N‐vanillyl‐6‐nonenamide) being developed as a nonopioid, long‐lasting, site‐specific treatment for postsurgical pain management. The objective of these studies was to examine the safety and tolerability of vocacapsaicin in an osteotomy model in two animal species and to evaluate bone healing parameters. Rats undergoing unilateral femoral osteotomy received a single perioperative administration (by instillation) of vocacapsaicin . Rabbits undergoing unilateral ulnar osteotomy received a single perioperative administration (by infiltration and instillation) of vocacapsaicin  alone or in combination with 0.5% ropivacaine. Clinical signs, body weights, food consumption, radiography, histopathologic examinations, ex vivo bone mineral density measurements (rats only), and biomechanical testing were evaluated at 4 and 8 weeks in rats and at 2 and 10 weeks in rabbits. Plasma samples were also collected in rabbits. There were no vocacapsaicin‐related effects on mortality, clinical observations, body weight, or food consumption in either species. Systemic exposure to vocacapsaicin and its metabolites, including capsaicin, was transient. In rats, vocacapsaicin was devoid of deleterious effects on bone healing parameters, and there was a trend for enhanced bone healing in rats treated with the mid‐dose. In rabbits, vocacapsaicin administered alone or in combination with ropivacaine did not adversely affect bone healing parameters. In conclusion, a single perioperative administration of vocacapsaicin in unilateral osteotomy models was well tolerated, locally and systemically, supporting its continued development as a novel, nonopioid treatment for postsurgical pain management.Vocacapsaicin is a novel prodrug of trans‐capsaicin (trans‐8‐methyl‐ A cornerstone of this approach is multimodal analgesia, which utilizes a range of mechanistically distinct pharmacological treatments to inhibit nociceptive processing at multiple levels of the neuroaxis.N‐vanillyl‐6‐nonenamide) being developed as a nonopioid, site‐specific treatment for postsurgical pain management. It is designed to provide long‐lasting (at least 96 h) postsurgical analgesia following a single intraoperative administration. The free base form of vocacapsaicin rapidly cyclizes under physiological conditions to yield active capsaicin and CA‐101 (a cyclic urea) as the sole conversion products  13–14 weeks of age and weighing 230–330 g were individually housed in a room maintained at 19–25°C on a 12‐h light/dark cycle. Food and water were available ad libitum. The study plan was reviewed and approved by Charles River MTL Institutional Animal Care and Use Committee (IACUC).Rats were anesthetized with isoflurane gas and prepared for surgery. An incision was made on the lateral aspect of the right femur. The fascia, separating the tensor fascia lata and biceps femoris muscles, was identified and incised. The vastus lateralis muscle was elevated off the lateral border of the femur, from the greater trochanter to the lateral femoral trochanter condyle, with care to preserve the overlying periosteum. A fixation kit that included a connecting polyether ether ketone plate and four metallic screws was used. An osteotomy was performed at the midshaft using an oscillating saw. The osteotomy site was thoroughly irrigated with sterile fluids during the osteotomy to avoid any microdamage caused by heat. A cerclage wire was used to stabilize the bone and the site was irrigated with saline to remove bone debris. The soft tissues were meticulously closed in layers. At least 30 min before surgery, subcutaneous injections of buprenorphine (0.1 mg/kg), meloxicam (2 mg/kg), and trimethoprim sulfa (30 mg/kg) were administered to each rat. Following surgery, three additional injections of buprenorphine and trimethoprim sulfa were administered at 12‐h intervals , and two additional injections of meloxicam were administered at approximate 24‐h intervals . Additional doses of buprenorphine and meloxicam could be administered thereafter at the discretion of the study director.n = 35–36/group): 0 , 0.15, 0.3, or 0.6 mg/kg vocacapsaicin. Each dosing group was comprised of a 4‐week and an 8‐week subset of animals (n = 17–18/subset)  4–5 months of age were individually housed in a room maintained at 16–22°C on a 12‐h light/dark cycle. At initiation of dosing, males weighed 2.2–3.1 kg and females weighed 2.0–3.2 kg. All animals received 120 g per day of a standard commercial laboratory diet  and water was available ad libitum. The study plan was reviewed and approved by Charles River MTL IACUC.Rabbits were anesthetized with ketamine and dexmedetomidine before surgical preparation and were maintained under isoflurane anesthesia for the entire surgery. An incision of approximately 4 cm in length was performed on the lateral aspect of the distal ulna of the right forelimb and the ulna was exposed extraperiosteally. The periosteum was cut longitudinally and detached from the bone along the incision. A cut the width of the saw blade was performed in the mid‐ulna using a pendular saw. The soft tissues were meticulously closed in layers. Each animal received subcutaneous injections of trimethoprim‐sulfa 24% (30 mg/kg), buprenorphine (0.05 mg/kg), and carprofen (4 mg/kg) 30 min before surgery. Following surgery, additional doses of trimethoprim‐sulfa 24% (q12 h for 2 days), buprenorphine SR , and carprofen (qd for 7 days) were administered.Vehicle, vocacapsaicin, ropivacaine, and vocacapsaicin/ropivacaine were administered with a syringe needle on the day of surgery (Day 1) as described in Table Mortality and moribundity checks were performed twice daily throughout the study. Detailed clinical observations were performed before treatment, daily from Day 1 to Day 10, and weekly thereafter. Food consumption was measured daily from Day −1 to the end of the study. Body weights were measured weekly starting at Week −3.2.1.3Blood (1 ml target volume) from rabbits was collected from an appropriate auricular vessel before dosing and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, and 96 h postdose. Samples were mixed gently, and placed on crushed wet ice, and centrifuged as per standard procedures within 30 min after collection. The resultant plasma was separated into two approximately equal aliquots. Plasma samples were frozen immediately over dry ice and transferred to a freezer maintained at −20°C within 90 min from the time of blood collection. Plasma samples were analyzed  by liquid chromatography with tandem mass spectrometry for concentration of CA‐008, CA‐101, and capsaicin using a validated analytical procedure.Tmax), the maximum observed concentration (Cmax), the area under the concentration versus time curve from dosing to time of last quantifiable concentration (AUCt0‐), AUCt0‐/dose, Cmax/dose, and half‐life (t1/2).PK parameters were estimated for CA‐008, CA‐101, and capsaicin using noncompartmental methods with Phoenix PK software. The following parameters were calculated for each analyte: time after dosing at which the maximum concentration was observed  were performed under isoflurane anesthesia on the day of surgery using Dragon Digital‐X‐ray System (Raytech Diagnostics) for all animals, at Week 4 for animals in the 4‐ and 8‐week subsets using Faxitron (Faxitron Bioptics LLC), and at Week 8 for animals in the 8‐week subset. Radiographs were evaluated semiquantitatively to determine healing. The radiographs were graded for the extent of callus formation and bone union according to three categories, adapted and modified from An et al.Radiographs of the ulna  were performed under isoflurane anesthesia using MTM Digital X‐ray System  on the day of surgery and at Weeks 6, 8, and 10, and evaluated semiquantitatively to determine healing. The radiographs were graded for the extent of callus formation and bone union as described above for rats.2.1.5The right femur, popliteal, and iliac lymph node from eight animals per dose group from the 4‐ and 8‐week subsets were embedded in paraffin, sectioned, and mounted on glass slides. For the femur, one section was stained with hematoxylin and eosin and another section was stained with Safranin O. Lymph nodes were stained with hematoxylin and eosin. Histopathological evaluation was performed by a board‐certified veterinary pathologist. Microscopic evaluation was performed to assess healing of the osteotomy site, using a semiquantitative grading scheme in which callus maturity was graded on a 1 –5 (severe) scale.For the histopathological population, the right ulna bone and the surrounding soft tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Histopathological evaluation was performed by a board‐certified veterinary pathologist.2.1.62), bone mineral content (mg/mm), and bone mineral density (mg/cm3) were reported for the total osteotomy site, the mature callus area, and the immature callus area, using averaged values from analyses of the three approved slices.Peripheral quantitative computed tomography (pQCT) scans of the osteotomy site were performed using an XCT Research SA bone scanner (Stratec Medizintechnik). The first slice was performed proximally and adjacent to the osteotomy site and four subsequent slices were then acquired distally to image as much of the osteotomy site as possible. The three sequential slices determined to most accurately span the osteotomy site were selected for analysis. The area (mm2.1.7·mm). The bone data and associated report were peer reviewed by an external expert.A 4‐point bending test was performed on the right femur of up to 10 terminally euthanized animals per dosing group, from the 4‐ and 8‐week subsets, using the Bose Electroforce® 3300 System (TA Instruments). The femur was positioned on the lower fixture in the most stable position. The upper fixture was positioned in a manner that the mid callus was in the middle of the upper and lower contact points. Testing parameters included: peak load (N), stiffness (N/mm), and work to failure . The ulna was carefully separated from the radius using a diamond coated water‐cooled and low‐speed saw before biomechanical testing. The cranial aspect of the ulna was positioned on the lower fixture in the most stable position. Upper fixture was positioned in a manner that the midcallus was in the middle of the upper and lower contacts points. Testing parameters included: peak load (N), stiffness (N/mm), and work to failure .2.1.8A stock solution of vocacapsaicin (2.4 mg/ml) at pH 3 was prepared and used to prepare individual dosing solutions. The appropriate amount of vocacapsaicin item was dissolved in pH 3.0 saline to meet necessary volume requirements, and the pH of the resulting solution was adjusted to 4.5 using 0.01 N NaOH. Vehicle control was saline for injection, USP.Vocacapsaicin was dissolved in citrate buffer and diluted with saline, with a final pH of 3.6– 3.8. Ropivacaine  was filtered, aliquoted, and then dispensed as received on the day of dosing for administration. Vehicle control consisted of 0.5 mM citrate buffer and 0.4 mg/ml mannitol, in saline, with a final pH of 3.5–3.8.2.1.9t‐test or Wilcoxon rank sum test, respectively. In the rat study, pairwise comparisons between each dose of vocacapsaicin versus placebo were conducted. In the rabbit study, pairwise comparisons of interest included: vocacapsaicin (both doses) versus vehicle; and vocacapsaicin (both doses) + 0.5% ropivacaine versus 0.5% ropivacaine alone. Adjustments for multiplicity were made based on the square root of the number of pairwise comparisons. All statistical tests were conducted at the 5% significance level.Descriptive statistics of mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential analyses were conducted for body weight, bone biomechanics, and densitometry (rats only) using a one‐way analysis of variance or the Kruskal–Wallis test when group variances were not homogenous. If significance was achieved, then pairwise comparisons were conducted using a two‐sided 33.13.1.1Across the dose range tested, 0.15 mg/kg (0.3 mg/ml) to 0.6 mg/kg (1.2 mg/ml), there were no vocacapsaicin‐related effects on mortality, clinical signs, body weights, or food consumption. During the first week of the study, several animals lost weight, an effect attributed to the surgery. Correspondingly, food intake was decreased during the first week of the study and subsequently increased during the second week and remained constant for the remainder of the study.3.1.2At Week 4 in both the 4‐ and 8‐week subsets of animals, there was a trend for increases in healing scores in animals dosed with 0.3 mg/kg (0.6 mg/ml) vocacapsaicin compared to vehicle controls Figure . Healing3.1.3Consistent with the radiographic findings, in the 4‐week subset, an increased number of rats dosed with 0.3 mg/kg (0.6 mg/ml) vocacapsaicin had mature or nearly mature calluses  in comparison to vehicle controls  for the femur in 4‐point bending in animals administered 0.3 mg/kg (0.6 mg/ml) compared to controls, suggestive of increased callus strength  and 0.52 mg (0.65 mg/ml), there were no vocacapsaicin‐related effects on mortality, clinical signs, body weights, or food consumption, with or without 0.5% ropivacaine administration. Two male rabbits were euthanized in an unscheduled manner. One male rabbit administered vocacapsaicin 0.256 mg + 0.5% ropivacaine was euthanized on Day 39 because of the limited usage of the right forelimb, which had persisted since Day 2. At the macroscopic examination, the proximal right ulna was bent at the osteotomy site. The exact contributory effect vocacapsaicin and ropivacaine remain uncertain due to the expected individual variability in the healing process in this model. A second male administered vocacapsaicin 0.256 mg was euthanized on Day 29 because of large cutaneous ulceration on the scapular region, which was unrelated to vocacapsaicin administration.3.2.2Tmax observed at the first sampling time (15 min) and concentrations decreasing below the limit of quantitation by 2 h postdose  of the osteotomy sites did not occur in any of the animals at this time point; however, a callus was present for each animal, as expected for this model. At 10 weeks postsurgery, all animals evidenced complete union at the osteotomy site as well as decreased size of the callus (composed exclusively of bone) compared to those observed 2 weeks postsurgery. The incidence and severity of the callus formation were comparable between control animals and animals administered vocacapsaicin with or without ropivacaine.3.2.5At Week 10, there were no vocacapsaicin‐related differences in ulnar 4‐point bending  in males or females administered any dose of vocacapsaicin, alone or in combination with ropivacaine, relative to their respective controls Figure .4The objective of these studies was to examine the safety and tolerability of a novel prodrug of trans‐capsaicin, vocacapsaicin, in an osteotomy model in rats and rabbits, and to evaluate radiographic, histopathologic, and biomechanical bone healing parameters in both species. Vocacapsaicin was administered on a single occasion by instillation (rats) or a combination of instillation and infiltration around the surgical site (rabbits). There were no effects of vocacapsaicin on mortality, clinical signs, body weights, or food consumption when tested up to human equivalent doses of 5.4 and 9.3 mg (free base) in rats and rabbits, respectively.In addition to the lack of adverse clinical signs, there were no deleterious effects of vocacapsaicin on bone healing after osteotomy surgery in rats and rabbits as assessed macroscopically or microscopically and by radiography, densitometry (rats only), and biomechanical testing. This was true in rats assessed 4 and 8 weeks following a single perioperative administration of vocacapsaicin and in rabbits assessed 2 and 10 weeks following a single perioperative administration of vocacapsaicin, with or without ropivacaine. While there was considerable variability within treatment groups across bone healing parameters in both species, there was no discernable dose–response relationship following vocacapsaicin treatment for any parameter. The lack of effect on bone healing with vocacapsaicin is concordant with preclinical observations with an investigational, purified formulation of capsaicin in bone and wound healing models.Interestingly, in rats treated with the mid‐dose of vocacapsaicin, 0.3 mg/kg (0.6 mg/ml), there was a trend for enhanced bone healing relative to control rats, as evidenced by increases in radiographic bone healing scores, percentage of animals with mature or nearly mature calluses, and AUCs (+78%) for the femur in 4‐point bending. Such trends were not observed; however, for the low or high dose in rats or for either dose in rabbits. A potential role of TRPV1 receptors in bone healing has been previously documented in rodent models. For example, in a unilateral open bone (femur) fracture model, TRPV1 knockout mice displayed delayed fracture healing and a reduction in osteoclast and osteoblast differentiation.To evaluate the relevance of any potential local or systemic toxicities following vocacapsaicin administration, blood was collected in rabbits for PK evaluation. Across the doses studied, there was a general dose‐dependent increase in systemic exposure for intact vocacapsaicin, CA‐101, and capsaicin. The delivery of vocacapsaicin to the systemic circulation and the production of CA‐101 and capsaicin from vocacapsaicin occurred rapidly. Plasma concentrations were below the limit of quantitation by 2 h for intact vocacapsaicin and by 4 h for CA‐101 and capsaicin. Therefore, systemic exposure to the intact prodrug and its metabolites, including capsaicin, was transient. These findings are consistent with the lack of overt effects of vocacapsaicin on mortality, clinical signs, food consumption, and body weight. Additionally, in single‐ and repeat‐dose toxicology studies conducted in rats and rabbits, where full systemic histopathology and clinical pathology were specifically evaluated, there were no clinically relevant findings and the no‐adverse‐effect level was equal to or higher than the highest concentrations tested in the current study (unpublished data). When vocacapsaicin was administered in combination with ropivacaine, systemic exposure to intact vocacapsaicin, CA‐101, and capsaicin were somewhat lower relative to vocacapsaicin administration alone. Due to the small number of animals tested, it is not possible to ascertain whether this is due to interanimal variability or to a modest effect of ropivacaine on exposure to these analytes.In summary, a single perioperative administration of vocacapsaicin in rat and rabbit unilateral osteotomy models was well tolerated, locally and systemically, as demonstrated by the absence of mortality directly related to vocacapsaicin, adverse clinical observations, or effect on body weight and food consumption. Systemic exposure to vocacapsaicin and its metabolites, including capsaicin, was transient. In rats, vocacapsaicin was devoid of deleterious effects on bone healing as assessed microscopically and by radiography and biomechanical testing, and there was a trend for enhanced bone healing in rats treated with the mid‐dose, but not low or high doses, of vocacapsaicin. In rabbits, vocacapsaicin was also devoid of adverse effects on bone healing parameters and coadministration of ropivacaine did not alter clinical observations or bone healing findings. The collective findings are consistent with a product that could potentially provide a well‐tolerated, nonopioid, site‐specific treatment for long‐lasting postsurgical pain management.Tarra Knotts, Susan Kramer, and John Donovan are employees and shareholders of Concentric Analgesics. Other authors declare no conflict of interest.Tarra Knotts, Kirsten Mease, Lakshmi Sangameswaran, and John Donovan contributed to the design, analysis, and interpretation of data, and revising the manuscript. Susan Kramer contributed to the analysis and interpretation of data and revising the manuscript. All authors approved the final version and agree to be accountable for all aspects of the work.


Pregnancy has been described as a ‘teachable moment’ for behaviour change, which presents an important opportunity for health promotion within antenatal care settings. However, no pregnancy‐specific model has been developed or tested in the context of health behaviour change during pregnancy. This study aimed to investigate and compare the utility of the Capability‐Opportunity‐Motivation Behaviour (COM‐B) and Teachable Moments (TM) models, to explain health behaviour change during pregnancy, within the context of eating behaviour.Longitudinal cohort study.Five hundred and sixteen women completed a survey at between 12–16 weeks gestation (T1). Follow‐up data were collected at 20–24 weeks (T2), 36–40 weeks (T3), and 6–12 weeks postnatally (T4). The primary outcome was eating behaviour. To assess the utility of the COM‐B model, perceived capability, opportunity, and motivation to eat healthily were measured. To assess the utility of the TM model, risk perceptions, self‐image, and affective response were measured.Overall, the COM‐B model explained 18.4% of the variance in eating behaviour, whilst the TM model explained 9%. Both models explained the most variance in eating behaviour at T1 and T3, compared with T2 and T4. Small changes were observed in eating behaviour and the model constructs over the time period studied, although these were not clinically meaningful.Neither the COM‐B nor TM model provide a satisfactory explanation of eating behaviour during pregnancy, however the findings suggest that certain stages of pregnancy may create more salient opportunities for behaviour change. The findings also support claims that motivation may not play a key role in directing eating behaviour during pregnancy. Further research is needed to explore the role of timing in antenatal behaviour change. The development of a pregnancy‐specific model is necessary to optimise understanding of pregnancy as a teachable moment for behaviour change. What is already known on this subject?Pregnancy is regarded as a ‘teachable moment’ for health behaviour change.Existing models used to understand pregnancy as a teachable moment have not been developed, nor widely tested, in a pregnant population.The development of a pregnancy‐specific model of behaviour change is necessary.What does this study add?The COM‐B and TM models provide an insufficient explanation of eating behaviour during pregnancy.Certain stages of pregnancy may provide more salient opportunities for behaviour change than others.Motivation may not play a key role in directing eating behaviour during pregnancy.Pregnancy has been suggested to be an opportune time for health professionals to support women to initiate, or maintain, healthy behaviours. Increased contact with health professionals during this time and increased receptivity to health messages means that pregnancy is often described as a ‘teachable moment’ Phelan, . That isMcBride et al.  first atHowever, Olander, Darwin, Atkinson, Smith, and Gardner  argue thWhilst both the COM‐B and TM models appear to explain pregnancy as a teachable moment, neither has been tested prospectively in a population of pregnant women. Given that both models are used to explain antenatal behaviour change (e.g. eating behaviour), it is important to understand the efficacy of these models within this context. Further to this, pregnancy is often conceptualized as one teachable moment in and of itself , social media/forum posts , university mailing lists, and recruitment websites.Women were eligible to participate if they were over the age of 18 and between 12 and 16 weeks pregnant at the time of recruitment. Women also needed to be receiving maternity care in the United Kingdom, with a singleton pregnancy, and able to read and write in English to participate. Women unable to provide informed consent were not eligible to participate.As an incentive, participants were offered the opportunity to be entered into a prize draw to win a £100 shopping voucher, for each of the four surveys they completed. Women were provided with necessary details about study participation, but no additional information on the topic under investigation was provided.Data were collected at four time‐points: between 12 and 16 weeks of pregnancy (T1), 20–24 weeks (T2), 36–40 weeks (T3), and 6–12 weeks postnatally (T4). Informed consent was gained at each time‐point. T1 surveys were completed online using Research Electronic Data Capture (REDCap) tools hosted at the sponsoring university  and the NHS Research Ethics Committee (REC) .Unless specified, items were generated for the present study. Details of the measures used are presented in Table t‐tests and chi‐squared tests were conducted to compare variable means at T1 for different demographic groups, to further explore the data and check for differences between groups. Mixed regression models were performed to compare changes in scores over time in order to make use of all available data at each time‐point. Multiple linear regression models were used to identify which model better explains eating behaviour, controlling for time, age, ethnicity, BMI, education, IMD decile, parity, gestational diabetes, prior pregnancy complications, experience of sickness/nausea, and pre/post‐lockdown recruitment. Controlling for the same potential confounders (without time), individual regression models built for each time‐point were then used to assess the associations between model constructs and dietary quality, to identify if certain time‐points act as more salient teachable moments than others during pregnancy. Experience of sickness/nausea and perceived risk to baby were not included for the T4 analysis as these were not measured at this time‐point. Adjusted R2 is reported for all regression analyses.Data were analysed using IBM SPSS Statistics, version 25. Data were cleaned and composite scores created, where <20% data were missing, for all model constructs and the PUQE items. Frequencies and descriptive statistics were generated to assess sample characteristics and mean scores at each time‐point for all available data. Independent n = 302, 58.5%) were recruited prior to lockdown.Five hundred and sixteen participants completed the survey at the first time‐point (T1). Two hundred and sixteen (41.9%) participants were recruited online and 300 (58.1%) via NHS maternity services. Three hundred and two (58.5%) participants participated using the online survey and 214 (41.5%) using the paper version. Most participants , 210 (40.7%), and 198 (38.4%) participants providing data at T2, T3, and T4, respectively  of participants were recruited post‐lockdown. Compared to those recruited pre‐lockdown, these participants perceived greater risk to their own health , p < .001) and to the health of their baby , p < .001). Greater levels of worry about their health and that of their baby , p < .001), as well as greater perceived capability to eat healthily , p = .004) were also reported.41.5%  reported greater risk perceptions about their own health , p < .001) and that of their baby , p < .001), higher levels of worry about their health and that of the baby , p < .001), and higher levels of negative affect , p = .009) than those recruited in maternity care settings. This is congruent with the above findings relating to lockdown recruitment, as the majority of participants completing online surveys were also recruited post‐lockdown.Participants who were recruited online  reported higher levels of perceived risk about their own health during pregnancy , p = .049) and greater levels of worry about their own health and that of their baby during pregnancy , p = .03) than participants who did not complete all four surveys. No association was found between survey completeness and age (p = .09), ethnicity , or prior pregnancy difficulties or complications (X2 (1) = 0.35, p = .62), and parity (X2 (1) = 1.05, p = .33). However, participants who were educated to a postgraduate or higher level were more likely to provide data at all four time‐points , as were those who were recruited online (X2 (1) = 5.44, p = .02), and those from higher IMD quintiles (X2 (4) = 11.47, p = .02).At T1, participants who provided data at all four time‐points . The majority of participants were from White backgrounds , married , and in full‐time employment . Most participants had obtained a higher or postgraduate education level qualification , and more participants lived in the most deprived 10% of areas in England  than in any other decile. At T1, 41.1% of participants had a healthy BMI (n = 212).Participants were aged between 18 and 45 years (SD = 1.2), 20.7 weeks , and 36.4 weeks  at T1, T2, and T3, respectively, and a mean of 6.5 weeks  postpartum at T4. The largest proportion of participants had no existing children .Participants had a mean gestational age of 13.5 weeks  and 5.0% (n = 26) had used assisted reproductive technology to conceive. These figures are broadly reflective of those reported in the general population  and 1.4% had experienced a stillbirth (n = 7), which appears to be higher than general population rates  reported higher levels of worry about their own health and that of their baby during their pregnancy , p = .03) than those participants who had not experienced this.Participants who reported experiencing any type of prior pregnancy difficulties or complications  or moderate  symptoms of nausea and vomiting. At T2 and T3, the majority of participants reported experiencing no symptoms . See Table At T1, most participants reported experiencing mild (n = 16) of participants reported developing gestational diabetes. This is similar to rates of gestational diabetes reported in the general population of pregnant women in Europe  than participants without gestational diabetes at T3.By T3, 7.6% (p = .03), T2 , p < .001), and T3  p < .001).Participants’ perceived capability to eat healthily was significantly lower at T4 than at T1 , p < .001), T2  p < .001), and T3 , p < .001).Similarly, perceived opportunity to eat healthily was significantly less at T4 than at T1  There were no statistically significant differences in participants’ motivation to eat healthily from T1 through to T4.p = .01).There were no statistically significant differences in participants’ perceived risk to their own health from T1 through to T4. However, participants’ perceived risk towards the health of their baby was significantly higher at T2 than T1 , There were no statistically significant differences in participants’ self‐image from T1 through to T4.p = .002), and from T2 to T4 , p < .001).Participants’ levels of worry about their health and that of the baby reduced significantly from T1 to T4 , p < .001), T3 , p = .002), and T4 , p < .001), than at T1. Positive affect at T4 was also significantly higher than at T2 , p = .01) and T3 , p = .002). There were no statistically significant differences in negative affect at different time‐points.Positive affect significantly increased across time‐points. Levels of positive affect were significantly higher at T2 , M = 9.68, SD = 1.98) to T2 , dietary quality decreased over subsequent time‐points , with a significant decrease in quality from T2 to T4 , p = .03).After initially increasing from T1  and for every one unit increase in opportunity score, the dietary quality score will on average increase by 0.42 . There was no statistically significant association between motivation and dietary quality. These constructs account for 18.4% of the overall variance in dietary quality when including confounding variables.After including potential confounders, for every one unit increase in perceived capability score, the dietary quality score will on average increase by 0.30 . There was no statistically significant association between negative affect, worry, self‐image, or risk perception scores and dietary quality scores. These constructs account for 9% of the overall variance in dietary quality.After including potential confounders, for every one unit increase in positive affect score, the dietary quality score will on average increase by 0.29  and opportunity  p = .03), and dietary quality scores, at T1 and T3 respectively.Across time‐points, the amount of variance in dietary quality explained by the COM‐B model constructs was fairly similar, with 19.2%, 13.8%, 18.6%, and 16.9% of the overall variance in dietary quality explained at T1, T2, T3, and T4, respectively. In line with the previous findings, there was a significant association between perceived capability  p = .02). There were no other significant associations at any time‐point.The TM model constructs account for 9.1%, 2.7%, 12.5%, and 4.5% of the overall variance in dietary quality at T1, T2, T3, and T4, respectively. At T2, a significant association was found between positive affect and dietary quality scores  Although dietary quality scores remained relatively stable over time, the constructs of both models accounted for the most variability in dietary quality at T1 and T3. See Table This study investigated the extent to which the COM‐B and TM models explain health behaviour change during pregnancy, within the context of eating behaviour. The results reveal small changes in eating behaviour and the model constructs throughout pregnancy and the early postnatal period. Both models explained the most variance in eating behaviour in early‐ and late‐pregnancy compared with mid‐pregnancy and the postnatal period. Overall, the COM‐B model explained more of the variance in eating behaviour than the TM model.Whilst the COM‐B and TM models have been suggested to be useful models to understand pregnancy as a teachable moment, both assume that women’s behaviour will be influenced by simultaneous changes in the individual psychological constructs. The findings of this study revealed marginal changes in these individual constructs at different points throughout pregnancy; for example, women’s worries about their health and that of their baby decreased slightly over time, whilst levels of positive affect increased. Risk perceptions about the health of the baby also increased slightly from early‐ to mid‐pregnancy, and capability and opportunity were lowest in the postnatal period. Although these findings were statistically significant, the changes observed were arguably too small to be clinically meaningful. However, our findings suggest that there may be some fluidity in the extent to which these psychological constructs are experienced throughout pregnancy and the postnatal period. This is important as neither the COM‐B nor TM model consider that the salience of their respective constructs might change at different points in time. Consequently, previous research has suggested that these models may be limited when applied to the context of pregnancy, due to the changeable nature of the pregnancy experience and the static understanding of behaviour change that these models offer   and late‐pregnancy (36–40 weeks), compared with mid‐pregnancy (20–24 weeks) and the postnatal period (6–12 weeks postpartum). This suggests that there may be certain stages of pregnancy that present more effective opportunities, or teachable moments, for women to change their eating behaviour. These findings support claims made by Olander et al.  who suggThese findings strengthen the argument for the development of a pregnancy‐specific model of behaviour change that considers the potential for certain stages of pregnancy to provide more opportune moments for change. However, it is evident that further research is needed to better understand the influence of timing on women’s health behaviour. Utilizing a qualitative approach may provide an enhanced understanding of the different factors influencing women’s eating behaviour at different time‐points throughout pregnancy, that goes beyond that captured using a quantitative approach. Furthermore, it is important to consider the different influences on women’s eating behaviour during the antenatal and postnatal period, and associated implications for the development of behavioural support during these times.This is the first study to compare existing models of behaviour change in the context of antenatal eating behaviour. Longitudinal data were collected from a large sample of women throughout the duration of their pregnancies, providing new insight that has highlighted the insufficiency of existing models to explain eating behaviour during pregnancy. However, there are several limitations that must be considered.Firstly, the vast majority of participants in the sample were from a White background. Whilst a high proportion of residents in England and Wales identify as White  or being mindful of practical barriers faced by women from socioeconomically deprived backgrounds , such as high food costs, time constraints, or lack of availability of healthy food. From a clinical perspective, the findings highlight the importance of considering gestational stages when delivering interventions or health promotional advice. NICE guidelines  recommenWhilst the COM‐B model explained more variance in eating behaviour during pregnancy than the TM model, neither model provides a sufficient explanation. However, both models explained more variance in eating behaviour in early‐ and late‐pregnancy than in mid‐pregnancy or the postnatal period, suggesting that certain gestational stages might afford more effective teachable moments. Furthermore, motivation may not play a key role in eating behaviour change. Further research is required to better understand the influence of timing in eating behaviour during pregnancy, and for the development of a pregnancy‐specific model of behaviour change.This study was funded by the Medical Research Council under Grant MR/N013751/1 and supported by the NIHR Clinical Research Network Greater Manchester. The views expressed are those of the authors and not necessarily those of the Medical Research Council, NHS, NIHR, or the Department of Health and Social Care. AEPH is funded by Tommy’s Charity via a grant to the Maternal and Fetal Health Research Centre.All authors declare no conflict of interest.Table S1. Severity of sickness/nausea across time‐points.Click here for additional data file.


Menstrual cycle characteristics are linked to reproductive function and long-term health outcomes. This study aimed to evaluate menstrual cycle patterns, characterized by regularity and length, and associated factors among women in the Korea Nurses’ Health Study.A total of 9335 premenopausal women aged 22–45 years were included in this cross-sectional study. Regularity and length of menstrual cycles were self-reported, and their associations with reproductive, lifestyle, and occupational factors were examined using binomial and multinomial logistic regression models. Adjusted least-square means of menstrual distress, depressive symptoms, stress, fatigue, anxiety, and sleep problems were estimated according to menstrual cycle characteristics using generalized linear models.2 had higher odds of irregular  and long cycles  than those with BMI 18.5– < 23 kg/m2. Irregular cycles were less common in women performing vigorous physical activity, but more common in those with prolonged standing or frequent heavy lifting at work. Frequent rotating night shift was associated with irregular cycles among nulliparous women. Levels of menstrual and premenstrual distress, depressive symptoms, perceived stress, physical and mental fatigue, anxiety, and sleep problems were higher in women with irregular cycles than in those with regular cycles .Twenty-one percent of nurses reported having irregular menstrual cycles (variability > 7 days). Ten percent, 64%, and 26% had menstrual cycle length of < 26, 26–31, and 32–50 days, respectively. Variability and length of cycles decreased with age and increased with age at menarche. Parous women showed low tendency of irregular cycles. Women with body mass index (BMI) > 25 kg/mThe study suggests that irregular and long menstrual cycles are associated with reproductive, lifestyle, and occupational factors; also with menstrual distress and perceived health status. Our findings contribute to a better understanding of potential risk factors for menstrual dysfunction, and thus, may help improve women’s health.The online version contains supplementary material available at 10.1186/s12905-022-01947-z. The menstrual cycle is regulated by a complex interaction of hormones produced by the hypothalamus, pituitary, and ovaries . The menMenstrual disorders include disruption of menstrual patterns, ovarian dysfunction and menstrual pain, and are common in women of reproductive age . In prevIdentifying menstrual patterns and associated factors is the first step to understand women’s health and examine the long-term effects of reproductive experience. Moreover, more data on menstrual cycle characteristics from different populations should be obtained to explore factors contributing to variations in menstrual patterns. Nursing is a physically and emotionally demanding profession; therefore, investigating factors affecting menstrual cycle characteristics of nurses may provide valuable insights regarding reproductive health of working women.This study aimed to examine the association of reproductive, lifestyle, and occupational factors with menstrual cycle regularity and length in nurses enrolled to the KNHS. Furthermore, we investigated whether the levels of menstrual distress and perceived health statuses differed according to menstrual cycle characteristics.The KNHS is a web-based cohort study of Korean female nurses aged 20–45 years at enrollment, initiated in 2013 [n = 275) or those who had reached menopause (n = 75) at the time of answering the module 3. We also excluded women who were pregnant (n = 497), had given birth or breastfed within the last 6 months (n = 310), had undergone hysterectomy (n = 126) or oophorectomy (n = 180), who currently used hormonal contraceptives (n = 438), reported no periods (n = 156), were diagnosed with cancer (n = 248), or had missing data on menstrual cycle characteristics (n = 17). Of 10,882 eligible participants, we further excluded women who reported endometriosis (n = 270), uterine fibroids (n = 727), or PCOS (n = 702) to reduce confounding of gynecological conditions. Finally, 9335 women were included in this cross-sectional analysis. When we examined association of potential risk factors with menstrual cycle length, we excluded women who reported cycles longer than 50 days or too irregular to estimate (n = 292); 97% of them had usually or always irregular menstrual cycles.In the module 3, questions on menstrual cycle characteristics were included and 12,851 women responded to the survey. The median time interval between modules 1 and 3 was two years. For the current analysis, we excluded women aged > 45 years to reduce variation from perimenopausal status , an ongoing prospective cohort study of the US and Canadian nurses , 20. In 2), and categorized using the World Health Organization Asia–Pacific criteria as: underweight (< 18.5 kg/m2), normal weight (18.5– < 23 kg/m2), overweight (23– < 25 kg/m2) and obese (≥ 25 kg/m2) [In the module 1–the initial baseline survey–participants were asked about demographic information, disease history, reproductive factors, current weight and height, work conditions, perceived health status, and lifestyle factors including smoking status. Body mass index (BMI) was calculated as a ratio of body weight (kg) to height squared (mht 23– <  kg/m2 an5 kg/m2) , Patient5 kg/m2) , Chalder5 kg/m2) , the six5 kg/m2) , and Jen5 kg/m2) , respectIn the module 2–the first follow-up survey–the frequency and amount of the three types of alcoholic beverages over the past year were collected in the food frequency questionnaire (FFQ). Total alcohol consumption (g/day) was calculated as the sum of frequency of drinking multiplied by the alcohol content (ethanol) of the individual alcoholic beverages. Coffee consumption (cups/day) on average over the past year was also calculated using the FFQ.2), vigorous physical activity (none or tertiles), and alcohol consumption . P for trend was calculated by assigning the midpoint of each category and using as a continuous variable in the model. Sensitivity analyses were performed by excluding women aged > 40 years or restricting analyses to nulliparous women. We also confirmed associations among participants with a time interval of < 2 years between module 1 and 3.Descriptive analyses of the characteristics of participants were performed according to the regularity and length of menstrual cycles. Menstrual cycle regularity was categorized as “regular (cycle variability within 7 days)” and “irregular” and menstrual cycle length was categorized as “short < 26 days),” “moderate 26–31 days),” and “long (32–50 days)”  days,” a. Logisti6 days,” p < 0.05 was considered to be statistically significant. Results of Bonferroni correction were additionally reported for multiple comparisons.All statistical analyses were performed using SAS statistical software version 9.4 . A two-sided The characteristics of 9335 premenopausal women included in the study are presented in Table 2 had significantly higher odds of irregular cycles than women with BMI 18.5– < 23 kg/m2 . In terms of physical activity, women engaging in vigorous physical activity were less likely to have irregular cycles . However, the probability of having irregular cycles increased with increasing hours of standing work  and frequent heavy lifting at work ; p for trend < 0.05 for each. There were no significant associations of smoking status, alcohol and coffee consumption, and rotating night shifts with menstrual cycle regularity , and lower in women with higher levels of vigorous physical activity . Higher alcohol consumption was associated with higher odds of having short cycles and lower odds of having long cycles. The odds of having long cycle lengths decreased with increasing coffee consumption (p for trend = 0.030). For working conditions, prolonged standing was significantly associated with short cycle lengths . There were no significant associations of parity, smoking status, rotating night shifts, and heavy lifting at work with menstrual cycle lengths.The multivariable-adjusted associations between potential risk factors and short or long menstrual cycle lengths are presented in Table For sensitivity analyses, we excluded women aged > 40 years or restricted analysis to nulliparous women; the results were similar to those of the main analysis . The levels of depressive symptoms, perceived stress, physical or mental fatigue, anxiety, and sleep problems were significantly higher in women with irregular cycles than in those with regular cycles (p < 0.001 for each). The adjusted LSmeans across the four levels of cycle regularity were significantly increased with increasing variability of cycles . The levels of perceived stress and mental fatigue were significantly higher in women with short cycle lengths than in those with moderate or long cycle lengths. The results of sensitivity analyses were similar to those of the main analysis . Multivariable-adjusted LSmeans of menstrual distress and perceived health status according to the menstrual cycle characteristics are presented in Table In this cross-sectional study, 21% of the women reported irregular menstrual cycles, while 10% and 26% of the women reported short and long menstrual cycle lengths, respectively. Irregular menstrual cycles were associated with young age, late age at menarche, nulliparous, high BMI, and low physical activity; also, with long hours of standing work and frequent heavy lifting. Similar associations were observed in the analyses of short or long cycle length. The levels of menstrual and premenstrual distress were higher in women with irregular and short or long cycle lengths than in women with regular and moderate cycle length. The scores of depressive symptoms, perceived stress, physical or mental fatigue, anxiety, and sleep problems were higher in women with irregular cycles than in those with regular cycles.To our knowledge, this is the largest study that described the regularity and length of menstrual cycles in young Korean women. The proportion of women reporting irregular cycles in our study was higher than that in the fifth Korea National Health and Nutrition Examination Survey (KNHANES) conducted between 2010 and 2012 (14%) , 39. TheThe length of menstrual cycle generally ranges from 25 to 30 (median 28) days in healthy women and it decreases with age until menopause as the length of follicular phase shortens; and menstrual cycles are often irregular immediately after menarche and shortly before menopause , 3, 43. 2 had higher odds of having long and irregular menstrual cycles than those with BMI 18.5– < 23 kg/m2, even in the absence of PCOS and other gynecological conditions. Being underweight is also associated with menstrual disorders, and J-shaped associations of body composition measures with long or irregular cycles have been reported in previous studies [The prevalence of overweight and obesity are increasing globally in all ages and both sexes ; thus, t studies , 49. In Weight-loss, athletic activity, and stress have been associated with functional hypothalamic amenorrhea which is characterized by the suppression of gonadotropin-releasing hormone pulsatility . HoweverAssociations between shift works and menstrual cycles have been examined based on the hypothesis that disrupted circadian rhythms may play a role in menstrual dysfunction. A recent meta-analysis suggested that women who worked rotating shifts were more likely to experience irregular cycles than those working fixed day shifts and this association was more pronounced among studies with the mean age of < 30 years . The dosSome studies suggest that cigarette smoking may be associated with short or irregular menstrual cycles mainly by disrupting endocrine function , 53. We Perceived stress , 37, 57,Our study has several limitations. First, the data on menstrual cycle characteristics were self-reported. Low to moderate agreement between reported and observed menstrual cycle lengths have been suggested in other validation studies , 60. HowOur study provides evidence on menstrual cycle patterns and associated factors in female nurses in Korea. Irregular and long menstrual cycles were associated with young age, late age at menarche, high BMI, and low physical activity. Work conditions, including prolonged standing and frequent heavy lifting, were associated with short and/or irregular menstrual cycles. Women with irregular cycles had higher levels of menstrual distress, depressive symptoms, perceived stress, physical or mental fatigue, anxiety, and sleep problems than those with regular cycles. The findings are generally consistent with the existing literature and may contribute to a better understanding of modifiable risk factors for menstrual dysfunction. The results of this study may serve as a basis for further research on reproductive health.Additional file 1. Supplementary data. Tables S1 to S4.


Pongo pygmaeus morio) to investigate the feeding ecology of wild orangutans. Compared with African great ape species, orangutans are adapted to environments with chronic lower nutrition. But the usefulness of stable isotope analysis in the study of wild orangutan feeding ecology has not been fully explored.We applied stable carbon and nitrogen isotope analyses to wild Bornean orangutans . Elemental composition and stable isotope ratios of orangutan feces do not systematically vary by orangutans' sex and age classes, although fecal stable isotope ratios showed seasonal fluctuations. No isotopic contribution of breast milk was found in fecal samples collected from individuals at 2.7–6.5 years of age.This study revealed key characteristics of the stable isotope ecology of wild orangutans living in a primary lowland forest. Although there was little isotopic variation among plant foods and orangutan individuals, seasonal fluctuations in baseline isotope ratios or orangutans' diet were found in Danum valley. Flanged male orangutan eating bark. Stable isotope analysis has been used to investigate the diet of extant primate species and to reconstruct the paleoenvironment using fossil materials  to be up to ~5‰ lower than those in open habitats    increases δ15N  and stable isotope ratios. For example, lignin and cellulose are primary structural compounds of plant tissues, and their proportion is generally greater in hard plant parts, such as bark. The δ13C and δ15N values of primate tissues  will show significantly different stable isotope ratios as can be seen in African and Asian tropical forests  to characterize their feeding ecology. We tested the following five hypotheses to validate the utility of stable isotope analysis in studying the feeding ecology of wild orangutans.13C values of herbivore hard tissues, such as teeth and bones, because δ13C values in herbivore tissues systematically vary with the dietary proportion of C4 plants and openness of habitat  and JKM/MBS.1000‐2/2 JLD.4 (87)). Export of fecal and plant samples to Japan was granted by the Sabah state government (Transfer License JKM/MBS.1000‐2/3 JLD.3 (46)).2.22 located in northeast Borneo, Sabah, Malaysia. The area is predominantly lowland dipterocarp forest interspersed with some lower‐hill dipterocarp forest and is characterized by comparatively low fruit availability among rain forests in the world . The lodge caters to tourists and is managed by the Yayasan Sabah Group. The mean ± standard deviation (SD)  for monthly rainfall recorded at Danum Valley Field Research Center (DVFC) of the DVCA (~15 km south from BRL) during the study period was 242.6 ± 108.8 mm .DVCA is ~438 kmP. pygmaeus morio) in the DVCA has been ongoing since 2004, and > 50 orangutan individuals have been identified and monitored  during the study period in BRL, as well as the difficulty in the collection of tiny infant feces that dropped from the higher canopy, only one fecal sample from an infant was available for this study. Two samples with outlier values were excluded from the data set  to avoid bias resulting from vertical stratification and difference in developmental stages  and δ15N (‰), with Vienna Pee Dee Belemnite (VPDB) and atmospheric nitrogen (AIR) as the standard for carbon and nitrogen, respectively. Isotope ratios were calibrated using three laboratory standards: L‐tyrosine , L‐proline , and DL‐alanine . These isotope compositions were calibrated with authentic standards  coupled to an isotope ratio mass spectrometer  through a continuous flow interface  with modification specifically made to improve the sensitivity of the analysis  and 0.7 ± 1.4‰ (from −2.6‰ to 3.6‰ in the full range), respectively (n = 71)  and 2.1 ± 0.8‰  , %N , δ13C , and δ15N  values between the infant/juveniles (2.7–6.5 years) and mothers . Fecal δ13C values obtained from infant/juvenile individuals in DVCA were used for comparison because enamel δ13C values represent dietary input during orangutan tooth formation from ~0 to 8 years of age , using the Bonferroni method  and Middle to Late Pleistocene and Modern . No significant difference was seen between dietary δ13C values calculated from Holocene and Modern samples .Dietary δe Figure . A Krusk44.113C and δ15N values , an understory plant sometimes consumed by orangutans. A single data point for ginger is included in this data set, but its δ13C value (−28.9‰) is rather greater than the overall mean δ13C value of plant samples (−30.3 ± 1.4 ‰). A canopy effect is lacking for ginger or perhaps absent in the DVCA. Other understory plants were not sampled, and these alternatives cannot be distinguished.Orangutans are arboreal animals, rarely moving to the ground, and thus consume plant foods higher in the canopy , and homogenous stable isotope ratios of food sources observed in the DVCA limits the utility of stable isotope analysis to differentiate intrapopulation dietary differences in a primary forest. We did not find systematic variations in δ13C and δ15N values of orangutan feces were found , and the periodic fluctuations in δnd Table . Therefo Heaton, , the obs13C, and δ15N values do not differ significantly between the one infant, as well as juveniles older than 2.7 years old, and their mothers  and mothers, which is contrary to Hypotheses 2 , 4 (isotopic effect of breastfeeding), and 5 . However, fecal δ13C and δ15N values fluctuated with rainfall, which is consistent with Hypothesis 3 . Several other aspects of isotopic ecology in orangutans, such as data from other types of habitats, data from mast fruiting periods, relationship with behavioral and ecological factors, a large sample size of orangutan age‐sex classes, and anthropogenic impacts, need to be investigated further to evaluate the usefulness of stable isotope analysis in ecological, evolutionary, and conservation studies of wild orangutans.Carbon and nitrogen stable isotope analysis was applied to feces and plant foods of wild Bornean orangutans in DVCA to characterize their feeding ecology. Stable carbon and nitrogen isotope ratios of plant species consumed by wild Bornean orangutans were homogenous, contrary to Hypothesis 1 (isotopic variation among plant parts). No significant differences were observed in %C, %N, δTakumi Tsutaya: Conceptualization (lead); formal analysis (lead); funding acquisition ; investigation ; methodology ; project administration (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Anna Wong: Conceptualization (supporting); project administration (supporting); resources ; writing – original draft (supporting). Peter T. Malim: Conceptualization ; resources . Henry Bernard: Conceptualization ; resources ; writing – original draft (supporting). Nanako O. Ogawa: Funding acquisition ; investigation ; visualization (supporting); writing – original draft (supporting). Naohiko Ohkouchi: Funding acquisition ; investigation ; validation (supporting); writing – original draft . Shun Hongo: Formal analysis ; visualization (supporting); writing – review and editing . Tomoyuki Tajima: Funding acquisition ; resources ; writing – original draft (supporting); writing – review and editing (supporting). Tomoko Kanamori: Conceptualization ; data curation ; funding acquisition ; methodology ; resources ; writing – original draft (supporting); writing – review and editing (supporting). Noko Kuze: Conceptualization ; data curation ; funding acquisition ; methodology ; resources ; writing – original draft (supporting); writing – review and editing .The authors declare no conflicts of interest.Supplementary Figure S1 Comparison of (a) %C, (b) %N, (c) δ13C, and (d) δ15N values of paired plant samples obtained from the same species + parts and collected during different periods.Supplementary Figure S2. Comparison of plant (a) %C, (b) %N, (c) δ13 C, and (d) δ15N values obtained from different parts of the same species.Supplementary Figure S3. Age change in infant/juvenile–mother difference in (a) %C, (b) %N, (c) δ13 C, and (d) δ15N values of the paired infant/juvenile–mother fecal samples collected within the same month. Raw data were used instead of average data of infant fecal samples collected from the same individual during the same month.Supplementary Table S1. Individual information of the subject orangutans in this study. The estimated age of orangutan individuals, as of September 1st, 2015, with information on the relatively precise birth date.Supplementary Table S4. Summary of the plant samples collected during different periods.Supplementary Table S5. Results of the paired Mann–Whitney U tests for the same plant species + parts collected in different periods.Supplementary Table S6. Explanatory variables and intercept in the linear mixed models for fecal %N of wild orangutans in DVCA. Significant fixed effects are shown in bold.Click here for additional data file.Supplementary Table S2 Carbon and nitrogen stable isotope ratios of the analyzed fecal samples. Excluded samples as an outlier are marked with an asterisk.Click here for additional data file.Supplementary Table S3 Carbon and nitrogen stable isotope ratios of the analyzed food samples. Orangutan individual that consumed the plant sample was also shown. Excluded samples as an outlier are marked with an asterisk.Click here for additional data file.


Accelerated cervical cancer control will require widespread human papillomavirus (HPV) vaccination and screening. For screening, sensitive HPV testing with an option of self‐collection is increasingly desirable. HPV typing predicts risk of precancer/cancer, which could be useful in management, but most current typing assays are expensive and/or complicated. An existing 15‐type isothermal amplification assay  was redesigned as a 13‐type assay (ScreenFire) for public health use. The redesigned assay groups HPV types into four channels with differential cervical cancer risk: (a) HPV16, (b) HPV18/45, (c) HPV31/33/35/52/58 and (d) HPV39/51/56/59/68. Since the assay will be most useful in resource‐limited settings, we chose a stratified random sample of 453 provider‐collected samples from a population‐based screening study in rural Nigeria that had been initially tested with MY09‐MY11‐based PCR with oligonucleotide hybridization genotyping. Frozen residual specimens were masked and retested at Atila Biosystems. Agreement on positivity between ScreenFire and prior PCR testing was very high for each of the channels. When we simulated intended use, that is, a hierarchical result in order of clinical importance of the type groups (HPV16 > 18/45 > 31/33/35/52/58 > 39/51/56/59/68), the weighted kappa for ScreenFire vs PCR was 0.90 (95% CI: 0.86‐0.93). The ScreenFire assay is mobile, relatively simple, rapid (results within 20‐60 minutes) and agrees well with reference testing particularly for the HPV types of greatest carcinogenic risk. If confirmed, ScreenFire or similar isothermal amplification assays could be useful as part of risk‐based screening and management. What's new?Due to cost and perceived complexity, most existing human papillomavirus (HPV) assays for cervical cancer screening are designed to yield a pooled result for the carcinogenic HPV types. Here, to promote rapid, affordable and risk‐based cervical screening, an existing isothermal DNA amplification test was redesigned to group the carcinogenic HPV types into four channels based on clinical importance . In masked retesting of 453 Nigerian specimens, the new ScreenFire assay showed good‐to‐excellent type group agreement with prior PCR testing. When validated, the redesigned test could support risk‐based screening in resource‐limited settings. ANOVAanalysis of varianceCEConformité EuropéenneCIconfidence intervalCIN2+cervical intraepithelial neoplasia grade 2+COVID‐19Coronavirus Disease of 2019ctcycle thresholdHPVhuman papillomavirusHRHigh‐riskIARCInternational Agency for Research on CancerIQRinterquartile rangeIRBInstitutional Review BoardsLBCliquid‐based cytologyNCINational Cancer InstitutePCRpolymerase chain reactionSPSSStatistical Package for Social SciencesWHOWorld Health Organization1Cervical cancer incidence and mortality are highest in lower‐resource regions of the world.,Nevertheless, HPV infections are too common and benign in many high‐risk regions to treat all infected women.Due to cost and perceived complexity, most existing HPV assays are designed to yield a pooled result for the carcinogenic HPV types with no or only limited genotyping (mainly of HPV16 and HPV18), obscuring the finer risk‐stratifications within the other HR HPV types . Moreover, many existing assays also include HPV66 and a few include HPV53 . The latest version of the IARC monograph, rectifying the mistake of the previous versions, reclassifies HPV66 along with HPV53 as only possibly and rarely carcinogenic to humans .Additionally, HPV assays need to be rapid, low‐cost and practical for scale‐up in cervical cancer screening programs in resource‐limited settings. An isothermal amplification technology‐based AmpFire HPV assay  shows promise in this regard.We collaborated with the scientists at Atila Biosystems to redesign the AmpFire HPV assay into a single‐step 13‐type assay providing risk‐based, extended HPV genotyping in four channels in a single tube as follows: (a) HPV16, (b) HPV18/45, (c) HPV31/33/35/52/58 and (d) HPV39/51/56/59/68. These efforts were undertaken for public health benefit. The current article aims to independently validate the redesigned AmpFire HPV assay (ScreenFire HPV RS hereafter referred to as ScreenFire) compared against a well‐validated MY09‐MY11‐based polymerase chain reaction (PCR) assay with genotyping.22.1Our study utilized 1339 residual provider‐collected cervical samples from a population‐based screening study of 16 to 88 year‐old (mean [SD] = 44.0 [15.8]) women in rural Nigeria. Project Itoju was conducted during 2009 to 2010; residual specimens were stored frozen in Preservcyt solution . The study methodology has been described in detail previously.One milliliter aliquots of the frozen residual cytology samples were tested in the United States (Albert Einstein Cancer Center) for HPV using a AmpliTaq Gold MY09‐MY11 PCR‐based test that included additional type‐specific primers, and primers to amplify a cellular beta‐globin fragment as an internal control (IC) for amplification as previously described.Of the 1339 such residual samples, valid PCR results were available for 1305 samples. A small set (n = 8) of typed samples positive for only low‐risk HPV types was unblinded and provided to the Atila Biosystems laboratory for the modification of the AmpFire assay chemistry. Of the remaining 1297 masked samples with valid results, we chose all 356 samples that had tested positive for any HPV types, including low‐risk types, by either PCR or HPV16/18 Luminex, and 100 randomly selected specimens testing HPV negative for all HPV types including low‐risk, by both PCR and Luminex. These specimens were provided to Atila BioSystems with masked labeling and retested there with the ScreenFire HPV assay.The first masked comparison generated fair to good agreement (data not shown). There was evidence of competition for reagents in the presence of multiple HPV infections, and slightly diminished sensitivity for some types. We provided the summary results to the Atila Biosystems team and re‐randomized the specimens under NCI direct supervision to permit another valid masked testing round. After primer redesign and reagent optimization were completed, the Atila Biosystems team “locked” their new format, which emphasized more sensitive detection particularly of HPV16 and HPV18/45. The masked testing of the specimen set was then completely repeated. This article reports the independent analysis of results from the second round of testing.2.2The assay is intended to be performed on “dry swabs”; thus, centrifugation was needed to process the 0.5 mL of cervical cell suspension in Preservcyt. Specifically, the suspension was transferred to a 1.5 mL tube for centrifugation at maximum speed for 20 minutes and the supernatant was discarded, followed by the addition of 50 μL of ×1 lysis buffer and vortexing thoroughly to resuspend the cell pellet. The resuspended cell pellets were then transferred to 96‐well PCR plates for incubation at 95°C for 15 minutes, cooled at room temperature and briefly spun. After this initial sample preparation, 5 μL of this prepared specimen was mixed with 20 μL of freshly prepared master mix (including reaction mix and primer mix) into a 96‐well PCR plate using hand pipetting. Additionally, positive and negative template controls were included in each 96‐well plate as well. Next, the plates were sealed with an optical compatible film, vortexed gently for mixing the reagents and centrifuged to bring down all liquid to the bottom of the wells. The plates were then loaded into the Bio‐Rad CFX96 real‐time PCR machine on the isothermal program mode run at 1 minute per cycle at 60°C for 60 cycles with fluorescence obtained from CY5 (for HPV16), ROX (for HPV18/45), CY5.5 (for HPV31/33/35/52/58), FAM (for HPV39/51/56/59/68) and HEX . A sample was considered positive for an HPV channel if the signal was detected within 60 minutes in the channel, regardless of the signal in the HEX channel. If no signal was detected for any of the four HPV channels within 60 minutes, then a signal was required in the HEX channel for the batch run to be called a valid negative.2.3Valid ScreenFire HPV results were available for 453 of the 456 samples and were included in the analysis. HPV results from PCR were categorized a posteriori into the same four risk groups .First, HPV group results were considered nonhierarchically, recognizing that a given specimen could test positive for more than one channel. Agreement statistics and McNemar's test for asymmetry were calculated for each of the channels. The analysis accounted for sampling of HPV‐negatives by reweighting to the original sample size for point estimates, but maintained standard errors derived from the subset tested. Box and whisker plots were examined for time to detection  of the positive samples for each channel by the ScreenFire HPV assay. Outlier positive samples were identified as the ones with “time to detect positive” of greater than Q3 (third quartile of detection times) + 1.5 times interquartile range (IQR) in minutes. For the samples that tested positive by both the ScreenFire and PCR assay, an association between the time to detect positive in minutes by the ScreenFire assay and the signal strength by PCR assay was examined by Analysis of Variance (ANOVA) and nonparametric ranked test for trend in a pooled analysis combining the channels, including only samples positive by PCR for a single HR13 HPV type and positive by ScreenFire for a single HPV channel (n = 127). The PCR signal strength variableSecond, the two tests were also compared using risk‐based hierarchical HPV group types, considering that varying risks of cervical cancer associated with different risk groups are usefully interpreted according to the highest‐risk result obtained to permit risk‐based management. Thus, the hierarchical risk‐groups were considered as HPV16 positive, else positive for HPV18 or HPV45 (if HPV16 was not present), else positive for HPV31, HPV33, HPV35, HPV52 or HPV58 , else positive for HPV39, HPV51, HPV56, HPV59 or HPV68, else negative. Agreement on positive results and kappa values were calculated for the hierarchical analysis.As a supplementary analysis, the investigational Luminex testing for HPV16 and HPV18 was examined to look for clues as to the meaning of interassay disagreement. Results from this assay are presented because they revealed interesting patterns but are outside the a priori comparison.All analyses were conducted using Microsoft Excel, SPSS  (Build 1.0.0.1089) and R (version 3.6.2).33.1There was good to excellent agreement between the ScreenFire assay and the PCR assay for all the channels on nonhierarchical analyses Table . No majoWhenever there was an agreement between PCR and the investigational Luminex assay for HPV16, ScreenFire always agreed with them. When there was discrepancy between PCR and Luminex assay (n = 19), ScreenFire agreed with Luminex for 57.9% (11 of 19) times and with PCR for 42.1% (8 of 19) times. Whenever there was an agreement between PCR for HPV18/45 and investigational Luminex assay for HPV18, ScreenFire for HPV 18/45 agreed with them for all but one time. When there was discrepancy between PCR and Luminex assay (n = 14), ScreenFire agreed with Luminex for 21.4% (3 of 14) times and with PCR for 78.6% (11 of 14) times. This is likely due to lack of HPV45 in Luminex assay   (data not shown). However, a significant negative correlation was found between the time to detect positive HPV signals and the time to detect positive internal control (HEX) signal on the ScreenFire HPV assay (Pearson's correlation coefficient = (−0.25), P‐value =.001; Spearman's correlation coefficient = (−0.38), P‐value <.001), suggesting some reagent competition.A significant association was found between the time to detect positive samples on the ScreenFire HPV assay and the signal strength by PCR  Figure . No sign3.2On the hierarchical analysis, which would be useful for risk‐based clinical management of the women, there was an overall 96.8%  agreement between the ScreenFire and PCR assay  Table . OverallData on valid liquid‐based cytology and histopathology were available for only 420 and 172 samples, respectively. Of these, for the 12 histopathologic high‐grade Cervical intraepithelial neoplasia grade 2+ (CIN2+) cases, 10 were detected by both the ScreenFire and PCR assay, both assays missed one case and the ScreenFire assay picked up one additional CIN2 on the HPV39/51/56/59/68 channel. Nevertheless, both assays were positive for all five CIN3 cases. In fact, out of 36 cytologic or histopathologic high‐grade samples, there was only one discordant result , simple  and less expensive than most other HPV typing tests .The assay could help to address an identified public health need. In resource‐limited settings, which account for 90% of cervical cancer deaths, vaccine coverage and particularly organized screening are minimal.As a result of this satisfactory pilot evaluation, the ScreenFire HPV assay is being evaluated in two independent US laboratories, for clinical accuracy against both prior HPV reference testing and histopathologic outcome, based on an extensively‐validated set of >2000 provider‐collected cervical samples.5In conclusion, the redesigned ScreenFire HPV assay was shown to provide accurate risk‐based genotype grouping. If the ongoing large validation study in two independent laboratories yields similar results, the assay using self‐collected dry swabs will be deployed for field evaluation in multiple international cervical cancer screening sites. The longer‐range public health goal is implementation in resource‐limited settings.The work reported in the article has been performed by the authors, unless clearly specified in the text. Conceptualization, Mark Schiffman, Silvia de Sanjose, Kanan T. Desai; methodology, Mark Schiffman, Kanan T. Desai, Silvia de Sanjose, Didem Egemen; formal analysis, Kanan T. Desai, Mark Schiffman, Didem Egemen; Itoju field investigation, Mark Schiffman, Kayode O. Ajenifuja, Clement A. Adepiti, Julia C. Gage, Nicolas Wentzensen, Robert D. Burk; original PCR and Luminex testing, Robert D. Burk; resources, Mark Schiffman, Kayode O. Ajenifuja, Clement A. Adepiti, Nicolas Wentzensen; data curation, Kayode O. Ajenifuja, Julia C. Gage, Clement A. Adepiti, Kanan T. Desai; writing—original draft preparation, Kanan T. Desai; writing—review and editing, all. All authors have read and agreed to the published version of the article.The authors declare no conflict of interest. Atila BioSystems had no role in the design, analysis, interpretation or drafting of this article.Both Nigerian and National Cancer Institute  institutional review boards (IRBs) (NCT 00804466) approved the original study protocol that included consent for specimen storage for future research.


The prevalence of obstructive sleep apnea (OSA) in patients with suspected coronary heart disease (CHD) is yet to be clarified. This study aimed to investigate the prevalence of OSA before coronary angiography (CAG).We retrospectively evaluated patients with suspected CHD admitted to the Department of Geriatric Cardiology of our hospital between July 2019 and July 2021. OSA was screened using the level III home sleep apnea test before CAG. The prevalence of OSA was then compared between the CHD and non‐CHD groups. CHD severity was determined using the Gensini score of CAG results, and OSA severity was graded using the apnea‐hypopnea index (AHI).P < 0.01) and higher AHI . The Gensini score was positively correlated with OSA severity in patients with CHD, and AHI ≥ 20 was a risk factor for CHD .Among the 327 patients, 211 had CHD. In total, 264 patients were diagnosed with OSA (80.7%) . The CHD group had a significantly higher prevalence of OSA (OSA screening before CAG revealed a higher prevalence in CHD patients than in non‐CHD patients. The degree of coronary artery obstruction is positively correlated with AHI, and AHI ≥ 20 is a risk factor for CHD. Therefore, attention should be paid to OSA screening and management before CAG in patients with suspected CHD. OSA screening for patients with suspected coronary heart disease before CAG showed that the prevalence of OSA in CHD patients was higher than that in non‐CHD patients. AHI ≥ 20 is a risk factor for CHD. Therefore, OSA screening and management should be paid attention to before CAG in patients with suspected CHD. All patients underwent CAG because of a suspected CHD for the first time. The exclusion criteria were as follows: (1) left ventricular ejection fraction <40%; (2) central and mixed sleep apnea; (3) history of upper airway surgery or current continuous positive pressure ventilation (CPAP) therapy and (4) within 3 months use of oral sedative sleeping pills.This study was approved by the ethics committee of Guangdong Provincial People's Hospital (No. GDREC2019757H).2.2Basic patient data, including sex, age, body mass index (BMI), personal history, family history and complications, were collected. On day 2 of admission, fasting blood samples were collected, and blood cell analysis, liver function, renal function, blood lipid and blood sugar levels were measured before CAG was performed.2.32) using a level III HST  one night before angiography. The AHI was defined as the average number of apnea and hypopnea events per hour. OSA was diagnosed according to symptoms of sleep apnea, such as habitual snoring, interrupted breathing or awakening from sleep due to holding breath, and apnea or hypopnea occurring more than five times per hour, with a minimum SpO2 at night as a reference, following the American Academy of Sleep Medicine Clinical Practice Guideline2 at night being 85–90%; moderate, 80–84%; and severe, <80%. The CHD group was divided into a simple CHD group (AHI < 5 times/h), mild OSA group (5 ≤ AHI < 15 times/h), moderate OSA group (15 ≤ AHI < 30 times/h) and severe OSA group (AHI ≥ 30 times/h).All patients were monitored for the sleep apnea‐hypopnea index (AHI) and oxygen saturation , and the score without a coronary artery lesion was defined as 0. Coronary artery scores were divided into three grades: normal (0), grade 1 (1–80) and grade 2 (81–160).2.5−x ± s) and were compared between groups using the t test. Meanwhile, enumeration data are expressed as rate (%) and were compared between groups using the χ2 test. Pearson's test was used to determine the correlation between variables. Risk factors for CHD were analysed using logistic regression analyses. All statistical analyses were performed using SPSS 20.0 software . Statistical significance was set at P < 0.05.Normally distributed measurement data are expressed as mean ± standard deviation (33.1P > 0.05). The CHD group was older (P < 0.01) and included a higher proportion of men and patients with chief complaints of palpitations, hypertension and diabetes . The patient characteristics are shown in Table In total, 327 patients were evaluated. Among them, 211 (64.53%) and 116 (35.47%) belonged to the CHD and non‐CHD groups, respectively. There were no significant between‐group differences in BMI and proportion of patients complaining of chest tightness, chest pain or shortness of breath between the two groups . However, creatinine levels were higher in the CHD group (P < 0.01), while the levels of total cholesterol, low‐density lipoprotein and high‐density lipoprotein were higher in the non‐CHD group (P < 0.01) . Meanwhile, the results of sleep respiration monitoring showed that there was no statistically significant difference in the time of blood oxygen <90% between the CHD and non‐CHD groups (P > 0.05). AHI was higher in the CHD group than in the non‐CHD group (P < 0.01), and both mean and minimum blood oxygen levels were higher in the non‐CHD group than in the CHD group (P < 0.01)  patients in the CHD group and 80 cases (69.0%) in the non‐CHD group, with the prevalence of OSA being significantly higher in the CHD group (3.3P = 0.149). The average Gensini scores of the severe OSA, moderate OSA, mild OSA and simple CHD groups were 38.18 ± 34.53 points, 24.67 ± 20.41 points, 24.91 ± 23.09 points and 26.15 ± 19.47 points, respectively. The Gensini score was significantly higher in the severe OSA group than in the other groups (P = 0.022) , and the proportion of patients with admission symptoms of palpitation in the non‐CHD group was higher than that in the CHD group (P < 0.01). After CAG excluded CHD post‐admission, the symptoms of chest tightness, chest pain, palpitation and shortness of breath in non‐CHD patients may be caused by OSA. Second, the selected subjects were all from the elderly cardiovascular department, and their age was high, with an average of 60.38 years. Previous studies have shown that the prevalence of OSA increases with age, and it is generally high in the elderly population.In this study, the prevalence of OSA in the non‐CHD group was significantly higher, which may be due to the following reasons: First, the selected patients were hospitalized for suspected CHD, and they generally complained of chest tightness, chest pain, palpitation, shortness of breath and other symptoms, which may be caused by CHD or OSA. In addition, the proportion of patients with admission symptoms of chest tightness, chest pain and shortness of breath in the non‐CHD group was not significantly different from that in the CHD group . All subjects signed an informed consent form and agreed to participate in the study.All authors contributed to the study conception and design. Data collection was performed by Qiong Ou, Guo Pei, Jiaoying Tan and Yongchi Chen. Data collection and analysis were performed by Guo Pei. The first draft of the manuscript was written by Guo Pei, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.