Geometric morphometric methods of capturing information about curves or outlines of organismal structures may be used in conjunction with canonical variates analysis (CVA) to assign specimens to groups or populations based on their shapes. This methodological paper examines approaches to optimizing the classification of specimens based on their outlines. This study examines the performance of four approaches to the mathematical representation of outlines and two different approaches to curve measurement as applied to a collection of feather outlines. A new approach to the dimension reduction necessary to carry out a CVA on this type of outline data with modest sample sizes is also presented, and its performance is compared to two other approaches to dimension reduction.Two semi-landmark-based methods, bending energy alignment and perpendicular projection, are shown to produce roughly equal rates of classification, as do elliptical Fourier methods and the extended eigenshape method of outline measurement. Rates of classification were not highly dependent on the number of points used to represent a curve or the manner in which those points were acquired. The new approach to dimensionality reduction, which utilizes a variable number of principal component (PC) axes, produced higher cross-validation assignment rates than either the standard approach of using a fixed number of PC axes or a partial least squares method.Classification of specimens based on feather shape was not highly dependent of the details of the method used to capture shape information. The choice of dimensionality reduction approach was more of a factor, and the cross validation rate of assignment may be optimized using the variable number of PC axes method presented herein. Quantitative morphometric methods have long been used to classify organisms. Discriminant function analysis (DFA) or canonical variates analysis (CVA) are often used to support the identification of distinct species, particularly in fossil lineages -4 and alTraditional quantitative morphometrics have made use of a variety of lengths, widths, angles, and ratios to capture information about shape. Geometric morphometric approaches to shape have focused on complete, uniform measurement of shape, retaining all geometric information throughout the analysis. Within this context, measurement of curves or outlines poses some challenges, since mathematically curves are infinite sets of points. The use of multivariate statistical methods  to classify specimens into groups requires that the curves or outlines on the specimens be represented by a limited number of measured variables. The linear CVA requires a matrix inversion of the pooled covariance matrix requiring more specimens than the sum of the number of groups and measurements per specimen. Classification of specimens based on outlines thus poses a challenge, in that accurate representation of a curve requires many measurements accurately, but this increase in parameters dramatically increases the sample sizes necessary to carry out the CVA.A variety of geometric morphometric approaches to curves have been used, but comprehensive assessment of their performance in a CVA has been limited. Curves have been represented by mathematical functions ,7-11 or a priori by some rule ; (2) manual tracing of curves, in which points are selected by eye as the curve is traced; and (3) automated curve tracing, in which software is used to detect differences in color or brightness to delimit the curve. Additionally, semi-landmark methods (bending energy minimization or perpendicular projection), elliptical Fourier analysis, and extended eigenshape analysis approach the alignment process differently. The interaction between digitization and alignment may affect the ability to discriminate among shapes.Applying DFA or CVA to outline data requires first digitizing the structure, then aligning the structures to compensate for any arbitrary decision in the digitizing process, and finally extracting a discriminant function or a set of canonical variate axes from the data. The impact of alternative approaches to digitizing and aligning the structure on the detection of differences in mean shape or discrimination among groups has not been established. Among the methods for digitizing the curves are (1) template- or fan-based methods, in which a set of points is defined Once the data are collected and aligned, yet another potential methodological question must be addressed because methods like CVA and DFA require that there be more specimens than variables. The linear CVA requires a matrix inversion of the pooled within-group variance-covariance matrix, requiring that it be of full rank, which in turn requires more measured specimens than the sum of measurements per specimen and groups. If this condition is not met, there are more degrees of freedom in the measurements than in the specimens. The quadratic form of CVA requires independently estimated covariance matrices for each group and thus places even greater demands on the data. Fortunately, the linear method is quite robust and often outperforms the quadratic method even when the covariance matrices are unequal ,21.x- and y-) when there is only one degree of freedom per point. While points along the curve are originally measured as a pair of Cartesian coordinates, only one degree of freedom remains after the semi-landmark alignment procedure is used. Principal components analysis (PCA) may be used to reduce the dimensionality of the data by analyzing a limited number of PC scores of the specimens instead of the original data. This reduction poses a need for an objective criterion to determine the number of PC scores used. The simplest approach is to use as many PC axes as possible, given the degrees of freedom in the data, i.e., retaining all PC axes with non-zero eigenvalues.The use of outline methods thus poses difficulties for CVA, both due to the large number of semi-landmarks needed per specimen to describe outlines and due to the representation of semi-landmark points by two coordinates  is known to be biased upwards , since tIn addition to methods of dimensionality reduction based on PCA, a method based on a partial least square regression technique has also been proposed . In thisSince the statistic of interest in many studies is the rate of correct assignments based on cross-validation, an alternative approach is to choose the number of PC axes that result in the highest cross-validation rate of correct assignments. This may be done by calculating cross-validation rates for a wide range of differing numbers of PC axes and using the number of PC axes that optimizes the cross validation assignment rates. The bootstrapping approach outlined by Solow  is then Seiurus aurocapilla), belonging to different age categories. Age-related differences in feather shape are common in many species and provide a good data set for a methodological study because there is a known, but subtle, difference in tail shapes between birds that are under a year old and birds that are more than a year old. In the field, experienced bird banders are often able to discriminate between age categories of birds by visual inspection of rectrix (tail feather) tip shape , elliptical Fourier analysis (EFA) and the extended eigenshape method to classify specimens. Additionally, we compare several approaches to data acquisition, manual curve tracing, template-based digitization, and automatic edge detection, as well as assessing the dependence of these methods on the number of points used in the analysis. We used as a test case the rectrices (tail feathers) of a single species of bird, the ovenbird . Tra. TraSeiue  were used to reduce the dimensions of the data prior to the CVA, there was strong evidence of over-fitting Table . The resThe ability to correctly discriminate among specimens from the two age groups did not depend on the approach to digitization or on the alignment method Table . The optThe number of points included in the analysis had little impact on the results, although we should note that we could not readily vary the number of points using fan-based digitization. The error in the estimated length of the curve based on a reduced number of points seemed to be quite low over the range of points used in this study Table . The abiAn experienced bird bander correctly classified 38  of all individual rectrices, similar to the percentage correctly assigned by the geometric morphometric methods. When the class of indeterminate feathers was removed, the proportion correctly classified increased to 90.3% (28 of 31). The more traditional method of using all tail feathers resulted in a classification rate of 93.5% (43 of 46), which increased to 97.4% (37 of 38) without indeterminate specimens.Discrimination between age classes based on shape is robust to changes in data acquisition methods, semi-landmark alignment criteria, and method of shape analysis . We find no evidence for the superiority of curve-tracing or fan-based methods of data acquisition for semi-landmark alignment for the specific example of feather outlines. However, the average time to digitize feathers was lower for curve tracing than fan-based methods, and curve tracing also allowed for easy variation in the number of points used in the final analysis. Based on these results, we would recommend curve tracing over the use of fans or templates.Methods of semi-landmark alignment can be directly compared with respect to repeatability, whereas it does not seem legitimate to directly compare numerical results of partial Procrustes distances or summed squared distances based on BEM with those based on PP. Although the specimens may be in the same linear tangent space, they were not projected into that space in exactly the same manner. Since a single curve projects into different locations under the two alignment schemes, the difference in the two methods is not linear with respect to distances measured in the two groups. Thus, rather than using raw variance measures, ratios of variance of repeated measures of a single specimen to the whole population variance were used to compare BEM and PP. BEM produced a larger ratio of variation in repeated measures of a single specimen to the variation in the entire sample than PP whether using a fan or curve tracing. Curve tracing produced a lower ratio than fan-based digitization. Since the total variance in the sample is the sum of biological variance and measurement error, it appears that the BEM results in higher variation in measurements than PP for this data set.The method of shape analysis–specifically whether the analysis was based on semi-landmark methods, EFA, or extended eigenshape analysis–had very little effect on the results. The different methods produced very similar rates of successful discrimination, based on the bootstrap estimates of the 95% confidence interval of the cross-validation rate of correct assignments. The semi-landmark methods did provide slightly higher observed cross-validation rates, perhaps due to the semi-landmark alignment or to the use of centroid size rather than outline length as a measure of size. It should be noted that the semi-landmark methods do incorporate "sliding" of semi-landmark points along the estimated curves, and thus the semi-landmark processing methods are not related to one another, or to the Fourier and eigenshape methods, by a simple linear transformation of variables. It is reassuring to note that the method of capturing the outline did not strongly affect the results of the analysis, indicating that any of the methods could be reasonably used to study outlines. However, the semi-landmark methods do allow ready incorporation of landmark data points not along a curve into the same analysis as outline data, which may prove advantageous in studies where interior structure is of importance. While this study did not incorporate landmark data in the CVA, an earlier ontogenetic study  indicateThe approach to dimensionality reduction presented here yielded higher rates of cross-validation assignment than the simple approach of using a number of PC axes equal to the degrees of freedom in the system, or the PLS method. In the original discussion of the randomization test to determine the range of classification probabilities, Solow  made useWhile a clear statement of the superiority of one method of outline analysis over the others would make for a resounding conclusion, this was not the case for this study. The general consistency of our results and the characteristics of CVA indicate the promise offered by semi-landmark methods of integrating information about outlines or curves with landmark points. Semi-landmark methods were as effective in capturing the outlines of the feathers as the more established Fourier and eigenshape approaches. The variable number of PC axes method of dimensionality reduction does serve to optimize the cross-validation rates of correct assignment when used in conjunction with outline data. Furthermore, this method produces higher cross validation rates than either a fixed, maximal number of PC axes, or the partial least squares method. Manual curve tracing was the preferred method of digitization, in that it was as reproducible as other methods, offered flexibility in the number of semi-landmarks used, and proved to be slightly faster than template-based digitization.Forty-six known age ovenbird specimens from the Canisius College Vertebrate Collections were utilized in this study. Age determination of each specimen occurred by dissection and examination of skull pneumatization. Incomplete skull pneumatization indicates a young (hatch-year) bird, while adults have a fully pneumatized skull. In addition, a bird bander with extensive experience with ovenbirds (SRM) assigned each individual to an age category using two different criteria. The first characterization involved examination of the right, fifth rectrix, the feather that was digitized for this study. Typically, bird banders designate each individual as young, adult, or indeterminate. Indeterminate feathers normally would not have been assigned an age category in the field, but in this study were further subdivided into either adult or young categories to match the two categories used by the CVA. Additionally, the bander examined all the feathers of the tail, which is what is typically done in the field, using the same initial classifications of young, adult, or indeterminate (subsequently subdivided into young and adult). The right fifth rectrix was removed from each specimen, and the upper surface was scanned using a flat-bed scanner. Prior to scanning, the barbs and barbules of each feather were arranged in the typical interlocking manner, producing intact vanes. A blue background provided contrast and a ruler provided scaling information in each scanned image.Three different approaches to the digitization of points along curves were examined during this study. The simplest approach was to measure points spaced on the curve in an arbitrary manner, obtaining a dense sampling of points around the curve, and then use interpolation methods to reduce this set of points to some desired, fixed number of equally-spaced points ,8,9,12. Using the tpsDig program  for manuWe used a software tool, MakeFan6 [42], to plot fans on the image of each feather, digitizing points at the intersection of the curve and the lines of the fan. When constructing the fan, we anchored the ends of the fan at the tip of the feather and at the proximal end of the feather vane where it met the rachis Fig. . A varieUsing the automatic edge detection option in tpsDig, we automated the digitization. The uneven edges of the feather limited the effectiveness of automatic edge detection, making it necessary to trace the outline of the feather using the pen tool in tpsDig prior to automatic edge detection. This additional manual procedure removed many of the advantages of the automatic edge detection method, leaving little practical difference in the operation of the edge detection method and the manual curve-tracing method. These feather data thus did not provide a good test set for the comparison of automatic versus manual curve tracing, except to the extent that other features of interest may similarly require considerable image enhancement.Once a set of points around the curve was digitized, these semi-landmarks were processed using one of two alignment algorithms intended to reduce effects of the arbitrary selection of a limited number of points to represent the entire outline. Two different approaches, bending energy minimization  and After initial estimation of the mean shape of the specimens Fig. , semi-laBEM alignment was carried out using the tpsRelwarp program . The poSemi-landmark alignment based on the PP method was carried out using the SemiLand6 program [42]. The components of the differences in semi-landmark positions between the reference form and the target form that are tangent to the curve were mathematically removed. This procedure resulted in an alignment of the semi-landmarks on the target form along lines perpendicular to the curve passing through corresponding semi-landmarks on the reference form (see ). As lonOnce the semi-landmarks were aligned under one of the two criteria, they were treated as points in a landmark-based analysis carried out in the linear tangent space to the underlying curved shape space ,47. Statx- and y- coordinates of these points were obtained. To standardize specimen size and orientation, the length of the ellipse formed by the first harmonic was scaled to one, and this ellipse was oriented along the x-axis.Data gathered using the manual curve-tracing approach was analyzed using elliptical Fourier analysis (EFA), a fairly standard approach to outline data ,8,11,48.The standard eigenshape analysis of shape ,13 startA singular value decomposition of the variance-covariance matrix of the φ* values ,50 was uTo estimate digitizing error, a single image of a single specimen was digitized ten times by a single operator. Each of the ten images was then analyzed using each of the three measurement methods and subjected to both methods of semi-landmark alignment. The summed-squared partial Procrustes distances about the mean shape divided by the number of specimens minus one was used as a measure of the variation in the measurements. Resampling with replacement was used to estimate a confidence interval for this variation. Data aligned by BEM and data aligned by PP were not directly compared to one another because of the difference in semi-landmark alignment criteria. Instead of direct comparisons of variation, we compared the ratio of the variation in repeated measures of a single specimen to the variation in the entire sample .df (the number of degrees of freedom in the system) PC axes and used the number of axes that produced the highest cross-validation rate.To discriminate between age-classes by shape, we used CVA. For the semi-landmark data, partial warp and uniform component scores based on the thin-plate spline decomposition were used ,51,52. PA simple cross-validation protocol was used throughout the study. The cross validation assignment rate was determined by sequentially selecting a single specimen at a time as the test data. The CVA was carried out on the training set and the resulting CV axis was used to classify the test data. The success rate over all specimens forms the estimate of the cross validation rate. Each of 46 specimens was used sequentially as the cross validation specimen. Thus, there were 45 specimens in the training set and 1 specimen in the test set at time, producing a total of 46 possible cross-validation sets available under this protocol, More complex methods of forming test sets in cross validation are available, but the simple approach used here appears to yield reasonable and consistent results. The bootstrapping approach outlined by Solow .i)/N in the ith column of the classification if it is a member of the ith class, and a score of -ni/N in that column if it is not, so that the columns all have a mean of zero. This form of "dummy coding" is often used in multiple regression analysis. A singular value decomposition  of the The scores of the specimens along these SVD axes are then used as variables in the CVA analysis. This approach as a whole is very similar to a multiple regression analysis. Kemsley  notes thThe author(s) declare that they have no competing interests.HDS designed the study, wrote the IMP series software used in the analysis, performed most of the data analysis, helped draft the figures, and was the lead author of the draft and manuscript. KMC and JMP scanned and digitized feather outlines, performed some data analysis, and assisted in drafting the manuscript and preparing figures. SRM performed dissections, determined specimen ages via dissection and visual inspection of tail feather shape, prepared the final figures, and helped write and revise the manuscript.


Many women suffer from health problems after giving birth, which can lead to sick leave. About 30% of Dutch workers are on sick leave after maternity leave. Structural contact of supervisors with employees on maternity leave, supported by early medical advice of occupational physicians, may increase the chances of return-to-work after maternity leave. In addition, to understand the process of sick leave and return-to-work after childbirth it is important to gain insight into which factors hinder return-to-work after childbirth, as well, as which prognostic factors lead to the development of postpartum health complaints. In this paper, the design of the Mom@Work study is described.The Mom@Work study is simultaneously a randomised controlled trial and a cohort study. Pregnant women working for at least 12 hours a week at one of the 15 participating companies are eligible to participate. The supervisors of these pregnant employees are randomised at 35 weeks pregnancy into the intervention group or control group. During maternity leave, supervisors in the intervention group contact their employee six weeks after delivery using a structured interview. When employees do not expect to return to their jobs at the end of their scheduled maternity leave due to health problems, the supervisor offers early support of the occupational physician. Supervisors in the control group have no structural contact with their employees during maternity leave. Measurements take place at 30 weeks pregnancy and at 6, 12, 24 and 52 weeks postpartum. In addition, cost data are collected. For the RCT, primary outcome measures are sick leave and return-to-work, and secondary outcome measures are costs, health, satisfaction with intervention and global feelings of recovery. Outcome measures for the cohort are pregnancy-related pelvic girdle pain, fatigue and depression. Finally, a number of prognostic factors for return-to-work and for the development of complaints will be measured.The Mom@Work study will provide important information about return-to-work of employees after giving birth. Results will give insight in prognosis of postpartum sick leave and complaints. Also, the role of supervisors and occupational physicians in successful return-to-work after childbirth will be clarified. Many women experience health problems during the first year after childbirth -4. CommoIn The Netherlands, every pregnant employee has the right to receive paid maternity leave, also called pregnancy and delivery leave. The total duration of this leave is 16 weeks, divided over four to six weeks before the delivery term and 10–12 weeks thereafter. About 90% of Dutch working women had the intention to return-to-work (RTW) after the birth of their first child . A DutchThere are several possible explanations for this high sick leave and postponed RTW postpartum. The postpartum care for women is probably to fragmented. At six weeks postpartum, women have their last contact with the midwife or obstetrician, who have limited time and who give only little or no information about RTW. At Youth Health Care centres only the child's health is considered and due to limited time the mother's health is often neglected. If there are health problems, women consult their general practitioner just before the scheduled maternity leave ends. In addition, these women with postpartum health complaints may not receive timely medical attention of their occupational physician (OP), who is currently not involved until 12–18 weeks postpartum. This may be too late; health complaints may have existed for a considerable time, while early intervention after the onset of complaints seems to be important , becauseSupervisors often serve as a case-manager in case of sick leave in The Netherlands, however, they do not play a similarly important role in supporting the employee and recognizing problems for RTW that occur during maternity leave. Most companies in The Netherlands do not have a special policy for structured contact during maternity leave. Nieuwenhuijsen et al. recommended that supervisors should communicate frequently with employees during (prolonged) sick leave and should hold follow up meetings often in order to facilitate RTW in general . It seemA structural contact between supervisors and women on maternity leave, supported by early medical advice of occupational physicians during maternity leave may increase the chances of RTW after maternity leave or reduce the incidence of sick leave after maternity leave. This paper describes the design of the Mom@Work study, a randomised controlled trial (RCT) and cohort study, conducted at Body@Work, Research Centre Physical Activity, Work and Health, TNO-VUmc in Amsterdam, The Netherlands. The Mom@Work study has three aims. The first aim is to investigate the effectiveness and cost-effectiveness of an early intervention in reducing sick leave in women after maternity leave in a randomised clinical trial. Our hypothesis is that supervisors who have structural contact with their employees during maternity leave combined with occupational health care, when needed, will be more effective in reducing the number of sick leave days after maternity leave than supervisors who do not. The second aim of the study is to examine which factors contribute to RTW for women after childbirth. The third aim is to examine which factors contribute to the development of the following postpartum complaints: pregnancy-related pelvic girdle pain, depression and fatigue. The second and third aim are examined in the cohort study.The study is designed as a simultaneous randomised controlled trial (RCT), to assess effectiveness and cost-effectiveness of the intervention in reducing postpartum sick leave, and a cohort study, to assess incidence and determinants who contribute to postpartum RTW and to the development of postpartum health complaints. The Medical Ethics Committee of VU University Medical Centre at Amsterdam, The Netherlands approved the study design, study protocol, and informed consent procedure. All participants must provide a written informed consent.For practical reasons, large companies with a predominantly female workforce were preferred to enter the study. It has been estimated that annually about five percent of the women becomes pregnant in The Netherlands. Van Beukering (2002) found that health care companies had higher sick leave rates before and after maternity leave than other companies. Therefore, health care companies were over sampled in the study.Ninety-three companies were approached and ultimately 15 companies consented to participate in the study. The participating companies included university hospitals (n = 3), child care companies (n = 3), regional hospitals (n = 2), a health care group including a regional hospital, elderly care and home care (n = 1), a ministry of the Dutch government (n = 1), a chain of supermarkets (n = 1), a youth health care company (n = 1), an occupational health service provider (n = 1), a chain of special travel shops (n = 1) and a chain of pharmacists (n = 1). The company size ranged between 391 and 52,481 employees.All pregnant women who were working between January 1, 2004 and March 31, 2006 at one of the 15 participating companies in The Netherlands were able to participate in the Mom@Work study. When a woman submitted the request for maternity leave, a human resources (HR) staff member of the participating company sent a short information letter about the study, a study leaflet, two response cards and a postal envelope to this woman. If the woman was interested in participating, she was asked to complete the 'Yes' response card and mail it to the research staff. The 'Yes' response card contained screening questions for both the RCT and cohort.Pregnant women were eligible to enter the cohort study when they met the following inclusion criteria: age between 18 and 45 years; employment for a minimum of 12 hours a week before maternity leave and intention to RTW after maternity leave. Furthermore, they had to give written informed consent and be sufficiently fluent in the Dutch language. Women were not admitted to the RCT or cohort study if they met any of the following exclusion criteria: definitely not returning to work after maternity leave; miscarriage or delivery before 27 weeks; receiving a full disability benefit; or submitting an application for a full disability benefit.The inclusion criteria for the RCT were stricter than for the cohort study. Women could only enter the RCT if they met the criteria of the cohort study and the following inclusion criteria: employment for a minimum of 12 hours a week until six months after the delivery and working for the same employer before and after maternity leave.The specific exclusion criteria for the RCT were: a delivery before thirty-four weeks; return to the same employer after maternity leave is uncertain; and definitely not returning to the same employer after maternity leave. All participants of the RCT took also part in the cohort study. Women, who were not admitted to the RCT, could enter the cohort study if they met the inclusion criteria of the cohort study. Figure Due to the fact that most supervisors managed a team of employees, the possibility existed that more than one employee would participate in the Mom@Work study. Thus, randomisation took place at the level of the supervisor rather than the participating woman. For each participating company, a randomisation list was computer-generated by an independent statistician. Because it was not known how many employees of each company would be participating in the study and some companies had a small number of employees, random allocation in blocks of four was chosen. Each block contained two intervention and two control group allocations in a random sequence. The details of the randomisation were printed on tickets and sealed into envelopes. The outside of the envelope was marked with the company's number and a number indicating the rang order. Supervisors were randomised when the participant was 35 weeks pregnant. When the researcher was informed in time that the participant gave birth before 34 weeks, the participant was not randomised, but still included in the cohort study.Each week during the enrolment period, a list of supervisors who were to be randomised, was printed. The researcher allocated the supervisors of the women to the intervention group or to the control group by opening the next numbered envelope. The research assistant was responsible for sending emails and information to the supervisors who were randomised into the intervention group. The supervisors who were randomised into the control group did not receive any information and were not informed that their employee was participating in the study.The intervention concerns a minimal intervention strategy. The participant receives a phone call from her supervisor six weeks after childbirth and, if needed, support of the occupational health service providers (OHSP). The aim of the intervention is to prevent sick leave by only one phone call and by instigating earlier help by OHSP for the health problems. When health problems are pointed out during the phone call from the supervisor, the women can receive support of the OHSP at least five to seven weeks earlier than usual.During the structured telephone contact the supervisor uses a standardised interview in the form of a checklist, and asks questions about the women's well being, the child's well being, child care, breast feeding, anticipated work problems, and the expectation of returning to work after maternity leave. When a woman expects that she is not returning to work after maternity leave, or has doubts about it, she is offered support of the OHSP by the supervisor. Only when she wants this support, the OHSP is called in. If requested by the participant, another representative of the company such as another supervisor, executive, line-manager, head of department or HR-advisor initiates this contact.When a woman does not anticipate any problem with RTW, the supervisor does not offer further support, and he or she only asks when the woman will start working again. Women who do not expect any problem and who do not receive any early help of the OHSP, but who ultimately delay returning to work after maternity leave due to illness, receive the usual sick leave care.If indicated during the phone call, the supervisor, participant or research team calls the OHSP that the woman needs their support on behalf of the study. A regular, but accelerated procedure for sick leave is initiated by the OHSP. In most cases, an appointment for early medical advice of the occupational physician with the participant is made within one week, instead of the usual six. The OP follows an OHSP complaint specific protocol, if available.The supervisors receive a thorough instruction to carry out the structural contact. First, they receive an email to check if they are actually the supervisor of the participant and to check their personal data like work address and phone number. Next, they receive an information package. This package contains a study leaflet explaining the background of the study and instructions on when and how to perform the structured interview with the participant. An instruction card and checklist for this interview are also included. Then, they receive an instruction phone call from the research team four weeks after delivery. Finally, an email is sent to respective supervisors six weeks after delivery as a reminder for the phone-call. The supervisors are asked to perform the structured interview and to complete the accompanying checklist in the sixth week after delivery. They are requested to return the checklist by fax or email to the research team thereafter as soon as possible. If the checklist is not received at seven weeks after delivery, a reminder is sent by e-mail or made by telephone.In the intervention group, supervisors are free to give their usual attention to employees who gave birth such as making home visits, calls or sending cards. If available, the policy of the company for pregnant employees or new mothers is allowed to be carried out during the study. For example, bringing a visit to the new mother with the "company's' bear" several weeks after childbirth.Supervisors in the control group perform their common practice of acknowledging the arrival of their employees' newborn, for example, by making a home visit, calling or sending cards. If the participant is not able to RTW after maternity leave has ended, the standard sick-listing procedure is followed. If participants experience medical complaints during maternity leave, there is always the regular possibility of calling their supervisor or OHSP. Some OP's have reported before the start of the study that they routinely ask employees to contact them a few weeks before the end of maternity leave, in case of serious complaints during pregnancy.Given the nature of the intervention, only the control group supervisors and participants are blinded to the intervention. The intervention group supervisors and participants cannot be blinded for the allocated treatment. Furthermore, the researcher and research assistant cannot be blinded for the allocated treatment. However, the data entry assistants are blinded for the allocated treatment. Data on sick leave and RTW are extracted from the routine databases of the company or the OHSP, thus, blinding does not play a role. The data will be blinded for the performance of the analysis. To guarantee blinding of the data for the analysis the participants, supervisors and companies are coded.All measurements, except sick leave data of OHSP or company, are performed by questionnaires. The baseline measurement consists of two parts: The first part (T0) when the employee makes herself known as participant; and the second part at 30 weeks pregnancy (T1). There is a one-year follow-up with assessments at six weeks (T2), 12 weeks (T3), 24 weeks (T4) and 52 weeks (T5) after the delivery date.Units of health care use and productivity loss which are used to calculate costs are measured at six weeks (except work performance), 12 weeks (except work performance), 18 weeks, 24 weeks and 52 weeks after the delivery date. If a woman takes sick leave, has health care costs or has decreased work performance at 24 weeks, she receives a questionnaire on units of health care use and productivity loss again at 30 weeks, and if indicated by the same criteria also at 36, 42 and 48 weeks after the delivery date. Self-reported sick leave is assessed as part of the cost measurements, and objective sick leave data is obtained from the databases of the OHSP and, if available, from the databases of the company.The primary outcome measure for the RCT is work-status. Work-status is defined as sick leave and RTW. In this study, the following indicators are measured:1) Time to partial and to full RTW, meaning number of calendar days between end of maternity leave and first day at work2) Time to full RTW corrected for partial RTW3) Partial and full RTW rate at 12, 24 and 52 weeks follow-up4) Incidence of sick leave in the first year after delivery, following full RTW5) Time to first recurrent sick leave in the first year after delivery, following full RTW6) Total days of sick leave in the first year after delivery7) Frequency of sick leave periods in the first year after deliveryIn addition, data will be collected on the following items:1. Costs are assessed from a societal perspective. Units of direct health care utilisation, both within and outside the health care sector, as well as productivity loss due to absenteeism and presenteeism are collected by cost diaries. Examples of direct health care units within the health care sector include consultations with the general practitioner, hospitalisations, and medications, and examples of direct health care units outside the health care sector include over-the-counter medication, informal care, and consultations with alternative health professionals. Costs will be calculated by multiplying the volume of resource use by cost prices, following the Dutch guidelines for economic evaluations in health care to estimate costs .2. Health is measured with the RAND-36, a Dutch translation of the 36-Item Short Form Health Survey (SF-36) ,17 and t3. Satisfaction with the intervention is measured with the question, "Are you satisfied with the contact 6 weeks after delivery?", which can be answered on a six-point Likert scale. This question will be asked at 12 weeks after delivery.4. Self-reported feelings of recovery from complaints, also called global perceived effect (GPE) is measured with the question, "To what extent have your complaints changed since 6 weeks after the delivery?", which can be answered on a seven-point Likert scale, with a range from "1" (completely recovered) to "7" (worse than ever). Again, this question will be asked at 12 weeks after delivery.In the cohort study, data will be collected on whether certain complaints develop or if RTW occurs among the participants.1. The first outcome measure for the cohort is the presence of complaints of pregnancy-related pelvic girdle pain (PPGP). Two aspects on PPGP are measured: pain intensity and functional status. Pain intensity is measured with the same method as used in the Maastricht PPGP cohort study . It is a2. The second outcome measure for the cohort is fatigue, measured in two ways. Firstly, self-reported fatigue is rated on an eleven-point scale anchored by "0" for "no complaints" and "ten" for "many complaints". Secondly, fatigue is measured with the Checklist Individual Strength (CIS) questionnaire . The CIS3. The third outcome measure is depressive complaints. Three instruments are used to measure several aspects of depressive complaints. First, self-reported depressive feelings are rated on an eleven-point scale anchored by zero for "no complaints" and ten for "much complaints. Second, the Four Dimensional Symptom Questionnaire (4DSQ) is used under the assumption that mothers may experience distress as well as somatization. The 4DSQ is a Dutch self-rating questionnaire measuring four dimensions of common psychopathology: depression (severe anhedonia and depressive thoughts), anxiety , distress (non-specific symptoms of psychopathology), and somatization . The 4DSInformation is gathered on a number of variables that are considered to be prognostic factors for sick leave, RTW or for the development of complaints such as pregnancy-related pelvic girdle pain, fatigue and depression. Several categories of prognostic factors are distinguished: pregnancy-related, work-related, child-related, psychosocial-related and health-related prognostic factors. For each category a number of variables is measured with questionnaires. These variables are measured at different moments in time during the follow-up period. Some variables are measured with questions that have been developed specifically for the purpose of this study, while others are measured with validated measurement instruments. Table One-third of the participants and supervisors in the intervention group receive a short questionnaire in order to evaluate the process of the intervention. Questions about their experience of the intervention are asked as well as which factors influenced according to them the effect of the intervention. One-third of the participants in the control group also receive a short questionnaire that asks about anticipated problems related to returning to work and whether these were discussed with their supervisor. Finally, at 12 weeks after delivery, all participants are asked if they had received any attention of their supervisor, whether their supervisor had talked about RTW and whether they were satisfied about this contact. They are also asked if they had received support of the OHSP and if they were satisfied about this support.Van Beukering found that in 2002 about 30% of the women were taking sick leave after the maternity leave has ended. In the For the RCT, data will be analysed using statistical tests such as linear regression analysis and Cox regression analysis. The dependency of the observations will be taken into account by performing a multilevel analysis. The multilevel analysis will be performed with a four-level data structure where time, participants, supervisors and companies will be defined as levels 1, 2, 3 and 4, respectively. The analyses will be adjusted for baseline and prognostic dissimilarities, if necessary. Both an intention-to-treat analysis and a per-protocol analysis will be performed. For the cohort study, multilevel analysis will be performed with a three-level data structure where time will be defined as level 1; participants, level 2; and the companies, level 3.The economic evaluation, which will be performed from a societal perspective, will consist of an analysis of differences in the total, direct and productivity loss costs between the intervention and control group, as well as a cost-effectiveness and cost-utility analysis of the intervention compared to common practice. In the cost-effectiveness analysis, sick leave will be the effect measure and the total costs will comprise all costs except those of productivity loss. Utilities will be based on the Euroqol questionnaire . QualityThe Mom@Work study is designed to provide information about RTW of employees after giving birth. The effect of structural contact during maternity leave on sick leave will be evaluated, and factors that contribute to the development of complaints after childbirth will be assessed. By prospectively following a group of 550 pregnant workers until one year after delivery, much information will be gathered. A discussion of methodological issues of the Mom@Work study follows below.In the intervention group, the supervisor contacts the participant six weeks after the delivery. An assumption was made that six weeks is a good moment to perform the intervention. In The Netherlands, most women have their last consult with their midwife, obstetrician or general practitioner at six weeks postpartum. It is assumed that, in general, women would be physically rehabilitated from delivery after 6 weeks. In The Netherlands, women have between 10–12 weeks of maternity leave after the delivery, before they have to start working again. When problems occur at six weeks after delivery, there is still sufficient time to solve those problems or to start prevention of long-term sick leave after maternity leave. Early identification of employees on sick leave who are at risk for long-term sick leave and work disability is important for starting early interventions. Therefore, it is crucial to seek early contact with those employees -11.It may be argued that another time point than six weeks after delivery would be a better moment to intervene. Some women extend their leave by taking vacation, fulltime unpaid leave, or fulltime parental leave for a few days up to three months. At six weeks, postpartum it may be difficult to estimate the ability to resume work on an advanced date. Even when a woman does not extend her leave, it may be hard to estimate if one can truly start working again four to six weeks later, because during this period changes can occur to the mother or to the child, or one might feel healthy, but not healthy enough to work.The supervisor is the person who contacts the participant in the intervention group. In our opinion, the supervisor is the most appropriate person to talk about RTW after maternity leave with their employees. The supervisor usually represents the employer in case the employee gets sick. Most companies have a policy that in case of sickness the supervisor keeps in contact with the employee. Although maternity leave is a special period, because the employee is not sick listed, employees are absent from work for at least 16 weeks.The relationship between supervisor and employee may be important for the success of the intervention. It is possible that eligible employees refuse to participate, because of a poor relationship with their supervisor. In case employees and supervisors have a 'good' relationship they probably communicate better, than employees and supervisors who have a 'bad' relationship. This better communication may have the same effect as the structural contact in the intervention group, which may lead to a smaller difference between the intervention and control group. Therefore, all women will be asked if they had any conversation with their supervisor about RTW during maternity leave.For practical reasons, we invited large companies with mostly female employees, and in particular, health care companies. This may have led to selection-bias and the results may probably not be generalisable to all (pregnant) working women. On the other hand, the results of the Mom@Work study will be most interesting for companies were a large number of women is working, and will also give an indication for problems and solutions for smaller companies or companies with a small number of female employees.This study will provide important information about RTW of employees after giving birth. Results will give insight into the prognosis of sick leave and complaints after childbirth. Also, the role of the supervisor and the occupational physician in prevention of sick leave after childbirth will be clarified.EPDS Edinburgh Postnatal Depression Scale4DSQ Four Dimensional Symptom QuestionnaireHR Human Resource departmentOP Occupational PhysicianOHSP Occupational Health Service ProviderPPGP Pregnancy-related Pelvic Girdle PainQALY Quality Adjusted Life YearsRAND-36 36-Item Short Form Health SurveyRCT Randomised Controlled TrialRDQ-24 Roland-Morris Disability QuestionnaireRTW Return-to-WorkCIS Checklist Individual StrengthThe author(s) declare that they have no competing interests.SGMS is performing the data collection and drafted the manuscript. MNMvP originated the idea for the study and is project-leader of the study. KU and MCdB are responsible for the cost-effectiveness data and analysis. SGMS, MNMvP, IJMH, DJB en WvM participated in the design of the study and research protocol. All authors read and corrected draft versions of the manuscripts and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The LTR  retrotransposons of higher plants are replicated by a mutagenic life cycle containing transcription and reverse transcription steps. The DNA copies are often subject to recombination once integrated into the genome. Complex elements, where two elements share an LTR, are not uncommon. They are thought to result from heterologous recombination between two adjacent elements that occurs following their integration.Here, we present evidence for another potential mechanism for the creation of complex elements, involving abnormal template switching during reverse transcription. The template switching creates a large, complex daughter element, formed by the fusion of two parent sequences, which is then inserted into the genome.Poaceae, especially in the Triticeae, but not of Arabidopsis. Hence, retrotransposon dynamics shaping the genome are lineage-specific.Those complex elements are part of the genome structure of plants in the  HIV) replication. Higher plant genomes, especially of the grasses , harbor a large number of these elements, which form the vast majority of the nuclear DNA. Retrotransposition involves a reverse transcription step, where cDNA is synthesized from an RNA template. Reverse transcription is catalyzed by reverse transcriptase, which is generally encoded by the retrotransposon being copied, and the cDNA is inserted into a new genomic location by the integrase, which is also self-encoded [Long Terminal Repeat (LTR) retrotransposons are Class I transposable elements that replicate by a "Copy-and-Paste" mechanism, called retrotransposition, which is quite similar to lentivirus  complex in position 11808–29240 . Accession AY368673  containPoaceae sequences for the occurrence of such complex structures. We carried out an ab initio scan of the rice pseudomolecules and all the available genomic sequences from maize, using the LTR_STRUC software [Squiq subfamily, with CAAAC as the TSD sequence. The second detected complex is a member of the Opie family in the maize BAC AY078063 [Based on these observations, we checked within other software  for deteAY078063 , in posiA model that can explain complex insertions such as these involves abnormal template switching as a part of reverse transcription. Immediately preceding reverse transcription, the RNA matrix forms a loop, using the high homology between the two R regions (5' and 3') within the LTRs to buckle the two ends of a single template together. This allows the (-)-strand cDNA, which otherwise cannot proceed once it reaches the 5' end of the RNA template, to jump across to the 3' end and continue. The jump is called template switching. The process leads to perfect identity between the 5' and the 3' LTR of the newly synthesized element, because the R and U3 segments of the 5' LTR and the U5 segment of 3' LTR in the RNA are copied into both LTRs of the cDNA. The cDNA is ultimately inserted into a new genomic location by the integrase. The enzyme ligates the cDNA to one strand of the asymmetric double-strand break in the host DNA, which is formed concomitantly with the ligation. The repairing of this break leads to the TSD , the phenomenon is undetectable because there are no major modifications to the resulting cDNA. However, if two different RNAs are packaged in the same virus-like particle, a jump to the other template during reverse transcription leads to abnormal or new elements, opening a new mode for LTR retrotransposon evolution. The VejuL  and BAREL [BARE2  elementsvia standard integrase catalysis, leading to a new genomic insertion harboring TSDs on either side , two template-switching complexes were identified out of the 20 recombinant complex elements recognized  BACs were re-analyzed as in . The updively by , and [10ively by . The scaively by  and reprab initio identification of LTR retrotransposons within the rice and maize sequences was performed by the LTR_STRUC software [Python script according to their size, and the 1416 candidates meeting the criterion of >10 kb length were then manually checked using Dotter [Dotter [ClustalX [The software  using stg Dotter  for the  [Dotter , and theClustalX , after mLTR, long terminal repeatTSD, target-site duplication.FS designed and performed the research and wrote the manuscript. AHS directed the research as well as edited and contributed to the manuscript. Both authors read and approved the final manuscript.ClustalX alignments of the LTRs from the two Angela complexes from Triticeae.Click here for fileDotter and ClustalX alignments of the LTRs from the Rice and Maize complexes.Click here for file


For the moment, scientific evaluation of programs on treatment of pregnancy-related pelvic girdle and/or low back pain after delivery is hardly available with only one study with a positive result, suggesting uncertainty about the optimal approach. Investigators draw particular attention to biomedical factors but there is growing evidence that biopsychosocial factors appear to be even more important as a basis of an intervention program.We studied the effectiveness of a tailor-made program with respect to biopsychosocial factors (intervention group) in women with pregnancy-related pelvic girdle and/ or low back pain versus usual care based on a pain contingent basis (control group) shortly after delivery in a randomized controlled trial. Women with severe complaints shortly after delivery were selected from a longitudinal prospective cohort study (n = 7526), aimed at pregnancy-related pelvic girdle and/or low back pain in the Netherlands. A concealed block randomization was performed after collecting baseline data. Researchers were blinded to treatment assignment. Outcomes were evaluated within the domains of the biopsychosocial approach. Primary outcome concerned limitations in activities (RDQ). Follow-up measurements were performed 12 weeks after delivery.Since May 2001 until July 2003, 869 women out of the cohort made a request for treatment by a physiotherapist, 10 days after delivery. Because of a quick recovery in two weeks time, we included only 126 women three weeks after delivery. There was a statistically significant and clinically relevant difference in improvement on the primary outcome (RDQ) between the two groups in favor of the experimental intervention.The results favored the hypotheses. Women's worries about their condition were major targets in the experimental intervention. The prognosis after delivery, especially in de first weeks, turned out to be favorable. Effective therapy depends to a large extent on the adequate interpretation of early symptoms and the outcome of the diagnostic process . These oAlso on behalf of historical factors, pregnancy-related pelvic girdle pain is approached as a syndrome separate from low back pain outside pregnancy in the Netherlands. A pain contingent approach focused on biomedical elucidations is firmly entrenched among physiotherapists and patients. Currently, the intervention program for this group of patients in the Netherlands aims at increasing disease-specific education and encouraging compliance with prescribed exercises and a pain contingent regime of relative (bed) rest and avoiding and limiting several day-to-day activities such as using the stairs, bending, twisting, lifting and cycling. Therapists rely heavily on reported pain duration and intensity during goal setting for intervention. Moreover, the approach of a physiotherapist more often includes an exercise program to address the muscle imbalance and alignment of the pelvic girdle ,8. HowevAt the moment, several investigators in this field draw particular attention to biomedical factors but there is growing evidence that important prognostic problems such as biopsychosocial factors appear to be even more important as a basis in an intervention program ,11. In tWe performed a double-blind randomized controlled trial in primary care to determine the effectiveness of a tailor-made program with respect to biopsychosocial factors (intervention group) for women with pregnancy-related pelvic girdle pain after delivery versus usual care on a pain contingent basis (control group). The trial is embedded in a longitudinal, prospective cohort study , which sThe present article describes the short-term results. The study design has already been described elsewhere . Long-teThe medical ethics committee of the Maastricht University Hospital approved the intervention and cohort studies which were performed in the Southeast of the Netherlands. Midwives and gynecologists recruited the women during early pregnancy (10–14 weeks). Women were included in the cohort if they were at least 18 years old, pregnant and well versed in the Dutch language (n = 7526). Women were given written information explaining the aims and contents of the cohort and intervention studies before they decided to participate. Concerning the intervention study they were told that according to current knowledge, the two investigated treatment options are considered to be equally effective. The moment of inclusion for the intervention study was about three weeks after delivery. An individual woman entered the intervention study after signing informed consent for both the cohort and the intervention studies during early pregnancy and meeting the in-and exclusion criteria of the intervention study shortly after delivery. A basic principle was that inclusion criteria must have a meaningful influence in goal setting for treatment. Women were included when having pain in the pelvic girdle (including the lower back) with an onset during pregnancy or within two weeks after delivery (cohort data), were restricted in their normal daily activities because of pelvic girdle and/or low back pain and if there was a delay in recuperation (unable to participate satisfactorily in housekeeping and care of children because of the complaints under investigation). The severity of symptoms should vary with physical activities and time during the day. In the absence of a clear definition and reference standard to diagnose pregnancy-related pelvic girdle pain, the outcomes of a number of currently used diagnostic classification strategies not only led to different selections of women having complaints, the prognostic importance of these subgroups remained also unclear. Therefore we used an extensive description of pregnancy-related pelvic girdle pain. Women diagnosed with relevant specific pathology  that affects pain and activities of daily life were excluded. Exclusion also occurred in case of severe family-related or psychosocial problems or when a disablement procedure was not yet finished. Final aspects for in-/exclusion were the willingness of a woman to participate in the study or having a clear treatment preference. We only included women who did not indicate such a preference .An experienced research-physiotherapist visited the women at home, about three weeks after delivery. This visit was called for on the basis of a short self-administered questionnaire or initiated by midwives. A positive answer from a participating woman from the cohort or her midwife: "Is an intervention by a physiotherapist necessary?" took a central position in these questionnaires. Preceding a possible home visit, a short history taking by telephone took place about two weeks after delivery. The telephone interview focused on exclusion criteria such as willingness to participate, specific pathology and inclusion criteria such as limitations in daily life caused by pregnancy-related pelvic girdle pain and a delay in recuperation. During the home visit, a standardized history was taken and physical examination to exclude specific pathology was performed. Self-administered questionnaires were used to asses pain, limitations in activities, restrictions in participation, pain-related fear, pain catastrophizing, positive and negative affectivity, depression, expectancy of treatment result and quality of life. The questionnaires contained clear instructions to complete them with no help or support from others. If a woman met the selection criteria, she was informed about the aim and method of the intervention study and if she was willing to participate, the informed consent procedure was completed. The research-physiotherapist collecting the baseline data was trained at performing the measurements in a standardized way and was blinded for treatment assignment.Randomization took place after collecting the baseline data and obtaining informed consent. An independent research assistant (unaware of the base line data) carried out the concealed randomization procedure using a random computer-generated list. Block size for the randomization was four. When a woman was allocated to the intervention group, a physiotherapist trained in the experimental intervention practicing in the neighborhood of the woman was contacted and we ensured that treatment could start within one week. Treatment costs were covered for all participants in the intervention group. Women, allocated to the usual care group, were free to choose usual care treatment by a physiotherapist who was not trained in the experimental intervention protocol.Women were blinded to a certain extent to the allocated intervention option because they were kept naïve of the exact content of both intervention options. Physiotherapists were not involved in the baseline and effect measurements. Researchers dealing with the baseline and outcome data were blinded to the intervention assignments.Women, allocated to the intervention group, were referred to a participating physiotherapist in their own neighborhood. These physiotherapists received an educational program about the treatment protocol prior and during the study. All physiotherapists were already experienced and specialized in treating women with pregnancy-related pelvic girdle pain prior to the study. The content of the experimental intervention was based on the latest literature findings. It focused particularly on patient-therapist relationship, education and the promotion of an active life style rather than avoiding activities. Results of interviews with women from the cohort and group discussions with experienced physiotherapists prior to the trial further defined the program.Theoretical concepts of self-management ,21 and fGenerally, a time contingent policy was followed in which women set the pace by means of action plans. The expertise of the physiotherapists of the condition in general and of the women about their own specific condition and lives were equally important .Women, allocated to the usual care group, were free to choose usual care treatment by an experienced physiotherapist who did not followed the educational program about the treatment protocol, guidance by a general practitioner or do nothing. Information about the option chosen by the women was collected by means of questionnaires in the follow-up period. When a woman opted in favor of treatment, the sessions started within one week.Prior to the trial, detailed information was gathered about the contents of the current treatment options. Part of the information was gathered by means of group discussions with experienced physiotherapists and occupational therapists and interviews on an individual basis with women out of the cohort. The most striking characteristics of the current treatment were the character of the therapist-patient relation and the way of goal setting, focusing on disease management . There wThe contrast between the experimental intervention and usual care was an important issue in this study. Major features that underscore the contrast were the contents of the patient information, character of patient-therapist relationship, pain-contingent versus time-contingent intervention and compliance to a regime of avoiding and limiting activities versus action planning and personal goal setting by the women themselves. Among therapists, the approach of the experimental intervention is not widespread at all in the Netherlands. The participating therapists were explicitly asked not to give any information about the contents of the experimental treatment to therapists who did not participate in the experimental intervention.During the visit at home a standardized history was taken  and physAfter history taking, a short standardized clinical examination program was performed, which included tests of nerve root radiation (exclusion). The resPrimary domain Table  for imprLimitations in activities were measured with the Dutch translation of the Roland Disability Questionnaire (RDQ) . We replThe Quebec Back Pain Disability Scale (QBPDS) ,33 was iSubjective measurements like global feeling of recovery  and severity of the main complaints (MC) reflecting a patient-specific approach were also selected. Global Perceived Effect  (GPE) waPain was measured with two Visual Analog rating Scales (VAS) of the McGill Pain Questionnaire (MPQ-DLV) ,37 to reThe impact on participation and autonomy (IPA) measured person-perceived restriction in participation and autonomy ,39. The Fear of movement was measured by the Dutch translation of the Tampa Scale for Kinesiophobia (TSK),41. We uThe Short-Form 36 (SF-36)  evaluatePain catastrophizing is measured by the Pain Catastrophizing Scale (PCS),47.The Beck Depression Inventory (BDI)  was usedTo measure the experience of negative affect we used the 14-item Negative Emotionality Scale . To measTreatment expectancy  was measThe Pain Behavior Scale (PBS),30 is anWomen were asked to complete a follow-up questionnaire Table  at 12 weThe follow-up questionnaire Table  asked alThe baseline status of the study groups was compared with respect to the distribution of all variables known at baseline. For the outcome measures recorded at baseline and follow-up, we computed the mean difference between the baseline and follow-up score for each woman. Differences between group and 95% CI's were calculated for each outcome measure according to the intention-to-treat approach . PrimaryFrom November 2000 until November 2002, 7526 women were included in the cohort study.Results of the self-administrated question: "Is an intervention by a physiotherapist necessary?" resulted in 869 eligible participants; 384 times indicated by midwives, 682 times indicated by women themselves and 197 times both the women and midwives responded positively.  and 62 to the experimental intervention group (EI group). In all patients, baseline data were complete. In the usual care group, for 56 patients (88%) data were available for all outcome measures at 12 weeks after delivery. In the experimental intervention group, the corresponding number was 58 (94%). In the usual care group 8 patients were lost to follow-up because of quick recovery within the first weeks after randomization and did not want the fill out the outcome assessments anymore (n = 3), lack of time (n = 4), or not motivated to fill out the outcome assessments (n = 1). In the experimental intervention group 4 patients were lost to follow-up because of family reasons (n = 1), discontinued intervention (n = 1) and quick recovery within the first weeks after randomization (n = 2). All patients who were lost to follow-up in both study groups were contacted by telephone.In the experimental intervention group, two patients discontinued intervention. One woman because of lack of motivation and one woman because she could not endorse the basic principle of the experimental intervention (preferred a pain contingent treatment option on second thought). Nevertheless, the last mentioned patient completed all the outcome questionnaires and did not receive another treatment during the investigation period.In the usual care group 22 women were treated by a physiotherapist, 4 women by a manual therapist, and 4 women were guided by their general practitioner. The other (n = 34) chose not to seek help for their complaints during the same period. Most reported reasons for not visiting a physiotherapist after all were:" too busy with housekeeping and care of the children," "progressing improvement after randomization" and "attention focused on the newborn baby, the low back and/ or pelvic girdle pain did not bother enough anymore to search for help."In the experimental group 6 women used pain medication, one woman used an elastic belt and X-ray examined 2 women. In the control group 5 women used pain medication, 9 women an elastic belt and 5 women were examined by X-ray.The outcome data were normally distributed. Measurement at 12 weeks after delivery showed a consistent difference in favor of the experimental intervention but only the primary outcome limitations in activities measured with the RDQ reached statistical significance Table . More woTherefore, we performed a subgroup analysis to explore a possible different intervention effect in subgroups of women with severe limitations in activities at baseline. We expected that women with severe limitations in activities benefited more from the experimental intervention than from the usual care. We performed a median split on the baseline data of the RDQ and compared the both subgroups (baseline score RDQ < 13 versus ≥ 13). We found a statistically significant and clinically relevant difference of 4.3 points in favor of the experimental intervention in the subgroup RDQ ≥ 13  in limitations in activities measured with the RDQ. Other outcomes, such as global perceived effect, main complaint, pain, fear-avoidance and general health supported our main finding but did not reach statistical significance.Although a substantial number of the participants of the cohort (n = 869) reported complaints and limitations in activities to the extent that treatment seems to be necessary during pregnancy, the prognosis (when not receiving treatment) is very good in the first weeks after delivery. For that reason the recruitment of women with persistent complaints and limitations in activities after delivery was lower than expected. We used the maximum of participants out of the cohort (n = 7526) and still included only 126 women. Nevertheless, we reached sufficient statistical power for the primary outcome, limitations in activities. But the smaller than planned study sample could have influenced the results of the other outcomes who were in line with the primary outcome but failed to reach statistical significance.The favorable prognosis after delivery influenced probably also the improvement in both study groups. The recovery rates of both study groups were still relatively high, 12 weeks after delivery. The reasons why a relatively large number of women in the control group abandoned the current treatment option on second thought, leads to the suspect that the favorable prognosis and distraction of the mind by environmental factors (the new born baby and the change in family life) influenced the ask for help by a physiotherapist.Longitudinal data of our cohort study (n = 7526) two weeks after delivery showed also a considerable drop in the number of women that reported pain in the lumbo-pelvic region. In literature there is some consensus about a good prognosis after delivery but not in the same extending as in our cohort. However, we could investigate the course of pregnancy-related pelvic girdle and/or low back pain more in detail and could combine it with the ask for help (physiotherapist) during pregnancy and after delivery in a very large group of women (n = 7526). Focusing only on pain did not answer our question if an intervention by a physiotherapist is advisable for whom and when, satisfactory. A reasonable number of women reported still some pain after delivery but they did not want to be referred to a physiotherapist anymore. Still having some pain was obviously not a deciding factor for many women to search for help. Other factors interfered such as a delay in becoming active again after delivery. Details about the enrollment out of the cohort into the trial showed that the start of the experimental intervention (three weeks after delivery) was reasonably well timed.Subgroup analysis showed that women with moderate limitations in activities (score RDQ at baseline <13 points) improved quite well and nearly in the same extent in both study groups. However, in the subgroups with severe limitations in activities (score RDQ at baseline ≥ 13 points) the improvement favored the experimental intervention group compared to women in the usual care group. This finding underscored the main conclusion of the study clearly that women who received the experimental intervention benefited more from treatment than women who received usual care.In general we did not deviate from the original study design, which was described independently of the study results . At randBecause of the nature of the experimental intervention, blinding of the therapists and women was not possible however the latter were kept naïve of the exact content of both intervention options. A clear treatment or therapist preference could influence responses regarding outcome measures. Therefore women who had a priori a treatment preference for a special treatment or therapist were excluded (n= 50).Prior to the trial, we arranged interviews on an individual basis with women out of the cohort and groups' discussions with experienced physiotherapists and occupational therapists. The outcomes of those interviews were compared with literature findings and the outcomes of the group discussions. The answers of women about their perception of the contents of the current treatment approach and the patient-therapist relationship formed a sharp contrast with the view of the therapists about those topics. It made clear that worries about their condition in this population of women are not minor concerns. The worries referred to an unremitting source of anxiety about the etiology and prognosis. The degree of distress may contribute to avoidance of picking up the full range of activities again after delivery and the feeling that a wrong movement could lead to a serious problem, making individual concerns and worries an important target of care. These aspects and the contrast in perception between therapist and patient were important reasons to introduce, within the scope of a biopsychosocial approach, the theoretical concepts of self-management and fear-avoidance. The developed standardized patient information focused therefore on positive attitudes, an active response to the complaints and a physiological rationale about pain. It did not include information about a possible vulnerability of the spine or pelvis to damage and a relation with pain.In general, women were willing to accept the responsibility to manage their own condition and solve their problems with guidance from the physiotherapists. They picked up the time contingent approach quickly. Nevertheless, there was some hesitation of a number of participating physiotherapists about their role in the intervention protocol during the educational training prior to the trial. Once they were be engaged with patients, they liked their supporting role of the women to create plans that will help her to achieve their goals. The shift from a pain contingent approach to a time contingent approach and the radical change in the character of the patient-therapist relationship seemed to be easier for the women than for the physiotherapists. Finally, both the women and the physiotherapists found the intervention protocol attractive and useful.The results of the trial supported the hypothesis. Although the biopsychosocial approach as a basis for intervention already is largely investigated in several musculoskeletal disorders and generated significant results, in the field of pregnancy-related pelvic girdle and/or low back pain it is a new approach. Women's individual worries and concerns about their condition were major targets in the experimental intervention. By adopting action plans as the most important act for goal setting, women's own values and the role of the therapist were included in the approach. The considerable drop of pain shortly after delivery influenced clearly the number of women that want to be referred to a physiotherapist. Because of the high level of improvement in both groups (even without treatment), it seems likely that the large improvement in the first weeks after delivery continued for a substantial deal in the first three months after delivery.The author(s) declare that they have no competing interests.CHGB: first author is involved in the design, data collection, statistical analysis, development of the experimental intervention, training the participating physiotherapist and the performance of the trial.RAdeB: has made substantial contributions to conception of the study, obtained grants, participated in the design, coordination, statistical analyses, has made substantial contribution to the development of the experimental intervention and is involved in revising the article for important intellectual content.PMJCW: participated in the design, has made substantial contribution to the development of the experimental intervention and is involved in revising the article for important intellectual content.PL: participated in the statistical analyses and is involved in revising the article for important intellectual content.JWSV: has made substantial contributions the development of the experimental intervention and choose of the measurements and is involved in revising the article for important intellectual content.FS: participated as research-physician in this study and is involved in revising the article for important intellectual content.JMB: participated in the design and statistical analyses and is involved in revising the article for important intellectual content.GGME and PvdB: have made substantial contributions to conception, design and experimental intervention and revising the article critically for important intellectual content.The pre-publication history for this paper can be accessed here:


The nucleotide sequence flanking the translation initiation codon (start codon context) affects the translational efficiency of eukaryotic mRNAs, and may indicate the presence of an alternative translation initiation site (TIS) to produce proteins with different properties. Multi-targeting may reflect the translational variability of these other protein forms. In this paper we present a web server that performs computations to investigate the usage of alternative translation initiation sites for the synthesis of new protein variants that might have different functions.Translation Initiation Sites and Secretory Targets) evaluates putative translation initiation sites and indicates the prediction of a signal peptide of the protein encoded from this site. The TISs-ST web server is freely available to both academic and commercial users and can be accessed at .An efficient web-based tool entitled TISs-ST (The program can be used to evaluate alternative translation initiation site consensus with user-specified sequences, based on their composition or on many position weight matrix models. TISs-ST provides analytical and visualization tools for evaluating the periodic frequency, the consensus pattern and the total information content of a sequence data set. A search option allows for the identification of signal peptides from predicted proteins using the PrediSi software. AUGG was observed to be a consensus sequence [AUGGC for monocots and A(A/C)aAUGGC for eudicots [Translation by cytosolic ribosomes generally occurs at the first AUG in the transcript. However, in eukaryotic mRNAs, efficient recognition of an AUG codon as a translation initiation site (TIS) depends on several factors, such as the nucleotide sequence that flanks the site -3. Theresequence . For plaeudicots ,13.coding sequences (CDS), may play a role in generating protein diversity [Upstream out-frame AUG may severely affect the translation of a gene, even if surrounded by a poor context , suggestiversity . The usaiversity -17. Sinciversity , the proIn silico determination of the sub-cellular localization of the proteins can provide information on their function, and is dependent on the correct identification of the first AUG and their potential N-terminally polymorphic forms. This translational polymorphism may serve as an important source of diversity in both cytoplasmic and organelle proteomes [roteomes ,17.Proteins must be localized correctly at the sub-cellular level to have normal biological functions . When thTranslation Initiation Sites and Secretory Targets) web server that investigated the usage of alternative TISs for synthesis of new protein variants possibly possessing different functions. This server deployed previously annotated complete CDSs retrieved from the NCBI UniGene database [In this paper, we present the TISs-ST  the web interface system, which is written in the Perl and HTML languages and (ii) the background process system, which is written in the Perl and R statistical languages. The web interface subsystem mainly deals with the task of receiving information from the user and checking the validity of the data submitted. The background processing subsystem computes all the analytical and prediction tasks: extracts features from the sequences, computes and displays the consensus sequences and total information content, and predicts the signal peptides of the user sequences. We used PrediSi for the prediction of signal peptides in our implementation of TISs-ST.The web interface allows for the easy evaluation of sequences (provided in FASTA format), for the presence of putative translation initiation sites and for the prediction of signal peptides. The TISs-ST interface is designed in such a way that the user specifies all necessary parameters in the initial page, and provides many possibilities to work with the sequence files. The content of a file can be pasted in the input window, or taken from a directory on a local computer. The user has the option of setting several parameters manually.TISs-ST takes the following input data:(i) DNA sequence data. This contains one or several sequences in FASTA format. The sequences can be in a nucleotide format, where the first ATG codon after the first 15 base pairs will be considered to be the one that encodes the initial Methionine. Since the analysis calculates the nucleotide frequencies in a context position, if the user does not choose to run the analysis with a pre-defined PWM, the amount of sequences must be bigger in order to obtain a significant result. A representative sample data is available on the website.(ii) An optional predefined PWM model for species or species group data sets. Currently, 32 species data sets are available on the web server Table  and thes(iii) A filter for the AUG sites.(iv) An option for signal peptide prediction in the protein deduced from the submitted sequence(s). This option requires the selection of the genetic code used to translate the DNA sequences.After submitting the data to the server, the TISs-ST program searches the consensus sequences according to the parameters selected. While the analyses are running, the web server shows a checklist of the steps finalized, from which the user can estimate the total time required for the analysis. An example of the output of the TISs-ST program is shown in Figure In addition to the tabulated output, the result page shows a graphic representation of the consensus and the information content found at each site analysed. There are also hyperlinks for each sequence pattern from the user sequences, the sequence header providing information about the probability of alternative TIS based on the frequency of AUG codons, and hyperlinks to frequency tables of this pattern generated in each analysis Figure .A typical consensus graphical representation concentrates the following information into a single graph: the general consensus of the sequences; the predominance order of the nucleotides at every position ; the relative frequencies of every nucleotide at every position; and the amount of information present at every position in the sequence .TISs-ST uses two different ways to display the graphical representation of consensus sequences: (i) the information content required to describe the pattern, which is the total information content (measured in bits) at every position in a site, and (ii) a useful graphical representation for displaying the global patterns in a set of aligned sequences, to focus on the periodic frequencies of the nucleotides in the consensus pattern.Non-redundant data sets of nucleotide sequences were compiled from the NCBI UniGene . Following the removal of sequences not annotated as 'complete CDS' (not identified previously as putatively and complete coding sequence), only sequences that had termination codon and had 15 bp before the first ATG annotated, remained in the data set. For this set, sequences were grouped according to species, based on their taxonomic classification in the Integrated Taxonomic Information System on-line database . SpeciesData sets of fragments flanking the AUG were created from the -15 to +15 nucleotides of each AUG in every sequence. All fragments were grouped and the consensus sequences were determined separately for each AUG  for each species and group of species using the 50/75 consensus rule described by Cavener . The reaThe consensus at a position is computed according to the following rules, with decreasing order of priority: (i) if a nucleotide at that position has a relative frequency greater than 50% and greater than twice the relative frequency of the second most frequent nucleotide, the nucleotide is given consensus status that is indicated in uppercase; (ii) if the sum of the relative frequencies of a pair of nucleotides exceeds 75%, these two nucleotides are given co-consensus status, indicated in uppercase; (iii) if there is a single most frequent nucleotide, it is given dominant status, indicated in lowercase; (iv) if two bases have the same highest frequency, they are given co-dominant status that is indicated in lowercase.The method begins by calculating a weight matrix from the frequencies of each nucleotide at each position of the aligned sequences. This matrix is then applied to the sequences to determine the sequence conservation of each individual site. Additionally we considered the nucleotide bias in genomes by using a linear noise correction .Riw , is created by using an aligned training set consisting of sequences from the nucleotides databases described before. The PWM is computed using the widely accepted information theoretical approach with some modifications [n as follows: Riw = A PWM model of first AUG site, called ications ,29. In Tications . If a paaaa@30E5@. Since taaa@30E5@.jth sequence by a matrix Sj  that contains only 0's and 1's. The individual information content of a base (Ri (l)), given by some manipulation of the Ri (j) [In a set of submitted sequences, we represent the e Ri (j) , is the Ri:And the total information content of the sites is the The prediction of signal peptides was evaluated by the PrediSi prediction program , which aArabidopsis data set (n = 14525) were used. Each of these data sets was used for training and testing the program. The total sequences were divided into two equal sets, training and testing. The training set was cross-validated by testing with the testing data set.The maize data set (n = 518) and Using the confusion matrix, various measurements of quality such as accuracy, specificity, sensitivity and the Matthews correlation coefficient (MCC)  were detaccuracy (AC): proportion of correct predictions of the total predictions.specificity (SP): proportion of true negatives to the total negatives.sensitivity (SN): proportion of true positives to the total positives.MCC: This is regarded as a more rigorous measurement to evaluate the performance of class prediction methods. MCC equals 1 for perfect predictions, whilst it is zero for completely random predictions .Prediction can be performed at different levels of specificity and sensitivity by defining various thresholds for the final score. At each threshold, the numbers for the TPs, TNs, FPs and FNs are calculated, and based on these values parameters such as accuracy, specificity and sensitivity are determined using equations (3–5). As seen in Table Specificity and sensitivity are two competing quality measurements for any two-classifier method. Table In the literature, purines are usually claimed to be important at position -3. This was the case for maize and tobacco suspension cells . AlthougWe also performed analyses on the rice and tomato data sets, and the results of a TISs-ST search of the context surrounding the first translated AUG are shown in Figure The current method was applied to a real data set of different mRNA variants produced by differential splicing in the human phosphodiesterase 9A gene. The PDE9A gene encodes a cGMP-specific high-affinity phosphodiesterase, and the physiological implication of the isoforms produced by this gene is not yet known ,32. At lWe performed analyses on the data set composed of splice variants that used the first start codon in exon 1 (7 isoforms) and the possible start codon present in exon 8 (5 isoforms). The results of the analyses of these sites are shown in Figure The observation that sub-optimal AUG contexts are present in many genes suggests the hypothesis that this context might be involved in modulation of gene expression. This might be the case for transcripts encoding two proteins that differ at their N-terminal end , which wSeveral molecular mechanisms might provide for efficient translation of the mRNAs containing upstream AUGs including leaky scanning and reinitiation or internal initiation of the translation. The contributions of these mechanisms remain uncertain but several recent studies suggest that the impact of at least some of them might be substantial ,7,34-36.Many databases in this research area are available on the web. Examples include Transterm, an information resource devoted to contexts of translation initiation and termination sites , and UTR• Project name: TISs-ST• Project home page: • Operating systems(s): Platform independent• Programming language: Perl and R• Other requirements: to build a local version of the web-service it is necessary to have a web server that allows CGI and Perl.• License: under the GNU General Public LicenseRV conceived the study, conducted the work and drafted the manuscript. MM participated in the design and coordination of the study and helped draft the manuscript. All the authors read and approved the manuscript.