Functional characteristics of patients with retinal dystrophy that manifest abnormal parafoveal annuli of high density fundus autofluorescence ; a review and update Purpose To examine the presence and functional significance of annular fundus autofluorescence abnormalities in patients with different retinal dystrophies . Methods Eighty one patients were ascertained who had a parafoveal ring of high density on fundus autofluorescence imaging . Sixty two had had a clinical diagnosis of retinitis pigmentosa -LRB- RP -RRB- or Usher syndrome with normal visual acuity . Others included a case of Leber congenital amaurosis and genetically confirmed cases of cone or cone-rod dystrophy -LRB- GUCA1A , RPGR , RIMS1 -RRB- , `` cone dystrophy with supernormal rod ERG '' -LRB- KCNV2 -RRB- and X-linked retinoschisis -LRB- RS1 -RRB- . International-standard full-field and pattern electroretinography -LRB- ERG ; PERG -RRB- were performed . Some patients with rod-cone or cone-rod dystrophy underwent multifocal ERG -LRB- mfERG -RRB- testing and photopic and scotopic fine matrix mapping -LRB- FMM -RRB- . Results In patients with RP , the radius of the parafoveal ring of high density correlated with PERG P50 -LRB- R = 0.83 , P < 0.0005 , N = 62 -RRB- and encircled areas of preserved photopic function . In the other patients , AF rings either resembled those seen in RP or encircled an area of central atrophy . Ring radius was inversely related to the PERG P50 component in 4 of 18 cases with a detectable response . FMM showed that arcs of high density were associated with a gradient of sensitivity change . Conclusions Parafoveal rings of high density autofluorescence are a non-specific manifestation of retinal dysfunction that can occur in different retinal dystrophies . Electrophysiology remains essential for accurate diagnosis . The high correlation of autofluorescence with PERG , mfERG and FMM demonstrates that AF abnormalities have functional significance and may help identify suitable patients and retinal areas amenable to future therapeutic intervention . Introduction Lipofuscin is derived from the shed and degraded photoreceptor outer segments and normally accumulates in the retinal pigment epithelium -LRB- RPE -RRB- with age -LSB- 1 -- 4 -RSB- . An abnormal increase or depletion of lipofuscin manifests as high or low density areas respectively in fundus autofluorescence -LRB- AF -RRB- images -LSB- 5 -RSB- and such changes may aid in the detection and characterisation of a wide range of inherited retinal disorders , either by accentuating the visibility of fundus abnormalities or by revealing changes not accessible by routine examination or fluorescein angiography -LSB- 5 -- 13 -RSB- . Lipofuscin has been shown to fragment when exposed to light -LSB- 14 -RSB- and its presence suggests continuing metabolic demand -LSB- 5 -RSB- . Absence of autofluorescence suggests either blocking of the incident radiation , photoreceptor cell death and RPE atrophy -LSB- 5 , 6 -RSB- or disruption of the vitamin A cycle -LSB- 15 , 16 -RSB- . Some patients with genetically different forms of retinitis pigmentosa manifest a ring of high density AF representing abnormal parafoveal accumulation of lipofuscin which usually encircles preserved foveal areas -LSB- 17 -- 20 -RSB- . Similar parafoveal rings have recently been documented in other retinal dystrophies including some patients with Leber congenital amaurosis -LSB- 21 -RSB- , Best disease -LSB- 13 -RSB- , X-linked retinoschisis -LSB- 22 -RSB- and cone-rod dystrophy consequent upon mutation in GUCA1A -LSB- 23 -RSB- , GUCY2D -LSB- 24 -RSB- , RIMS1 -LSB- 25 , 26 -RSB- , or RPGRORF15 -LSB- 26 , 27 -RSB- . Abnormal annular AF has also been described in some cases of `` cone dystrophy with supernormal rod ERG '' consequent upon mutation in KCNV2 -LSB- 28 , 29 -RSB- . Unlike RP , these disorders often result in atrophic macular changes , manifesting as low density AF within the ring . The main aims of the current study were to review the electrophysiological phenotypes associated with annular AF abnormalities , and to examine two heterogeneous groups of patients in more detail by comparing indices of macular function associated with abnormal macular AF in rod-cone and cone-rod dystrophies . Materials and methods Autofluorescence imaging was performed using a scanning laser ophthalmoscope according to previously described techniques -LSB- 5 , 30 -RSB- . Eighty one patients with abnormal AF of the posterior pole in the form of a high density parafoveal ring were reviewed . Sixty two had a clinical diagnosis of retinitis pigmentosa or Usher syndrome with a visual acuity of 6/9 or better and included 30 cases described previously -LSB- 17 -RSB- . Nineteen other individuals were ascertained from previous studies -LSB- 21 -- 23 , 25 -- 29 -RSB- including 14 with cone-rod or cone dystrophy consequent upon mutation in RPGR ORF15 -LRB- 4 cases -RRB- , RIMS1 -LRB- 8 cases -RRB- or GUCA1A -LRB- 2 cases -RRB- . Two subjects had `` cone dystrophy with supernormal rod ERG '' consequent upon mutation in KCNV2 , two had RS1mutations and manifested rings in one or both eyes . One patient had Leber congenital amaurosis . Full-field ERGs were performed according to extended testing protocols incorporating the ISCEV minimum standard -LSB- 31 -RSB- in order to assess generalised retinal function . A stimulus 0.6 log units greater than the ISCEV maximum was also used , to demonstrate better the a-wave under conditions of dark adaptation . Pattern ERGs evoked by high contrast checkerboard reversal were recorded according to ISCEV recommendations -LSB- 32 -RSB- using standard parameters ; checkerboard size 12 × 15 degrees , check size 45 ′ , Michelson contrast 0.98 . The PERG P50 component was used as an index of macular function . Additional PERG testing was performed using a range of circular checkerboard fields ranging from 3 to 18 degrees in diameter presented in a random order -LSB- 17 -RSB- . Check size was constant at 45 ′ . International-standard mfERGs -LSB- 33 , 34 -RSB- and fine matrix mapping were performed in some cases . Fine matrix mapping measures rod and cone system sensitivity and has been described previously -LSB- 18 , 35 , 36 -RSB- . In brief , detection thresholds for a spot of light were determined at 1-degree intervals over selected 9 × 9-degree retinal areas . Data were presented both as sensitivity contours , illustrating the position and orientation of tested retinal locations and as three-dimensional threshold profiles , plotted using interpolated values at 0.25-degree intervals , obtained by Gaussian filtering . Results All patients were selected according to fundus autofluorescence that was characterised by an abnormal high density parafoveal ring -LRB- Figs. 1 , 2 , 4 -- 8 -RRB- . Sixty two with a clinical diagnosis of retinitis pigmentosa or Usher syndrome and a visual acuity of 6/9 or better had evidence of preserved RPE within the ring . Nineteen others had either cone or cone-rod dystrophy , `` cone-dystrophy with supernormal rod ERG '' , X-linked retinoschisis or Leber congenital amaurosis . Many of the older non-RP cases had evidence of macular RPE atrophy within the ring -LRB- Figs. 2b -- d , 7c , 8b -- c -RRB- . Conversely , AF imaging in some RP patients showed mild to moderate atrophic RPE changes within the vascular arcades but external to the ring -LRB- Figs. 4d and 5e -RRB- . Fig. 1Full-field ERGs , PERGs and AF in 3 patients with rod-cone dystrophy -LRB- a -- c -RRB- including a patient with RP18 -LRB- a -RRB- and Usher syndrome -LRB- b -RRB- . Row D shows full-field ERGs and AF in a case of Leber congenital amaurosis ; ERGs and the AF image were obtained in the presence of nystagmus and are consequently noisy . Normal examples are shown for comparison -LRB- e -RRB- . LU indicates log units greater -LRB- + -RRB- or less -LRB- − -RRB- than the ISCEV standard flashFig . 2Full-field ERGs , PERGs and AF in cone rod-dystrophy consequent upon mutation in RPGR -LRB- a -RRB- , RIMS1 -LRB- b -RRB- , `` cone dystrophy with supernormal rod ERG '' -LRB- c -RRB- and in a case of X-linked retinoschisis -LRB- RS1 ; d -RRB- . Normal examples are shown for comparison -LRB- e -RRB- . LU indicates log units greater -LRB- + -RRB- or less -LRB- − -RRB- than the ISCEV standard flash Figures 1 and 2 show representative full-field ERGs and PERGs in eight individuals with different retinal dystrophies . Figure 1 shows data from 3 subjects with rod-cone dystrophy and normal visual acuity including a case of autosomal dominant RP18 -LRB- Fig. 1a -RRB- and Usher syndrome -LRB- Fig. 1b -RRB- . Figure 1d shows undetectable ERGs in a patient with Leber congenital amaurosis ; ERGs are contaminated by the effects of nystagmus but no ERG is detectable , in keeping with severe generalised photoreceptor dysfunction . Figure 2 shows representative examples of cone-rod dystrophy consequent upon mutations in RPGR -LRB- Fig. 2a -RRB- , RIMS1 -LRB- Fig. 2b -RRB- and KCNV2 -LRB- `` cone dystrophy with supernormal rod ERG '' ; Fig. 2c -RRB- . Figure 2d shows the ERG and AF findings in a patient with X-linked retinoschisis . In the 62 RP cases PERG P50 components varied between normal -LRB- > 2 uV -RRB- and undetectable , consistent with varying degrees of macular involvement . There was no significant correlation with the bright flash ERG a-wave amplitude or 30 Hz flicker ERG amplitude . Figure 3 shows high positive correlation between PERG P50 and mean ring radius -LRB- r = 0.83 , P < 0.0005 -RRB- . Data are shown from one RP patient that was tested comprehensively -LRB- Fig. 4 -RRB- ; PERGs were normal to the smallest diameter checkerboard but minimal enlargement was seen as the stimulus field size was increased -LRB- Fig. 4b -RRB- . Multifocal ERGs showed widespread reduction with relative preservation of the response associated with the central stimulus element -LRB- Fig. 4a -RRB- , consistent with a central island of visual field preservation -LRB- Fig. 4c -RRB- . The internal edge of visual field constriction corresponded closely with the ring of high density , as shown by photopic FMM -LRB- Fig. 4d -RRB- . Scotopic fine matrix mapping revealed rod sensitivity losses that were severe and that encroached upon the central macula within the ring . Additional examples of mfERGs , visual fields and fine matrix mapping in RP patients are shown in Figs. 5 and 6 . Fig. 3Comparison of mean ring radius with PERG P50 in 62 patients with rod-cone dystrophy -LRB- RP ; broken linear regression line -RRB- and normal visual acuity and in 19 patients with other retinal dystrophies , including 4 cone or cone-rod dystrophy cases in which there was a detectable PERG -LRB- solid regression line -RRB- Fig. 4Multifocal ERGs -LRB- a -RRB- , small field PERGs -LRB- b -RRB- , Humphrey visual field -LRB- c -RRB- and photopic -LRB- d -RRB- and scotopic -LRB- e -RRB- fine matrix mapping in a patient with a clinical diagnosis of RP . Diamonds and error bars in -LRB- b -RRB- show mean values and standard deviations for 8 normal subjects ; triangles and squares show patient data from right and left eyes . Contour plots -LRB- d and e -RRB- show sensitivity gradients over tested retinal locations ; corresponding 3-D plots show retinal location -LRB- abscissa , degrees -RRB- and thresholds -LRB- ordinate , log units -RRB- . Labelling -LRB- x -RRB- shows correspondence between the orientation of contour and threshold plots . Normal photopic and scotopic values are plotted in Fig. 5a and dFig . 5Contour sensitivity plots -LRB- rows 1 and 3 -RRB- and 3-D threshold plots -LRB- rows 2 and 4 -RRB- obtained in representative normal subjects -LRB- a , d -RRB- and in 3 RP patients -LRB- b , e and c , f -RRB- . Subjects were tested under photopic -LRB- a -- c -RRB- and/or scotopic conditions -LRB- d -- f -RRB- . Labelling -LRB- x -RRB- shows correspondence between the orientation of contour and threshold plots . Abscissa shows retinal location -LRB- degrees -RRB- , ordinate axes show threshold -LRB- log units -RRB- . Corresponding photopic FMM in individual -LRB- e -RRB- has been published elsewhere -LSB- 18 -RSB- . Normal 3-D plots show averaged data from 14 -LRB- a -RRB- or 12 -LRB- d -RRB- normal subjectsFig . 6AF images , mfERGs and corresponding Humphrey visual fields in 3 patients with RP and normal visual acuity Pattern ERGs were detectable in only 4 patients with cone or cone-rod dystrophy -LRB- GUCA1A , RPGR or RIMS1 , Fig. 3 -RRB- . Pattern ERG P50 was inversely related to ring size in these subjects -LRB- Fig. 3 -RRB- . Data from one subject with cone-rod dystrophy -LRB- RIMS1 -RRB- are shown in Fig. 7 . Multifocal ERG showed widespread reduction with only relative preservation of the central response -LRB- Fig. 7a -RRB- . Standard Humphrey visual fields showed a central scotoma and some superior field loss -LRB- Fig. 7b -RRB- . Fine matrix mapping revealed severe threshold elevation across the macula but with relatively preserved photopic sensitivity over a central island of preserved RPE AF -LRB- Fig. 7c -RRB- . Threshold values inside the ring are maximally elevated over a concentric atrophic area and show a gradient of increasing sensitivity over the arc of high density -LRB- Fig. 7c and e -RRB- . The PERGs in this patient were undetectable -LRB- data not shown -RRB- . Additional examples of fine matrix mapping in RIMS1 and RPGR patients are illustrated in Fig. 8 . Central RPE atrophy was not always present -LRB- Figs. 2a , 8a -RRB- . Fig. 7Multifocal ERGs -LRB- a -RRB- , Humphrey visual field -LRB- b -RRB- and photopic -LRB- c , d -RRB- and scotopic -LRB- e , f -RRB- fine matrix mapping in a patient with cone-rod dystrophy consequent upon RIMS1 mutation . Labelling -LRB- x -RRB- shows correspondence between the orientation of contour and 3-D threshold plots . Abscissa shows retinal location -LRB- degrees -RRB- , ordinate axes show threshold -LRB- log units -RRB- . Threshold values for half the tested area have been removed from c and e , to expose the foveal values that would otherwise be obscuredFig . 8Contour sensitivity plots -LRB- rows 1 and 3 -RRB- and 3-D threshold plots -LRB- rows 2 and 4 -RRB- obtained in 3 patients with cone-rod dystrophy consequent upon RPGR -LRB- column 1 -RRB- or RIMS1 mutations -LRB- columns 2 and 3 -RRB- . Subjects were tested under photopic -LRB- a -- c -RRB- and scotopic conditions -LRB- d -- f -RRB- . Labelling -LRB- x -RRB- shows correspondence between the orientation of contour and threshold plots . Abscissa shows retinal location -LRB- degrees -RRB- , ordinate axes show threshold -LRB- log units -RRB- Discussion This study reviews a heterogeneous group of 81 patients with genetically-determined retinal diseases that manifest a common feature on fundus autofluorescence imaging in the form of a parafoveal ring of high density . Patients with RP and normal visual acuity had rings that encircled preserved central AF . Mild to moderate atrophic changes were occasionally detected within the vascular arcades but eccentric to the ring -LRB- Figs. 4d , 5e -RRB- . In the other retinal dystrophies that were examined in this and in previous studies -LSB- 25 -- 27 -RSB- , the ring could also encircle preserved central AF but in older subjects there was often central RPE atrophy within the annulus and preserved AF at more eccentric locations . As patients with RP or different forms of retinal dystrophy can have indistinguishable AF abnormalities , the AF appearance can not be used to establish a diagnosis in such cases . Non-specific annular increases in AF may be associated with a wide variety of distinctive or pathognomonic full-field ERG changes that are essential for accurate diagnosis and functional phenotyping -LRB- see below -RRB- . The data from 62 patients with RP and normal visual acuity show a high positive correlation between ring size and the PERG P50 component extending and confirming findings in a cohort of 30 of these cases -LSB- 17 -RSB- , demonstrating the robust nature of this relationship . The findings are corroborated by high spatial resolution fine matrix mapping , visual field data and mfERG testing that shows preserved photopic sensitivity within central areas bordered by the ring , consistent with previous reports -LSB- 18 -- 20 , 37 -RSB- . Scotopic sensitivity losses encroach upon central macular areas suggesting that rod-system dysfunction precedes abnormal parafoveal accumulation of lipofuscin and progressive visual field loss -LSB- 18 -RSB- . Serial data indicate that the rings may vary greatly in terms of their stability ; to date only 3 cases have been reported in which progressive ring constriction has occurred -LSB- 37 -RSB- . The rate of AF ring constriction may prove to be of prognostic value in predicting retention of visual acuity and visual field preservation , but further monitoring is required . In addition to RP , the parafoveal ring of high density may occur in cone or cone-rod dystrophy consequent upon mutation in GUCA1A -LSB- 23 -RSB- , GUCY2D -LSB- 24 -RSB- , RPGR -LSB- 25 , 26 -RSB- , RIMS1 -LSB- 26 , 27 -RSB- , in `` cone dystrophy with supernormal rod ERG '' -LRB- KCNV2 -RRB- -LSB- 28 -RSB- , in X-linked retinoschisis -LRB- RS1 -RRB- -LSB- 22 -RSB- and in Leber congenital amaurosis -LSB- 21 -RSB- . Similar AF findings have also been documented in Best Disease -LSB- 13 -RSB- and other maculopathies -LSB- 10 -RSB- . In young patients with cone-rod dystrophy , small rings may have preserved central AF and are similar to those seen in RP cases -LSB- 26 -RSB- . Older individuals tend to manifest central atrophic changes that are encircled by the ring and there may be a central island of RPE preservation . Fine matrix mapping suggests that the abnormal accumulation of lipofuscin in cases of cone-rod dystrophy represents a transitional stage between relatively preserved parafoveal function and severe central dysfunction that is likely to precede central atrophy -LSB- 26 , 27 -RSB- . In patients with cone-rod dystrophy consequent upon RPGR or RIMS1 mutations , serial studies have recently demonstrated ring expansion -LSB- 26 -RSB- , suggesting an expanding front of macular photoreceptor dysfunction . This contrasts with RP where the opposite occurs -LSB- 37 -RSB- . It is possible that rings associated with other causes of maculopathy may also expand with time as lesions become larger with age . It is noted that RPGR mutations are more commonly associated with X-linked retinitis pigmentosa -LSB- 38 -RSB- with visual acuity reduction -LSB- 39 -RSB- ; in the current study not all patients underwent genetic screening but none were known to have X-linked RP . Lipofuscin accumulation in the RPE is likely to reflect metabolic activity which is largely determined by outer segment renewal . There is evidence of light-induced degradation of lipofuscin -LSB- 14 -RSB- , so that its presence would depend upon continuing metabolic demand . Evidence from RCS rats indicates that failure of RPE cells to phagocytose outer segments results in reduced lipofuscin levels -LSB- 40 -RSB- . Lipofuscin formation is almost completely dependent on a normal visual cycle and on the availability of dietary vitamin A -LSB- 41 -RSB- . In the RPE65 knockout mouse , the visual cycle is impaired and fluorescence is reduced -LSB- 42 -RSB- . Similarly , patients with RPE65 mutations can have reduced autofluorescence in the presence of near-normal fundi and OCT findings that do not suggest reduction in the photoreceptor layer thickness -LSB- 15 , 16 -RSB- . It is widely accepted that photoreceptor degeneration results in atrophy and reduced rather than stable levels of autofluorescence and it is likely that autofluorescence is lost within a few weeks of photoreceptor loss . The presence of foveal or parafoveal AF in the photoreceptor dystrophies outlined in the current study , suggest that photoreceptors are likely to be intact and thus may be amenable to functional rescue . Recent developments , for example in the field of gene therapy , make early identification of candidate patients increasingly important . Patients with RP classically present with impaired night vision and visual field constriction , consistent with generalised retinal dysfunction involving rod more than cone photoreceptors . In the early stages the fundi may be near normal and rod-cone dystrophy is established by full-field ERG testing -LRB- Fig. 1 -RRB- . In patients with normal visual acuity , the degree of macular sparing can not be predicted from the severity of peripheral dysfunction . The cone and cone-rod dystrophies are typically characterised by progressive worsening of visual acuity , dyschromatopsia , and photophobia with eventual central scotomata and peripheral field abnormalities . Ophthalmoscopic abnormalities , when present , are generally confined to the macula . Photopic full-field ERGs are typically delayed and subnormal with milder scotopic ERG abnormalities -LRB- Fig. 2a and b -RRB- . A notable exception is the autosomal dominant cone dystrophy consequent upon mutation in GUCA1A ; cone-mediated ERGs show amplitude reduction without significant implicit time delay -LSB- 23 -RSB- and although not diagnostic , may enable focussed mutational screening . Patients with `` cone dystrophy with supernormal rod ERG '' exhibit a wide range of fundus AF abnormalities including ring-like or bull 's eye changes , central atrophy or increased foveal AF -LSB- 28 -RSB- . Full field ERGs are pathognomonic in this disorder -LRB- Fig. 2c -RRB- , have recently been shown to be consequent upon mutation in KCNV2 and have been described in detail -LSB- 28 , 29 -RSB- . X-linked retinoschisis is usually associated with an electronegative bright flash ERG in keeping with inner retinal dysfunction -LRB- Fig. 2d -RRB- ; two cases -LRB- aged 47 years and 49 years -RRB- with annular AF abnormalities have been documented within a series of seven atypical but genetically confirmed cases -LSB- 22 -RSB- . The rings of high density in these individuals surround areas of central atrophy and may represent an outer boundary of macular photoreceptor dysfunction in addition to generalised inner rather than outer retinal disease . Commonly , younger individuals with X-linked retinoschisis manifest stellate macular lesions that are also visible in AF images -LSB- 43 -RSB- . Conclusions A parafoveal ring of high density autofluorescence is a non-specific manifestation seen in different retinal dystrophies . Electrophysiology remains essential for accurate diagnosis . The high correlation of autofluorescence with PERG , mfERG and FMM demonstrates that AF abnormalities have functional significance and may be an important parameter in the monitoring of these patients . Autofluorescence may be of prognostic value and may help identify suitable patients and retinal areas amenable to future therapeutic intervention .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

electroretinography
retinitis pigmentosa
usher syndrome
autofluorescence imaging

cone-rod dystrophy
genotype-phenotype correlation


Endometrial expression of the insulin-like growth factor system during uterine involution in the postpartum dairy cow ☆ Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception . Expression patterns for members of the insulin-like growth factor -LRB- IGF -RRB- family were determined by in situ hybridisation at day 14 ± 0.4 postpartum -LRB- n = 12 cows -RRB- to investigate a potential role for IGFs in modulating uterine involution . Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn . IGF-I mRNA was localized to the sub-epithelial stroma -LRB- SES -RRB- of inter-caruncular and caruncular endometrium . Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma -LRB- DES -RRB- , the caruncular stroma and myometrium . IGFBP-2 , IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue , and in the DES and caruncular stroma , with IGFBP-4 mRNA additionally expressed in myometrium . IGFBP-3 mRNA was only detectable in luminal epithelium . IGFBP-5 mRNA was found in myometrium , inter-caruncular and caruncular SES and caruncular stroma . These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state . The differential expression of binding proteins between tissues -LRB- IGFBP-3 in epithelium , IGFBP-2 , -4 , -5 and -6 in stroma and IGFBP-4 and -5 in myometrium -RRB- suggest tight control of IGF activity within each compartment . Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling . 1 Introduction In dairy cows , the peri-partum period is critical to future milk production and fertility . Uterine involution involves extensive restructuring of the extracellular matrix alongside mitogenesis and apoptosis -LSB- 1 -- 3 -RSB- . Initial degeneration of placental cotyledons and maternal caruncles accumulate as tissue debris in the uterine lumen forming a lochial discharge -LSB- 4 -RSB- . Contractions of the myometrium aid expulsion of lochia , and also restore uterine size , shape and tone to that of a non-pregnant animal -LSB- 5,6 -RSB- . Whilst most of these changes have occurred within 2 -- 3 weeks postpartum , involution is not considered complete until about 40 -- 50 days postpartum -LSB- 1 -RSB- . The previously non-gravid uterine horn returns to a non-pregnant state 10 -- 15 days earlier than the previously gravid uterine horn -LSB- 7 -RSB- . Histological repair of the endometrium lags physical involution by 10 -- 20 days -LSB- 8 -RSB- , completing when caruncles regenerate epithelium -LSB- 4 -RSB- . Microbial contamination of the postpartum uterus is almost universal during the first week postpartum -LSB- 9 -RSB- . When pathogenic bacteria are not cleared the uterus becomes infected and inflamed and uterine involution is delayed -LSB- 1,10 -RSB- . Clinical endometritis is characterised by the continued presence of a purulent discharge beyond 21 days after calving -LSB- 1 -RSB- . Many processes involved in uterine repair are common to those of wound healing in other tissues -LRB- for a review see -LSB- 11 -RSB- -RRB- . Potential mediators of tissue turnover and remodelling in the uterus include cytokines , matrix-degrading enzymes and growth factors -LSB- 11,12 -RSB- . The insulin-like growth factors -LRB- IGF-I and IGF-II -RRB- function in such tissue repair processes . In healing-impaired wounds , the mRNA for IGF-I , IGF-1R , and IGFBP-3 is significantly reduced -LSB- 13 -RSB- . The administration of IGF-I to these wounds corrects defective tissue repair -LSB- 14 -RSB- and in combination with other growth factors it increases connective tissue regeneration and epithelialisation -LSB- 15 -RSB- . Components of the IGF system have been described in the uteri of a variety of species -LRB- e.g. humans -LSB- 16 -RSB- , rodents -LSB- 17 -RSB- , pigs -LSB- 18 -RSB- , cattle -LSB- 19 -RSB- , and sheep -LSB- 20 -RSB- -RRB- . The proliferative and differentiating effects of IGFs on uterine cells are thought to support the growth and regression of uterine tissue throughout the estrous cycle and also the regenerative processes in women following menstruation -LSB- 16,21 -RSB- . IGFBP-2 has also been shown to stimulate endometrial cell mitogenesis directly -LSB- 22 -RSB- . An increased rate of uterine involution is associated with earlier resumption of ovarian activity -LSB- 23 -RSB- , which is in turn important for increasing pregnancy rate to first service -LSB- 24 -RSB- . Conversely , endometrial damage associated with sub-clinical endometritis leads to prolonged intervals to conception , with many cows failing to conceive at all -LSB- 25 -RSB- . The mechanisms that regulate uterine involution are not completely understood and , to the best of our knowledge , no previous studies have investigated the uterine IGF system during involution in lactating dairy cows . We postulated that changes in IGF bioavailability may be implicated in the rate of postpartum uterine recovery and thus influence the calving to conception interval and reproductive efficiency . The objective of the study was to determine patterns of mRNA expression for the IGF system within the previously gravid -LRB- PG -RRB- and previously non-gravid -LRB- PNG -RRB- uterine horns during the early postpartum period . Samples were obtained at approximately 2 weeks after calving as we hypothesised that this represents a time by which a delay in the normal recovery process may predispose cows to the subsequent development of endometritis . 2 Materials and methods 2.1 Animals and tissue samples All procedures were carried out under license in accordance with the European Community Directive , 86-609-EC . Uteri were collected from 12 multiparous Holstein-Friesian dairy cows -LRB- mean parity 4.7 -RRB- following slaughter at day 14 ± 0.4 postpartum . The diameters of both horns were measured approximately 5 cm anterior to the bifurcation of the uterus . Samples of inter-caruncular and caruncular tissue were dissected from the previously gravid and non-gravid uterine horns approximately 1 cm anterior to the bifurcation of the uterus . A 5 cm square region was harvested , wrapped in aluminium foil , and frozen in liquid nitrogen-tempered isopentane . Samples were stored at − 80 °C until sectioning . 2.2 In situ hybridisation The in situ hybridisation procedure was performed as described previously -LSB- 26 -RSB- . All chemicals were purchased from Sigma -- Aldrich Company Ltd. -LRB- Poole , Dorset , UK -RRB- or VWR International Ltd. -LRB- Poole , Dorset , UK -RRB- unless otherwise specified . Briefly , sections of 10 μm were cut from each uterine tissue sample and thaw-mounted onto SuperFrost ® Plus or POLYSINE ™ microscope slides , fixed in 4 % -LRB- w/v -RRB- paraformaldehyde in 0.01 M PBS , washed in PBS , and sequentially dehydrated in 70 % and 95 % ethanol . The oligonucleotide probes for the IGF system were end-labelled with -LSB- 35S -RSB- dATP -LRB- Amersham Biosciences UK Ltd. , Buckinghamshire , England -RRB- using terminal deoxynucleotidyl transferase -LRB- Promega UK Ltd. , Southampton , England -RRB- . Tissue sections were subsequently treated with 100 000 cpm -LRB- 100 μl -RRB- − 1 hybridisation buffer and hybridised overnight at either 42 , 45 , or 52 °C -LRB- Table 1 -RRB- . Following incubation , slides were washed in a solution of 1 × SSC , 2 g l − 1 sodium thiosulphate at room temperature for 30 min followed by fresh 1 × SSC , 2 g l − 1 sodium thiosulphate at 60 °C for 60 min . Slides were then rinsed in solutions of 1 × SSC , 0.1 × SSC , 75 % ethanol and 95 % ethanol and air-dried before exposure to β-max hyperfilm -LRB- Kodak BioMax MR Film -RRB- for either 4 or 5 days . All uterine sections treated with a particular probe were hybridized in the same batch . Sense probes , which were identical in sequence to the respective mRNA targets , were always included as negative controls and any signal from these was regarded as non-specific . Each batch also contained an appropriate positive control tissue , based on previous studies . These were cross-sections of uterus from an estrous ewe for IGF-I and the type 1 IGF receptor -LSB- 20 -RSB- , IGFBP-1 -LSB- 27 -RSB- and IGFBP-6 -LSB- 28 -RSB- ; ovine placentome for IGF-II and IGFBPs-2 , -3 and -4 -LSB- 29 -RSB- and ovine intercotyledonary tissue for IGFBP-5 -LSB- 30 -RSB- . 2.3 Photographic emulsions To aid cellular localisation of hybridised probes , slides previously subject to autoradiography were coated with photographic emulsion LM1 -LRB- Amersham Biosciences UK Ltd. , Buckinghamshire , England -RRB- according to the manufacturer 's instructions and stored for 28 , 30 or 42 days at 4 °C in the dark -LRB- Table 1 -RRB- . The slides were developed in 20 % phenisol -LRB- ILFORD Imaging UK Ltd. , Cheshire , England -RRB- fixed in 1.9 M sodium thiosulphate and counterstained with haematoxylin and eosin . All other slides were also stained with haematoxylin and eosin to aid identification of tissue region . 2.4 Optical density measurements Readings were obtained from at least two sections per tissue for each of the antisense -LRB- AS -RRB- and sense -LRB- S -RRB- probes . Autoradiographs were scanned into a computer and optical density -LRB- OD -RRB- measurements were recorded from digital images . The relative expression of mRNA for components of the uterine IGF system was quantified from the autoradiographs using the public domain NIH ImageJ program -LRB- available through the NIH website -- http://www.nih.gov -RRB- , which calculated the average optical density -LRB- OD -RRB- over the selected area of film based on a linear grey scale of 0.01 -- 2.71 . The following tissue layers were each assessed separately : luminal epithelium , sub-epithelial stroma -LRB- a layer of dense connective tissue underlying the luminal epithelium -RRB- , caruncular stroma -LRB- the dense connective tissue forming the caruncles -RRB- , deep endometrial stroma -LRB- loose connective tissue between the sub-epithelial stroma and the myometrium -RRB- and myometrium . The latter two tissue types were only present in samples collected from the inter-caruncular region . Each tissue type was measured separately on each section . The background OD , from a blank area of film , was also measured and subtracted from both AS and S OD measurements . Finally the S values were subtracted from AS values to give an average OD value for specific hybridisation -LSB- 31 -RSB- . The detection limit was taken as an OD value of 0.01 . 2.5 Statistical analysis Statistical analyses were performed using Statistical Package for the Social Sciences -LRB- SPSS for Windows , V13 .0 -RRB- . Data for uterine diameter measurements at the time of tissue collection were analysed using Student 's t-test . OD measurements were obtained from four samples per cow , taken from each of the caruncular and inter-caruncular regions of the previously gravid and non-gravid horns . The effects of uterine horn and tissue region on the level of mRNA expression for each probe were analysed by general linear model analysis . Cow was entered as a random effect . For this purpose , data from uteri in which a particular probe showed no detectable specific hybridisation -LRB- OD of < 0.01 -RRB- were given an OD of 0.01 , which equated to the lower limit of detection . Results were considered statistically significant when P < 0.05 . 3 Results At the time of tissue collection , the diameter of the previously gravid uterine horn was larger than that of the previously non-gravid uterine horn -LRB- 56 ± 6.9 and 31 ± 3.1 mm , respectively , mean ± S.E.M. , P = 0.005 -RRB- . The spatial distribution of mRNA encoding components of the uterine IGF system is shown in Figs. 1 and 2 . The concentrations of mRNA in OD units are summarised in Table 2 according to uterine horn and tissue region and their two-way interactions are illustrated in Figs. 3 and 4 . The method used provided a semi quantitative measure of the intensity of mRNA expression in specific cell types . 3.1 Expression of the IGFs and IGF type 1 receptor IGF-I mRNA was localized to the sub-epithelial stroma -LRB- SES -RRB- of inter-caruncular and caruncular endometrium in both uterine horns -LRB- Figs. 1 -LRB- A -RRB- and 2A -RRB- . Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma -LRB- DES -RRB- , the caruncular stroma -LRB- not shown -RRB- and myometrium -LRB- Figs. 1 -LRB- C -RRB- , -LRB- E -RRB- and 2C , E -RRB- . Overall expression of IGF-I mRNA was higher in the inter-caruncular than caruncular SES -LRB- P = 0.001 , Table 2 -RRB- . There was a significant horn × region interaction -LRB- P = 0.032 -RRB- , with lower levels of IGF-I transcript in the inter-caruncular SES of the PG compared with the PNG horn -LRB- Fig. 3 -LRB- A -RRB- -RRB- . IGF-II expression was higher in the DES than in the caruncular stroma and myometrium -LRB- P ≤ 0.001 , Table 2 -RRB- . When data from tissue regions were pooled the concentration of IGF-II mRNA did not vary between the PNG and PG horns , but there was a significant horn × region interaction -LRB- P ≤ 0.001 -RRB- . Expression of IGF-II in the DES and caruncular stroma was lower in the PG than PNG horn , whereas within myometrium expression was higher in the PG than PNG horn -LRB- Fig. 3 -LRB- B -RRB- -RRB- . For the IGF-1R , expression was highest in myometrium and similar between DES and caruncular stroma -LRB- Table 2 and Fig. 3 -LRB- C -RRB- -RRB- . Overall , the level of IGF-1R transcript was higher -LRB- P = 0.030 -RRB- in the PNG than PG horn -LRB- Table 2 -RRB- . The horn × region interaction was not significant for uterine IGF-1R mRNA expression . 3.2 Expression of IGFBPs IGFBP-1 mRNA could not be detected in any uteri examined , despite expression being observed in the ovine estrous uterus which was used as positive control tissue -LRB- data not shown -RRB- . IGFBP-2 , IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue , and in the DES and caruncular stroma -LRB- Figs. 1 -LRB- G -RRB- , -LRB- K -RRB- , -LRB- O -RRB- and 2G , K , O -RRB- . IGFBP-4 mRNA was additionally expressed in myometrium . In contrast , IGFBP-3 mRNA expression was only detected in the luminal epithelium -LRB- LE -RRB- of both inter-caruncular and caruncular samples -LRB- Figs. 1 -LRB- I -RRB- and 2I -RRB- . IGFBP-5 mRNA was found in myometrium , inter-caruncular and caruncular SES and caruncular stroma -LRB- Figs. 1 -LRB- M -RRB- and 2M -RRB- . IGFBP-2 mRNA expression in inter-caruncular and caruncular SES was higher than in DES and caruncular stroma -LRB- P ≤ 0.001 , Table 2 -RRB- . There was no main effect of horn , but there was a horn × region interaction -LRB- P = 0.034 -RRB- . Within caruncular stroma only , the concentration of IGFBP-2 mRNA was higher in the PG than the PNG uterine horn -LRB- Fig. 4 -LRB- A -RRB- -RRB- . For IGFBP-3 mRNA the main effects of uterine horn and tissue region were not significant but there was an interaction -LRB- P ≤ 0.001 -RRB- . Expression in the inter-caruncular LE was higher in the PNG than PG horn , whereas in the caruncular LE expression was higher in the PG uterine horn -LRB- Fig. 4 -LRB- B -RRB- -RRB- . Expression levels of IGFBP-4 mRNA varied between tissue regions , with higher expression in the caruncular than inter-caruncular SES , lowest expression in myometrium , and intermediate signal intensity in the DES and caruncular stroma -LRB- Table 2 -RRB- . There was no difference in transcript levels between the PNG and PG uterine horns when regional data were combined -LRB- Table 2 -RRB- . Levels of IGFBP-4 mRNA expression were , however , affected by an interaction between uterine horn and tissue region -LRB- P = 0.024 -RRB- : within DES expression was lower in the PG than PNG uterine horn -LRB- Fig. 4 -LRB- C -RRB- -RRB- . Expression of IGFBP-5 mRNA was highest in myometrium , intermediate in the inter-caruncular and caruncular SES and lowest in caruncular stroma -LRB- P ≤ 0.001 , Table 2 -RRB- . When regional expression data were pooled , the PNG uterine horn expressed higher concentrations of IGFBP-5 mRNA than the PG horn -LRB- P ≤ 0.001 , Table 2 -RRB- . There was also a significant effect of the interaction between uterine horn and tissue region -LRB- P ≤ 0.001 -RRB- . Expression in both the inter-caruncular SES and the caruncular stroma was lower in the PG than PNG uterine horn whereas for the caruncular SES the reverse was true -LRB- Fig. 4 -LRB- D -RRB- -RRB- . IGFBP-6 mRNA was expressed at higher concentrations in the inter-caruncular and caruncular SES than in DES and caruncular stroma -LRB- P ≤ 0.001 , Table 2 -RRB- . Transcript levels were higher -LRB- P ≤ 0.001 -RRB- in the PNG than PG uterine horn when regional expression data were pooled -LRB- Table 2 -RRB- . The interaction between uterine horn and tissue region was significant -LRB- P = 0.045 -RRB- . Expression in each of the inter-caruncular SES , caruncular SES , DES and caruncular stroma was lower in the PG than PNG horn -LRB- Fig. 4 -LRB- E -RRB- -RRB- . 4 Discussion The rate of uterine involution is an important factor influencing the subsequent fertility of dairy cows -LSB- 24 -RSB- . In this study we have investigated for the first time a possible role for the IGF family of proteins in this event in lactating dairy cows . The timing of tissue collection at approximately day 14 postpartum occurred when the PG uterine horn in our group of multiparous cows was larger than the PNG horn . At this stage caruncular tissue is expected to have undergone degeneration and sloughing , but not to have completed re-epithelialisation -LSB- 2 -RSB- . In contrast , the inter-caruncular area does not lose its epithelial layer -LSB- 32 -RSB- and recovers from pregnancy more quickly -LSB- 2 -RSB- . The ongoing process of uterine involution at the time of tissue collection was thus expected to involve tissue regeneration alongside size recovery . An adequate recovery process may be crucial in preventing the uterus , which is heavily contaminated with bacteria following calving -LSB- 1 -RSB- , from developing endometritis . Samples were analysed using in situ hybridisation . Whilst this approach is considered only semi-quantitative , we have found the technique described here to be highly repeatable . Furthermore , it enables measurement of mRNA concentrations in individual cellular types . This is not feasible in a complex organ such as the uterus using alternative techniques such as RT-PCR , as it is not readily possible to separate different populations of epithelial and stromal cells for RNA extraction . IGF-I mRNA was localised to the SES , confirming earlier observations in the cow -LSB- 33 -RSB- and sheep -LSB- 20,34 -RSB- . Normal wound healing involves a sequence of inflammation , proliferation , and maturation or remodelling -LSB- 12 -RSB- and local IGF-I production increases as wound healing progresses -LSB- 35 -RSB- . Since IGF-I increases during the late proliferative phase of the human menstrual cycle -LSB- 36 -RSB- , and is known to stimulate cell proliferation and collagen synthesis during tissue regeneration -LSB- 14,35 -RSB- , we propose that IGF-I produced by SES may act in an autocrine and/or paracrine manner to stimulate the proliferation of uterine stroma and epithelium -LSB- 37,38 -RSB- during uterine involution . In early pregnancy the bovine endometrium synthesises IGF-II primarily within caruncular stroma -LSB- 33 -RSB- . The present study localised IGF-II mRNA at similar concentrations in both the caruncular stroma and myometrium . The strongest expression of IGF-II mRNA was , however , in the DES . Similar results were found in human endometrium -LSB- 36 -RSB- . IGF-1R was similarly localised to the DES , caruncular stroma and myometrium , confirming earlier observations in the bovine uterus -LSB- 33 -RSB- . Since the effects of IGF-II are probably mediated by the IGF-1R -LRB- for a review see -LSB- 39 -RSB- -RRB- , the co-localisation of IGF-II and IGF-1R transcripts supports a local action for IGF-II in uterine repair and regeneration within both endometrial and caruncular stroma -LSB- 16,21 -RSB- . Stromal IGF-II may also act in a paracrine manner to stimulate epithelial cell proliferation -LSB- 22 -RSB- . The interaction between IGFs and their receptors in muscle growth and regeneration has been comprehensively reviewed by Florini et al. -LSB- 40 -RSB- . In myometrium , IGF-II may stimulate muscle growth and regeneration -LSB- 40 -RSB- , and potentially increases muscle strength -LSB- 41 -RSB- . These actions would support myometrial contractions that return the uterus to its non-pregnant size , shape and tone -LSB- 5 -RSB- . Since the PG horn has to contract from a larger size at parturition , the higher concentration of IGF-II in the PG myometrium supports the proposal that IGF-II assists postpartum uterine size recovery . In rat myometrium the IGF-1R is up-regulated in the early postpartum period -LSB- 42 -RSB- . This study failed to detect IGFBP-1 mRNA in any postpartum uteri . During the estrous cycle IGFBP-1 expression is low at estrous and relatively higher during the luteal phase -LSB- 33 -RSB- concurrent with progesterone production . Since the cows in this study had yet to establish ovulatory cycles , then the uterus would not have been recently exposed to progesterone stimulation -LSB- 43 -RSB- . In ruminants IGFBP-1 appears to be involved in pregnancy recognition -LSB- 33,44 -RSB- rather than , as this study shows , postpartum uterine events . In agreement with previous studies -LSB- 33,44 -RSB- , the expression of uterine IGFBP-2 mRNA was localised to the SES and at relatively lower levels in the DES and caruncular stroma . In the cyclic cow IGFBP-2 mRNA levels increased during the luteal phase , concomitant with the highest levels of plasma progesterone -LSB- 19 -RSB- . Other studies have shown that human endometrial cells constitutively synthesise and secrete IGFBP-2 in vitro -LSB- 45 -RSB- and in response to estradiol -LSB- 46 -RSB- . In the bovine mammary gland -LSB- 47 -RSB- , IGF-I may stimulate IGFBP-2 expression and protein secretion , whereas in fetal visceral glomerular epithelial cells isolated from human kidneys IGFBP-2 production was stimulated by IGF-II -LSB- 48 -RSB- . The exact mechanisms regulating postpartum uterine IGFBP-2 mRNA expression thus requires further investigation . IGFBP-2 is presumably modulating uterine involution indirectly by regulating the bioavailability of IGF-I and IGF-II and the interaction of these ligands with their receptors -LSB- 39 -RSB- . The precise action of IGFBP-2 also remains uncertain . Both stimulatory -LSB- 22 -RSB- and inhibitory -LSB- 48 -RSB- actions of IGFBP-2 on IGF-stimulated epithelial cell proliferation have been suggested . Alternatively or additionally , IGFBP-2 could modulate uterine cell growth directly -LSB- 22 -RSB- . In contrast to all the other binding proteins investigated , the expression of IGFBP-3 mRNA was confined to the luminal epithelium , again agreeing with earlier work in the cyclic animal -LSB- 33 -RSB- . Epithelial IGFBP-3 may regulate local IGF bioavailability -LSB- 39 -RSB- or transport IGFs across this cell layer -LSB- 49,50 -RSB- for secretion into the uterine lumen . Removing excess IGF from the endometrium would prevent the IGF-1R from being down-regulated -LSB- 51 -RSB- . Alternatively , since IGFBP-3 associates with cell surfaces , then it may store IGFs -LSB- 52 -RSB- in the uterus and further promote IGF-stimulated tissue repair -LSB- 37,39 -RSB- as proposed for other physiological systems -LSB- 53 -RSB- . IGFBP-4 mRNA was detected in multiple uterine tissue compartments . The localisation in SES and caruncular stroma has also been found in the pregnant ewe -LSB- 54 -RSB- and synthesis in the DES and myometrium agrees with studies in the human and pregnant bovine uterus -LSB- 44,55 -RSB- . IGFBP-4 is generally considered inhibitory to IGF actions -LSB- 56 -RSB- . IGFBP-4 does not appear to bind to the cell surface or extracellular matrix , but can cross the endothelium -LSB- 39 -RSB- , indicating that IGFBP-4 may clear endometrial IGFs . IGFBP-5 mRNA was localised to caruncular stroma and myometrium , in agreement with previous studies in the cow -LSB- 33 -RSB- and sheep -LSB- 34 -RSB- , and transcript was also detected in the SES . With a lack of detectable IGFBP-2 , IGFBP-3 , and IGFBP-6 mRNA alongside relatively low levels of IGFBP-4 mRNA in the myometrium , the abundance of IGFBP-5 in this tissue suggests this binding protein as the primary regulator of local IGF bioavailability -LSB- 57 -RSB- . In the rat myometrium , IGFBP-5 mRNA is significantly up-regulated after parturition , which is suggested to support tissue remodelling during involution -LSB- 42 -RSB- . It is also possible that within the myometrium , IGFBP-5 is directly stimulating muscle cell survival during myogenesis -LSB- 57,58 -RSB- . In stromal fibroblasts , IGFBP-5 can adhere to the extracellular matrix , which decreases its affinity for IGF and can potentiate IGF-stimulated DNA synthesis -LSB- 39,59 -RSB- . Furthermore , IGFBP-5 may stimulate local tissue growth independently of IGF -LSB- 39,59 -RSB- . IGFBP-6 was localised to SES and at lower levels in DES and caruncular stroma , similar to the non-pregnant ovine uterus -LSB- 28 -RSB- . This expression pattern parallels that of IGFBP-2 mRNA and may suggest these two binding proteins are co-regulated -LSB- 60 -RSB- . IGFBP-6 has a markedly higher affinity for IGF-II -LSB- 61 -RSB- and so the major function of IGFBP-6 is probably to regulate IGF-II actions -LSB- 62,63 -RSB- . Furthermore , IGFBP-6 is generally considered to inhibit the effects of IGF-II , including cell proliferation and differentiation -LSB- 59 -RSB- . The importance of controlling uterine IGF bioactivity has been demonstrated in the human uterus where low levels of IGFBP-6 and higher levels of IGF-II are associated with uterine leiomyomas -LRB- fibroids -RRB- , compared with normal endometrium -LSB- 64 -RSB- . Many members of the IGF family showed differential expression between the two uterine horns . Expression of IGF-I , the IGF-1R and IGFBP-6 was at an overall lower level in the previously gravid horn whereas IGFBP-4 mRNA expression was lower in the DES only . IGF-II , IGFBP-3 and IGFBP-5 expression showed horn by region interactions , with mRNA concentrations reduced for some regions but increased for others . These differences may reflect the temporal misalignment of the horns in their rate of tissue remodelling , with the PG horn lagging behind the PNG horn by up to 15 days -LSB- 7 -RSB- . In conclusion , the IGF system is significant to uterine function and the synthesis of these growth factors in the postpartum uterus indicates a role in uterine involution . This study has shown that in the postpartum bovine uterus IGF-I synthesis was localised to sub-epithelial stroma , whilst maximum concentrations of IGF-II and IGF-1R mRNA were in the endometrial stroma and myometrium , respectively . The uterine tissue compartments expressed different profiles of IGF-binding proteins , indicating that IGF bioavailability and bioactivity is differentially regulated throughout the regenerating endometrium . IGFs are presumably supporting the tissue repair that follows parturition , similar to that of normal wound healing -LSB- 12 -RSB- . We propose that myometrial IGF-II synthesis stimulates tissue recovery in an autocrine manner , which may assist the uterus returning to its non-pregnant shape and size . Although IGFs may be key physiological mediators of endometrial repair , other growth factors and cytokines are undoubtedly also important in this process -LSB- 11,65 -RSB- . Conflicts of interest The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work .

O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


Medication

administered

to

children

from

to

years

in

the

Avon

Longitudinal

Study

of

Parents

and

Children

-LRB-

ALSPAC

-RRB-

Objective

To

present

data

on

the

parentally-reported

use

of

all

types

of

medicinal

products

in

children

from

to

years

in

a

large

cohort

in

south-west

England

Introduction

It

is

now

well

recognised

that

there

is

a

need

for

more

studies

to

obtain

paediatric

information

on

medicines

used

in

children

with

the

aim

of

reducing

the

amount

of

unlicenced

and

off-label

use

of

medicines

in

this

population

In

of

children

under

and

of

children

aged

were

reported

as

having

a

long-standing

illness

-LSB-

-RSB-

In

addition

young

children

may

experience

a

range

of

relatively

minor

ailments

requiring

medical

treatment

A

previous

report

using

data

from

this

cohort

showed

that

cold

cough

high

fever

colic/stomach

ache

diarrhoea

and

vomiting

were

very

common

symptoms

in

the

pre-school

years

and

that

cough

cold

earache

and

high

temperature

are

the

most

common

symptoms

to

present

to

medical

services

-LSB-

-RSB-

In

million

prescription

items

were

dispensed

in

the

community

for

children

under

at

a

cost

of

million

-LSB-

-RSB-

This

represents

an

average

of

items

per

head

in

the

age

range

years

-LSB-

-RSB-

The

categories

of

drugs

most

frequently

prescribed

for

children

are

antibacterials

analgesics

and

bronchodilators

-LSB-

-RSB-

Prescribing

outside

the

terms

of

the

product

licence

occurs

in

drugs

commonly

prescribed

in

general

practice

such

as

systemic

antibiotics

-LSB-

-RSB-

Although

children

are

entitled

to

free

medicines

on

a

National

Health

Service

prescription

in

the

UK

parents

may

also

choose

to

purchase

products

over-the-counter

-LRB-

OTC

-RRB-

for

various

reasons

such

as

convenience

and

confusion

over

their

entitlements

-LSB-

-RSB-

In

a

US

survey

involving

had

been

given

an

OTC

medication

in

the

last

days

most

commonly

paracetamol

and

cough/cold

medicines

-LSB-

-RSB-

and

a

Norwegian

study

concluded

that

paracetamol

was

an

important

tool

for

parents

in

managing

their

children

's

illnesses

-LSB-

-RSB-

A

significant

proportion

of

OTC

medicines

used

in

children

may

be

purchased

from

non-pharmacy

outlets

where

advice

on

appropriate

use

is

unavailable

-LSB-

-RSB-

This

study

describes

the

patterns

of

parentally-reported

use

of

all

types

of

medicinal

products

in

a

large

community

sample

of

young

children

at

four

data

collection

times

between

the

ages

of

weeks

and

years

Methods

The

data

for

this

study

were

collected

as

part

of

the

Avon

Longitudinal

Study

of

Parents

and

Children

-LRB-

ALSPAC

-RRB-

a

longitudinal

study

designed

to

investigate

the

health

and

development

of

children

-LSB-

-RSB-

The

study

was

approved

by

the

ALSPAC

law

and

ethics

committee

and

the

three

local

research

ethics

committees

Pregnant

women

resident

in

the

three

Bristol-based

health

districts

in

the

former

county

of

Avon

in

southwest

England

and

with

an

expected

delivery

date

falling

between

April

and

December

were

eligible

to

be

enrolled

The

core

ALSPAC

sample

consists

of

women

who

had

a

total

of

study

pregnancies

involving

fetuses

resulting

in

live

births

An

additional

children

from

eligible

pregnancies

not

included

in

the

core

sample

were

included

in

the

study

from

age

Cohort

retention

has

been

good

and

we

are

currently

in

regular

contact

with

approximately

families

Further

details

can

be

obtained

from

the

ALSPAC

website

From

various

studies

performed

at

the

planning

stage

the

Avon

population

was

considered

to

be

broadly

similar

to

that

of

the

whole

of

Great

Britain

After

delivery

the

representative

nature

of

the

sample

was

assessed

As

in

similar

studies

the

ALSPAC

cohort

has

a

slight

shortfall

in

the

less

affluent

families

and

a

shortfall

in

ethnic

minority

mothers

Birth

weights

and

lengths

and

growth

measures

in

ALSPAC

are

very

similar

to

national

standards

-LSB-

-RSB-

The

primary

source

of

data

collection

is

via

postal

questionnaires

about

the

health

development

and

home

environment

of

the

study

children

completed

by

each

child

's

main

carer

usually

the

mother

This

paper

presents

data

obtained

from

four

such

questionnaires

administered

when

the

study

children

were

weeks

and

months

old

Each

of

these

asked

questions

designed

by

the

ALSPAC

study

team

about

the

use

of

medicinal

products

The

questionnaire

-LRB-

sent

out

between

and

-RRB-

asked

the

baby

's

mother

to

Please

list

all

the

ointments

pills

and

medicines

that

have

been

given

to

your

baby

while

he/she

has

been

at

home

Check

have

you

included

ointments

to

prevent

nappy

rash

eyedrops

herbal

remedies

etc.

The

questionnaire

sent

out

between

and

when

the

child

was

months

of

age

-LRB-

years

-RRB-

asked

the

following

Children

often

have

accidents

or

illnesses

that

need

treatment

Please

indicate

which

of

the

following

have

been

given

to

your

child

since

he/she

was

years

old

Cough

medicineAntibiotics/penicillinThroat

medicineVitaminsParacetamol/calpolOintment

for

skinEye

ointmentDiarrhoea

mixture/pillsDimotapp/decongestantEar

dropsEye

dropsIronLaxativesHomeopathic

medicineHerbal

medicineOtherFor

each

category

the

carer

was

asked

to

tick

whether

the

response

was

Never

Yes

for

one

episode

only

or

Yes

for

or

more

episodes

and

If

yes

please

give

full

names

of

substances

if

you

can

Are

there

any

pills

ointments

or

medicines

that

he/she

has

taken

every

day

or

nearly

every

day

for

the

last

months

-LRB-

Include

vitamins

skin

cream

inhaler

laxatives

as

well

as

antibiotics

homeopathic

and

herbal

remedies

etc.

-RRB-

If

yes

please

describe

At

months

-LRB-

years

data

collection

-RRB-

and

months

-LRB-

years

data

collection

-RRB-

the

first

question

was

expanded

slightly

as

follows

Children

often

have

accidents

or

illnesses

that

need

treatment

Please

indicate

which

of

the

following

have

been

given

to

your

child

in

the

last

months

Cough

medicineAntibiotics/penicillinThroat

for

skinEye

ointmentDiarrhoea

mixture/pillsDimotapp

decongestantEar

dropsEye

dropsIronLaxativesHomeopathic

medicineHerbal

medicineAsthma

medicationVaporiserOther

-LRB-

active

ingredients

brompheniramine

phenylephrine

and

phenylpropanolamine

-RRB-

The

tick

box

response

options

the

request

for

full

name

of

the

product

and

the

second

question

were

the

same

as

at

months

After

a

questionnaire

was

sent

out

if

a

response

was

not

received

within

weeks

a

reminder

letter

was

sent

If

the

questionnaire

had

still

not

been

received

after

a

further

weeks

a

second

reminder

letter

was

sent

Finally

if

no

response

had

been

received

after

months

a

member

of

the

study

team

either

rang

the

mother

or

visited

her

home

and

encouraged

or

helped

her

to

complete

the

questionnaire

The

date

of

completion

is

included

on

each

questionnaire

so

whilst

all

returned

questionnaires

are

added

to

the

database

and

included

in

the

overall

response

rate

those

that

were

completed

after

a

defined

cut-off

date

can

be

excluded

from

individual

analyses

Most

of

the

self-completion

responses

on

the

questionnaires

are

self-coding

the

ticked

reply

box

contains

a

printed

number

that

can

be

directly

keyed

However

the

actual

names

of

medicinal

products

used

-LRB-

including

vitamins

and

supplements

herbal

products

homeopathic

remedies

over-the-counter

and

prescription

products

-RRB-

were

collected

as

free

text

responses

An

ALSPAC

drug

dictionary

has

been

developed

based

on

the

World

Health

Organisation

-LRB-

WHO

-RRB-

Drug

Dictionary

-LRB-

first

quarter

version

provided

free

of

charge

by

the

Uppsala

Monitoring

Centre

-RRB-

The

ALSPAC

dictionary

assigns

a

Drug

Name

and

Anatomical

Therapeutic

Chemical

-LRB-

ATC

-RRB-

classification

as

described

in

the

WHO

Drug

Dictionary

to

an

individual

product

The

latter

classification

groups

medications

according

to

the

organ

or

body

system

on

which

they

are

designed

to

act

and

their

chemical

pharmacological

and

therapeutic

properties

Some

drugs

have

several

ATC

codes

as

they

can

be

used

in

different

preparations

on

different

body

systems

The

ALSPAC

dictionary

codes

preparations

and

thus

a

single

ATC

has

been

assigned

to

each

product

In

the

few

cases

where

more

than

one

ATC

code

would

have

been

possible

a

decision

was

made

as

to

the

most

likely

use

in

this

population

Additional

details

about

the

WHO

Drug

Dictionary

can

be

found

on

the

WHO

website

In

cases

where

a

category

of

medicinal

product

was

ticked

as

having

been

used

but

no

further

details

were

provided

a

non-specific

code

for

that

category

has

been

added

to

the

database

of

text

responses

For

the

purposes

of

this

report

all

responses

indicating

that

a

product

in

the

defined

category

had

been

used

have

been

included

thus

indicating

ever

use

of

these

categories

of

medicinal

products

rather

than

taking

into

account

frequency

of

use

Results

The

response

rate

was

good

Table

gives

the

number

of

questionnaires

mailed

and

returned

at

each

age

The

majority

of

questionnaires

were

completed

at

or

close

to

the

intended

age

For

example

of

the

returned

month

questionnaires

had

been

completed

by

months

and

by

months

so

all

data

from

the

month

questionnaires

have

been

included

However

as

the

purpose

of

the

question

at

weeks

was

to

ascertain

what

medicinal

products

were

used

on

the

study

children

as

very

young

babies

a

cut-off

was

applied

so

that

only

questionnaires

completed

before

the

child

was

weeks

old

were

included

Using

this

criterion

at

least

one

questionnaire

was

completed

for

study

children

and

all

were

completed

for

children

Table

number

of

medicinal

products

recorded

per

child

at

each

age

weeks

-LRB-

weeks

-RRB-

monthsNo

of

questionnaires

sent

No

of

completed

Total

no

products

recorded

per

aCompleted

within

weeks

of

birth

For

most

of

the

defined

categories

the

number

of

tick

box

responses

not

supported

with

a

text

response

giving

the

name

of

the

product

increased

between

the

ages

of

and

months

-LRB-

at

weeks

responses

to

the

medication

question

were

all

collected

as

free

text

-RRB-

The

week

questionnaire

was

completed

before

the

child

was

weeks

of

age

in

cases

-LRB-

of

those

sent

of

those

returned

-RRB-

Around

three-quarters

of

study

children

were

exposed

to

some

form

of

medicinal

product

before

weeks

of

age

At

this

age

dermatological

products

were

most

commonly

reported

mainly

those

for

treatment

and

protection

against

nappy

rash

such

as

Sudocrem

a

proprietary

product

containing

zinc

oxide

used

in

of

babies

and

zinc

and

castor

oil

cream

used

in

Simeticone

-LRB-

activated

dimeticone

-RRB-

in

products

for

treatment

of

colic

and

flatulence

such

as

Infacol

was

given

to

of

babies

Gripe

water

-LRB-

terpeneless

dill

seed

oil

and

sodium

bicarbonate

-RRB-

was

used

in

of

the

cohort

Other

commonly

reported

products

included

oral

and

topical

antifungals

and

ophthalmic

antibiotics

-LRB-

mostly

chloramphenicol

-RRB-

A

few

babies

-LRB-

-RRB-

were

given

vitamin

drops

Some

were

given

herbal

drinks

-LRB-

-RRB-

homeopathic

products

-LRB-

-RRB-

and

a

variety

of

topical

complementary

and

alternative

medicine

products

such

as

calendula

and

camomile

creams

-LRB-

-RRB-

Several

OTC

products

not

licensed

for

use

in

this

age

group

were

reported

For

example

inhalant

decongestant

products

not

licensed

for

children

under

months

of

age

Snuffle-babe

Vapour

Rub

-LRB-

eucalyptus

oil

menthol

and

thyme

oil

-RRB-

Karvol

Decongestant

Capsules

for

inhalation

-LRB-

levomenthol

with

chlorobutanol

pine

oils

terpineol

and

thymol

-RRB-

and

Vicks

Vaporub

-LRB-

levomenthol

camphor

eucalyptus

oil

and

turpentine

oil

-RRB-

were

used

in

over

of

the

cohort

and

xylometazoline

nasal

spray

was

used

on

Calpol

-LRB-

paracetamol

-RRB-

which

is

licensed

from

months

was

given

to

over

when

they

were

under

weeks

old

At

the

older

ages

and

months

-LRB-

of

those

mailed

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

questionnaires

were

completed

and

returned

respectively

In

these

latter

questionnaires

relatively

few

children

were

reported

not

to

have

used

any

medicinal

products

in

the

preceding

months

At

months

-LRB-

years

-RRB-

only

children

-LRB-

of

those

returning

the

questionnaire

-RRB-

were

not

reported

as

having

used

a

medicinal

product

since

the

age

of

At

and

months

the

figures

were

-LRB-

-RRB-

and

-LRB-

-RRB-

respectively

Table

shows

the

number

of

products

reported

per

child

at

each

age

Table

shows

the

frequency

of

use

of

medicinal

products

by

ATC

code

at

each

data

collection

time

The

data

are

presented

as

the

total

number

of

drugs

reported

in

an

ATC

class

per

number

of

children

in

which

any

products

within

this

class

were

used

-LRB-

i.e.

more

than

one

product

in

a

class

may

have

been

reported

per

child

-RRB-

The

pattern

of

use

of

different

categories

of

medicinal

product

varied

with

age

Usage

of

several

product

categories

was

higher

at

months

than

at

or

months

For

example

at

months

children

-LRB-

-RRB-

were

reported

to

have

used

topical

steroids

-LRB-

mostly

hydrocortisone

-RRB-

compared

with

at

months

and

at

months

Paracetamol

-LRB-

as

a

single

ingredient

-RRB-

was

recorded

for

children

-LRB-

-RRB-

at

months

-LRB-

-RRB-

at

months

and

-LRB-

-RRB-

at

months

In

addition

systemic

antibiotics

-LRB-

mainly

amoxicillin

-RRB-

laxatives

-LRB-

mostly

lactulose

-RRB-

treatments

for

diarrhoea

-LRB-

mainly

rehydration

salts

-RRB-

various

cough

and

cold

products

emollient

and

protective

skin

preparations

-LRB-

mostly

paraffin-based

proprietary

products

-RRB-

topical

antipruritics

antihistamines

and

local

anaesthetic

products

were

all

greater

at

months

than

at

the

older

ages

Conversely

use

of

other

categories

such

as

throat

preparations

-LRB-

sprays

and

lozenges

-RRB-

and

anti-inflammatory

products

increased

with

increasing

age

Use

of

asthma

medication

-LRB-

mainly

salbutamol

and

beclomethasone

-RRB-

also

increased

with

increasing

age

at

months

of

the

cohort

reported

use

of

anti-asthmatics

by

months

this

had

increased

to

and

to

at

months

Of

the

children

reported

as

having

used

asthma

medication

at

months

-LRB-

-RRB-

had

previously

had

a

diagnosis

of

asthma

from

a

doctor

and

-LRB-

-RRB-

reported

having

asthma

in

the

past

months

Table

of

use

of

medicinal

products

by

ATC

code

at

each

age

Total

number

of

drugs

reported

in

a

class/number

of

children

in

which

this

class

of

product

was

used

-LRB-

individual

children

may

have

used

more

than

one

product

in

a

class

-RRB-

ATC

level

-LRB-

in

bold

-RRB-

includes

all

reported

cases

ATC

level

is

given

where

or

more

children

reported

a

product

in

this

category

in

at

least

one

questionnaireATC

code

months

months

months

yearsNo

of

questionnaires

returned

with

ATC

text

AGastrointestinal

drugs

for

peptic

ulcer

and

anticholinergics

and

intestinal

anti-inflammatories

and

and

blood

forming

DDermatologicals

for

dermatological

and

including

antihistamines

anaesthetics

and

chemotherapeutics

and

dermatological

system

and

sex

hormones

excl

sex

hypothalamic

hormones

and

for

systemic

anti-infectives

for

systemic

for

systemic

LAntineoplastic

and

immunomodulating

and

antirheumatic

-LRB-

antidepressants/stimulants

-RRB-

insecticides

and

scabicides

insecticides

and

cold

for

systemic

and

otological

other

therapeutic

products

-LRB-

including

CAM

-RRB-

other

non-therapeutic

products

-LRB-

eg

baby

toiletries

-RRB-

Treatments

for

less

common

conditions

were

also

reported

by

this

community

sample

By

months

children

were

using

insulin

reported

methylphenidate

-LRB-

for

attention

deficit

hyperactivity

disorder

-RRB-

and

reported

thyroid

therapy

At

months

children

reported

antiepileptic

medication

but

this

had

reduced

to

children

by

months

Between

and

months

a

very

small

number

of

children

were

reported

to

have

been

given

an

OTC

medicine

whilst

they

were

younger

than

the

age

specified

on

the

Patient

Information

Leaflet

or

Summary

Product

Characteristics

for

example

aspirin

Dequadin

lozenges

-LRB-

dequalinium

chloride

-RRB-

Night

Nurse

Liquid

-LRB-

paracetamol

pomethazine

hydrochloride

dextromethorphan

hydrobromide

-RRB-

and

Diocalm

-LRB-

morphine

hydrochloride

and

attapulgite

-RRB-

Discussion

The

advantage

of

collecting

data

on

children

's

use

of

medicinal

products

from

their

parents/carers

as

opposed

to

from

medical

records

and

prescription

databases

is

that

the

former

are

in

a

position

to

know

what

was

actually

given

to

their

children

rather

than

what

may

have

been

prescribed

but

not

collected

or

taken

by

the

child

In

addition

the

data

potentially

includes

medication

obtained

from

all

sources

GP

's

pharmacies

supermarkets

CAM

practitioners

friends

relatives

and

neighbours

However

potential

issues

include

the

accuracy

of

recall

-LRB-

especially

over

long

periods

of

months

as

used

at

the

older

ages

in

this

study

-RRB-

and

parental

knowledge

and

understanding

about

the

medicines

given

to

their

child

These

data

were

collected

over

the

period

Between

and

the

annual

number

of

children

aged

years

prescribed

antibacterial

drugs

ranged

from

to

per

-LSB-

-RSB-

This

compares

favourably

with

the

current

study

where

the

number

reporting

use

of

antibiotics

over

the

previous

months

was

per

at

years

of

age

At

years

the

rate

was

lower

at

per

but

these

latter

data

reflect

use

of

antibiotics

between

and

when

a

substantial

decline

occurred

in

the

prescribing

of

antibiotics

by

general

practitioners

in

an

effort

to

curb

the

spread

of

antibiotic

resistance

-LSB-

-RSB-

Children

under

are

known

to

be

the

greatest

users

of

antibiotics

-LSB-

-RSB-

and

the

data

from

this

study

are

consistent

with

this

in

that

antibiotic

use

reported

at

years

was

considerably

greater

than

at

and

years

The

number

of

children

aged

years

prescribed

bronchodilators

over

the

period

ranged

between

and

per

boys

and

per

girls

-LSB-

-RSB-

The

prevalence

of

treated

asthma

in

the

same

age

group

was

per

boys

and

per

girls

and

around

of

these

are

prescribed

bronchodilators

-LSB-

-RSB-

In

this

study

the

reported

use

of

all

asthma

medication

for

boys

and

girls

combined

was

per

at

years

and

per

at

years

Thus

the

figures

from

this

study

do

not

suggest

a

lack

of

recall

Indeed

at

years

the

reported

use

of

asthma

medication

is

slightly

higher

than

the

national

figures

However

as

the

latter

combine

data

over

a

age

range

-LRB-

years

-RRB-

any

differences

at

individual

ages

may

be

masked

Therefore

as

the

questionnaire

data

on

use

of

prescribed

antibiotics

and

anti-asthmatics

in

this

study

is

in

the

range

expected

from

prescription

data

parentally-reported

use

of

non-prescription

medication

in

this

cohort

is

also

likely

to

be

reasonably

reliable

The

findings

suggest

that

most

children

use

some

form

of

medication

from

a

very

young

age

Medicines

available

over-the-counter

were

widely

used

although

it

was

not

possible

to

determine

from

these

data

whether

they

were

actually

purchased

over-the-counter

or

prescribed

The

reason

for

use

was

not

collected

so

it

was

also

not

possible

to

assess

to

what

extent

products

were

appropriately

used

For

example

paracetamol

has

been

found

to

be

used

by

parents

to

calm

and

settle

their

children

and

aid

sleep

-LSB-

-RSB-

Some

products

were

used

in

children

younger

than

the

age

for

which

they

were

licensed

It

was

not

possible

to

assess

general

adherence

to

information

given

in

the

patient

information

leaflets

or

whether

advice

was

sought

prior

to

using

a

product

outside

the

specifications

but

parents

of

young

children

should

be

encouraged

to

follow

the

labelling

advice

or

seek

advice

from

a

health

professional

The

ALSPAC

database

provides

a

resource

to

investigate

the

use

of

commonly

used

medications

with

later

health

and

development

for

example

paracetamol

which

is

widely

used

and

may

be

associated

with

development

of

asthma

-LSB-

-RSB-

Future

work

may

also

examine

differences

in

usage

by

sex

of

the

child

and

socio-economic

status

of

the

parents

B O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O