/apo A1 ratio was significantly associated with CV, all-cause, and atherosclerotic CV mortality among patients receiving PD treatment. Additionally, subgroup analyses and formal interaction tests confirmed that the higher CV  and all-cause  mortality (Kaysen et al. recruited 433 prevalent patients treated with HD and concluded that apo B/apo A1 ratio had no association with CV mortality , while aortality , 18, whiortality . On the ortality , 24. Theortality , which mortality . Increasortality . When thortality , the patortality . Anemia,ortality , 28, 29.ortality . Two stuortality , 31. TheIn addition to etiology, follow-up time is another important factor . Our stuSeveral studies have mentioned that apo B/apo A1 ratio was closely related to diabetes , 36, butThere are several limitations of this study that cannot be ignored. First, as described in the study conducted by Little et al., the lipid profile of PD patients changed dynamically and its atherogenic property progressively worsened over time , only usIn summary, we concluded that elevated apo B/apo A1 ratio level was significantly associated with increased risk of CV, all-cause, and atherosclerotic CV mortality in patients undergoing PD. Moreover, the association was especially statistically significant in patients with diabetes. In the future, more multiple-center studies are needed to verify our conclusions and evaluate whether the reduction of apo B/apo A1 ratio levels can improve the survival of PD patients, especially those with diabetes.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study.JY, XX, and F-XH did the concept and design the study. JY, N-YH, and Y-GQ collected the data. JY, XX, and Y-GQ analyzed the data. JY, XX, N-YH, and WC performed statistical analyses. JY drafted the manuscript. XX, N-YH, XY, H-PM, WC, and F-XH revised and completed the manuscript. All authors critically revised the manuscript, approved the final version to be published, and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to investigate the role of cytokines as intermediates in the pathway from increased adiposity to disease.n = 8,293). Mendelian randomization (MR) was used to assess the impact of BMI on circulating cytokines and the impact of BMI‐driven cytokines on risk of obesity‐related diseases.BMI and circulating levels of up to 41 cytokines were measured in individuals from three Finnish cohort studies , and tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and were as ratios of geometric means 1.13 (95% CI: 1.08‐1.19), 1.08 (95% CI: 1.04‐1.14), and 1.13 (95% CI: 1.04‐1.21), respectively. TRAIL was associated with a small increase in the odds of coronary artery disease . There was inconsistent evidence for a protective role of MCP‐1 against inflammatory bowel diseases.Observational and MR estimates of the effect of BMI on cytokine levels were generally concordant. There was little evidence for an effect of raised levels of BMI‐driven cytokines on disease. These findings illustrate the challenges of MR when applied in the context of molecular mediation. Increased adiposity, frequently assessed using BMI, has been directly implicated in the development of diseases such as cardiovascular disease , diabeteChronic low‐grade inflammation as characterized by abnormal cytokine production and the activation of a network of inflammatory signaling pathways is a common feature of obesity and obesity‐related conditions, such as insulin resistance and type 2 diabetes . AlthougAs signaling molecules with a key role in regulating inflammation, circulating cytokine levels provide a useful readout of inflammatory function. Tumor necrosis factor alpha was the first proinflammatory cytokine to be associated with obesity‐linked insulin resistance . Since tMore commonly, MR studies have been undertaken looking at the downstream effects of specific inflammatory molecules. Because of the need for genetic variants to act as proxies for the exposure, such analyses have been limited to the study of cytokines and other inflammatory biomarkers for which these exist. CRP has been the focus of much attention, but despite its strong association with inflammation and its observational association with disease, evidence for a causal effect of CRP on disease is limited. One proposed explanation for this, given in the context of breast cancer, is that CRP is only linked to disease as a “cytokine reporter,” hence there is no relationship to non–“responsive‐mode” variation in CRP levels driven by polymorphisms in the gene . In contTherefore, although evidence on the possible role of inflammation in pathologies related to increased adiposity is accumulating, the relevance of many less well‐characterized cytokines is unclear. The recent publication of a genome‐wide association study (GWAS) for 41 cytokines  conducteThis study has three main components as outlined in Figure https://youngfinnsstudy.utu.fi/) is a multicenter follow‐up study with randomly chosen subjects from the Finnish cities of Helsinki, Kuopio, Oulu, Tampere, and Turku and their rural surroundings. FINRISK surveys  are population‐based cross‐sectional studies conducted every 5 years to monitor the levels of chronic disease risk factors in Finland. Each survey includes 25‐ to 74‐year‐old randomly chosen subjects from five geographical areas of Finland. For further details of the cohorts used, see the Supporting Information Methods. The present study includes unrelated individuals from these cohorts with both cytokine measurements and genome‐wide genotype data. Further details regarding cytokine quantification, genotyping, and subsequent imputation can be found in the Supporting Information Methods and are as described previously  and FINRISK 2002 , 48 cytokines were measured using Bio‐Rad’s premixed Bio‐Plex Pro Human Cytokine 27‐plex and 21‐plex assays. In FINRISK 1997 , a custom‐selected 20‐cytokine plex was used. Cytokines with >90% of values missing were excluded from all analyses. A full list of the measured cytokines is provided in Supporting Information Table Individual level data analyses  were selected from a meta‐GWAS for BMI   (P < 5 × 10−8) was first cross‐matched to the corresponding GWAS summary statistics for each disease outcome in turn. Then, taking the subset of SNPs that were present  in both data sets, an independent set of instruments was generated using an LD clumping procedure with an r2 threshold of 0.01 (see Supporting Information Methods for full details). This procedure was implemented using the TwoSampleMR R package (Ninety‐seven independent single‐nucleotide polymorphisms (SNPs) that were associated with BMI at genome‐wide significance 339,224) . The gen= 8,337) . To maxi . The geCRP gene associated with different circulating levels in populations of European descent  that has previously been shown to represent common variation within the  descent .P < 0.001 can be considered equivalent to P < 0.05). No such penalization was applied in the second step of the MR in which we took P < 0.05 as evidence of association.In the observational analysis and first step of the MR , analyses were rerun using natural log‐transformed values (adjusting for age and sex) to aid interpretation .A one‐sample MR analysis  evidence of BMI effect were taken forward. In this analysis, SNP‐exposure associations were taken from our rerun of the 41‐cytokine GWAS and for CRP, from a recent meta‐GWAS   . SNP‐outDiseases were considered relevant if there was existing observational evidence of their association with BMI and/or cytokines. From this selection, diseases were included in the analysis if there existed, within MR‐Base , summaryFor each instrument (SNP), causal estimates were derived using the Wald ratio method, and when multiple instruments were available, estimates were combined using the inverse‐variance weighted method to provide a single causal estimate of the exposure‐outcome association . Methods2, respectively.Baseline characteristics of the participants in YFS and in FINRISK 1997 and 2002 are shown in Table https://doi.org/10.5523/bris.3g3i5smgghp0s2uvm1doflkx9x. Removing BMI as a covariate in the model had a minor impact on the GWAS results. The correlations between betas derived from the two models (with and without BMI fitted) were between 0.77 and 1 (median 0.99). Of the 27 SNP‐cytokine associations previously reported  increase in BMI . Overall, we identified 3, 6, and 33 SNPs as instruments for HGF, MCP‐1, and TRAIL, respectively; effect estimates for these and the three CRP SNPs used are shown in Supporting Information Table P = 0.03) per unit change in natural log‐transformed CRP levels    was in the opposite direction to that observed using the Immunochip data set  using the CARDIoGRAMplusC4D GWAS data but no apparent effect when using the smaller European‐only CARDIoGRAM GWAS data   of 0.91  Figure .In this study, we compared estimates from observational analyses with those from MR (1) to allow inference to be drawn about the causal relationship between adiposity and inflammation and (2) to evaluate the subsequent impact of BMI‐driven cytokines on relevant disease outcomes. We can now expand our understanding of the impact of BMI on the inflammatory profile beyond the “usual suspects”  in a systematic and robust way that makes use of new data. Using a two‐step MR design, we were able to partially replicate previous associations and contribute new knowledge. Overall, we saw little evidence for a detrimental effect on health of raised levels of the BMI‐driven cytokines examined. However, we cannot rule out the possibility that these cytokines may have small effects on disease risk that are not detectable here because of limited power. Our findings contribute to the emerging literature that points toward a complex role for cytokines in immune‐mediated diseases  and provIn observational analyses, the majority of cytokines within the IL subclass showed a positive association, with BMI having the strongest signal observed for IL‐18. Outside of the IL subclass, strong effects were also observed for HGF, TRAIL, and MIP‐1β. Although the majority of cytokines were found to be positively associated with BMI, both eotaxin and CTACK (CCL27) showed decreased levels with increased BMI. In the one‐sample MR framework, evidence was strongest for a BMI effect on HGF, MCP‐1, and TRAIL. These effects are commensurate with reductions in both HGF  and MCP‐Restricting our attention to HGF, MCP‐1, and TRAIL, we went on to consider whether these cytokines could be causally related to BMI‐associated disease outcomes. Increased levels of TRAIL appeared to be associated with a small increase in the odds of CAD but only when using the larger CARDIoGRAM‐plusC4D GWAS result. Although marginal, particularly within the context of the number of tests performed, this effect is of interest given the complex relationship of TRAIL and its receptor with atherosclerosis and vascular biology. TRAIL and its receptors are expressed in both physiological and pathological arterial walls . There iMCP‐1 mRNA expression in inflamed tissues, which in some cases was considered to be evidence for a pathological role of MCP‐1 in the disease , such that increased levels of the cytokine appear to decrease odds of disease. IBDs are immunologically mediated diseases characterized by chronic relapsing inflammation of the intestine. MCP‐1 is a chemokine that plays an important role in the recruitment of monocytes and macrophages from the bloodstream to inflamed tissues. Early studies involving intestinal biopsy specimens from patients with IBD showed increased  disease , 42. How disease . This aptrans and as such, we presume their effect must go through several pathways, opening the possibility that those pathways influence the outcome independently of the target exposure (ACKR1 and rs2228467 in ACKR2) generated estimates in the opposite direction to most other variants leading to heterogeneity in the estimates (P < 0.10 for IBD and UC). As anticipated, given their function, we found evidence that rs12075 and rs2228467 are associated with eight and four other cytokines (P < 0.05) in our data, respectively. One potential strategy for reducing the impact of pleiotropy could be to restrict analyses to cis variants only, as these effects are likely closer to the biology of the molecular trait because of their genomic proximity. However, this would reduce the power of the MR because of the small number of instruments that remain.One specific issue potentially complicating interpretation is pleiotropy. The idea that genetic variants are associated with levels of single cytokines seems unrealistic and instead what we expect is that pleiotropy  is more likely. If pleiotropy is present in the form of independent pathway effects , our MR estimates can be biased in specific conditions . Many ofexposure . The incAlthough various strategies have been developed to assess and minimize the impact of pleiotropy in MR , this isN = 50,000 (our threshold for inclusion) and assuming a 1:2 ratio of cases to controls, we had 80% power to detect an OR of 1.21, 1.19, and 1.05 for HGF, MCP‐1, and TRAIL, respectively  Integrative Epidemiology Unit at the University of Bristol, which is supported by the MRC (MC_UU_00011) and the University of Bristol. NJT is a Wellcome Trust Investigator (202802/Z/16/Z) and works within the University of Bristol National Institute for Health Research Biomedical Research Centre. LJC is supported by NJT’s Wellcome Trust Investigator grant (202802/Z/16/Z). NJT and VYT are supported by the Cancer Research UK Integrative Cancer Epidemiology Programme (C18281/A19169). JK is supported by the Academy of Finland (297338 and 307247) and the Novo Nordisk Foundation (NNF17OC0026062). MK was funded by Biocenter Oulu. VS is supported by the Finnish Foundation for Cardiovascular Research. The Young Finns Study has been financially supported by the Academy of Finland ; the Social Insurance Institution of Finland; Competitive State Research Financing of the Expert Responsibility area of Kuopio, Tampere and Turku University Hospitals (grant X51001); Juho Vainio Foundation; Paavo Nurmi Foundation; Finnish Foundation for Cardiovascular Research; Finnish Cultural Foundation; The Sigrid Juselius Foundation; Tampere Tuberculosis Foundation; Emil Aaltonen Foundation; Yrjö Jahnsson Foundation; Signe and Ane Gyllenberg Foundation; Diabetes Research Foundation of Finnish Diabetes Association; EU Horizon 2020 (grant 755320 for TAXINOMISIS and grant 848146 for TO‐AITION); European Research Council (grant 742927 for MULTIEPIGEN project); Tampere University Hospital Supporting Foundation; and the Finnish Society of Clinical Chemistry. SR is supported by the Academy of Finland Center of Excellence in Complex Disease Genetics (grant 312062), Academy of Finland (grant 285380), the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation, and University of Helsinki HiLIFE Fellow grant. MS is funded by Academy of Finland (grant 141136).VS has consulted for Novo Nordisk and Sanofi and has received honoraria from these companies. He also has ongoing research collaboration with Bayer Ltd . The other authors declared no conflict of interest.Supplementary MaterialClick here for additional data file.Table S13‐S16Click here for additional data file.